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Modulation of ASIC1a function by sigma-1 receptors

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Title:
Modulation of ASIC1a function by sigma-1 receptors physiological and pathophysiological implications
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Herrera, Yelenis
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University of South Florida
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Calcineurin   ( mesh )
Receptors, N-Methyl-D-Aspartate   ( mesh )
Receptors, sigma   ( mesh )
Ion Channels   ( mesh )
Acidosis   ( mesh )
ASIC channel   ( mesh )
Ischemia   ( mesh )
Neuroprotective Agents   ( mesh )
Intracellular calcium
Whole-cell currents
AKAP150
Calcineurin
G protein
Neurotransmission
Glutamate
VGCC
NMDA receptor
Dissertations, Academic -- Molecular Pharmacology and Physiology -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: Acid-sensing ion channels (ASIC) are a class of ligand gated plasma membrane ion channels that are activated by low extracellular pH. During ischemia, ASIC1a are activated and contribute to the demise of neurons. Pharmacological block of ASIC1a provides neuroprotection at delayed time points. However, no endogenous receptors have been implicated in the modulation of ASIC1a activity. The hypothesis presented is that sigma receptor activation inhibits ASIC1a function and ASIC1a-induced Ca²⁺i elevations during acidosis and ischemia, which may be a mechanism by which sigma ligands provide neuroprotection following stroke. This hypothesis is based on the following observations: First, sigma receptors regulate multiple ion channels that become activated during ischemia. Second, ASIC1a remain functionally active hours beyond the ischemic insult and sigma receptors have been shown to be neuroprotective at delayed time points following stroke. ^Ratiometric Ca²⁺ fluorometry and whole-cell patch clamp experiments showed that sigma-1 receptor activation depresses elevations in Ca²⁺i and membrane currents mediated by ASIC1a channels in cortical neurons. Furthermore, most of the elevations in Ca²⁺i triggered by acidosis are the result of Ca²⁺ channels opening downstream of ASIC1a activation. Stimulation of sigma-1 receptors effectively suppressed these secondary Ca²⁺ fluxes both by inhibiting ASIC1a and the other channels directly. The signaling cascade linking sigma-1 receptors and ASIC1a was determined to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein 150/calcineurin complex, which resulted in a decrease of acid-induced Ca²⁺i elevations and ASIC1a-mediated currents. Furthermore, immunohistochemical studies confirmed that sigma-1 receptors, ASIC1a and AKAP150 colocalize in the plasma membrane of cortical neuron cell bodies and in the dendritic processes of these cells. Additionally, concurrent exposure to acidosis and ischemia resulted in synergistic potentiation of Ca²⁺i dysregulation. Although ASIC1a activation does not induce long-lived priming of synaptic vesicles for release, channel activation does have a temporal effect on ischemia-mediated Ca²⁺i increases after ischemia onset. Moreover, presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission and thus enhance postsynaptic Ca²⁺i elevations. Sigma-1 receptor activation decreased ischemia-mediated Ca²⁺ dysregulation at pH values of 7.4 - 6.0 and prevented the synergistic interaction between ischemia and acidosis.
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Dissertation (Ph.D.)--University of South Florida, 2009.
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Includes bibliographical references.
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by Yelenis Herrera.
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Document formatted into pages; contains 209 pages.
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Includes vita.

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ABSTRACT: Acid-sensing ion channels (ASIC) are a class of ligand gated plasma membrane ion channels that are activated by low extracellular pH. During ischemia, ASIC1a are activated and contribute to the demise of neurons. Pharmacological block of ASIC1a provides neuroprotection at delayed time points. However, no endogenous receptors have been implicated in the modulation of ASIC1a activity. The hypothesis presented is that sigma receptor activation inhibits ASIC1a function and ASIC1a-induced [Ca]i elevations during acidosis and ischemia, which may be a mechanism by which sigma ligands provide neuroprotection following stroke. This hypothesis is based on the following observations: First, sigma receptors regulate multiple ion channels that become activated during ischemia. Second, ASIC1a remain functionally active hours beyond the ischemic insult and sigma receptors have been shown to be neuroprotective at delayed time points following stroke. ^Ratiometric Ca fluorometry and whole-cell patch clamp experiments showed that sigma-1 receptor activation depresses elevations in [Ca]i and membrane currents mediated by ASIC1a channels in cortical neurons. Furthermore, most of the elevations in [Ca]i triggered by acidosis are the result of Ca channels opening downstream of ASIC1a activation. Stimulation of sigma-1 receptors effectively suppressed these secondary Ca fluxes both by inhibiting ASIC1a and the other channels directly. The signaling cascade linking sigma-1 receptors and ASIC1a was determined to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein 150/calcineurin complex, which resulted in a decrease of acid-induced [Ca]i elevations and ASIC1a-mediated currents. Furthermore, immunohistochemical studies confirmed that sigma-1 receptors, ASIC1a and AKAP150 colocalize in the plasma membrane of cortical neuron cell bodies and in the dendritic processes of these cells. Additionally, concurrent exposure to acidosis and ischemia resulted in synergistic potentiation of [Ca]i dysregulation. Although ASIC1a activation does not induce long-lived priming of synaptic vesicles for release, channel activation does have a temporal effect on ischemia-mediated [Ca]i increases after ischemia onset. Moreover, presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission and thus enhance postsynaptic [Ca]i elevations. Sigma-1 receptor activation decreased ischemia-mediated Ca dysregulation at pH values of 7.4 6.0 and prevented the synergistic interaction between ischemia and acidosis.
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Modulation of ASIC1a Function by Sigm a-1 Receptors: Physiological and Pathophysiological Implications by Yelenis Herrera A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences Department of Molecular P harmacology and Physiology College of Medicine University of South Florida Major Professor: Javier Cuevas, Ph.D. Keith R. Pennypacker, Ph.D. Eric S. Bennett, Ph.D. Jay B. Dean, Ph.D. Alison Willing, Ph.D. ZhiGang Xiong, Ph.D. Date of Approval: February 27, 2009 Keywords: intracellular calcium, whol e-cell currents, AKAP150, calcineurin, G protein, neurotransmi ssion, glutamate, VGCC, NMDA receptor Copyright 2009, Yelenis Herrera

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DEDICATION Those who have inspired me throughout my life and scientific career are those that I love the most. This disse rtation is dedicated to them my husband and family. I would like to thank my Mo m and Dad for giving me the opportunity to be free, succeed and pursue my educatio n. To my brother, you are the best and I am very proud of you; thank y ou for always being there and for your support. I would like to give a special t hanks to my husband, Roger, for his love and always putting a smile in my face, and for his understanding, support and encouragement in making this dream possi ble. This accomplishment would have not been feasible if it was not for all of you. Thank you all and I love you all very much. I would also like to dedicate this thesis to my baby, Dukey.

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ACKNOWLEDGMENTS First of all, I would to thank my ment or, Dr. Javier Cuevas, for giving me the opportunity to grow as a scient ist, for his support, inspiration and encouragement throughout the years. I would also like to thank my committee members, Drs. Keith R. Pennypacker, Eric S. Benne tt, Jay B. Dean and Alison Willing, for their scientific advice and support. I would like to extend my gratitude to my external examiner, Dr. ZhiGang Xiong, for accepting to chair my dissertation committee and for his construc tive comments on the dissertation. I would like to thank all the member s of Dr. Cuevas’ laboratory, Dr. Christopher Katnik, Trimetria Bonds, Cryst al Reed, Adam Behensky and Michelle Cortes-Salva, for their help and support all these years. Last but not least, I would like to thank the Chair, Dr. Bruce G. Lindsey, and staff, Bridget Shields and Barbara Nic holson, of the departm ent of Molecular Pharmacology and Physiology for all their assistance. Thank you all very much. This work was funded in part by a Mc Knight Doctoral Fellowship from the Florida Education Fund.

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i TABLE OF CONTENTS LIST OF FI GURES ............................................................................................... iii ABSTRACT ......................................................................................................... vii CHAPTER 1 INTR ODUCTION ............................................................................. 1 Significance ....................................................................................................... 1 Backgroun d ....................................................................................................... 1 Pathobiology of Stroke ...................................................................................... 4 Acid-Sensing Ion C hannels (ASI C) ................................................................... 8 ASIC1a Channels, Calcineurin an d Scaffolding Proteins ................................ 13 Sigma Rec eptors ............................................................................................ 16 In Vitro Chemical Ischemia Models ................................................................. 22 CHAPTER 2 SIGMA-1 RECEPT OR MODULATION OF ASIC1a CHANNELS AND ASIC1a-INDUCED Ca2+ INFLUX IN RAT CORTICAL NEURON S.......................................................................................................... 25 Introducti on ..................................................................................................... 25 Materials and Methods .................................................................................... 28 Primary Rat Cortical Neuron Prepar ation .................................................... 28 Calcium Imaging Me asurements ................................................................. 29 Electrophysiology Recordi ngs ..................................................................... 30 Solutions and Reagent s .............................................................................. 31 Data Anal ysis .............................................................................................. 31 Result s ............................................................................................................ 32 Discussion ....................................................................................................... 69 CHAPTER 3 SIGMA-1 RECEPTOR ACTIVATION INHIBITS ASIC1a CHANNELS VIA A PERTUS SIS TOXIN SENSITIVE G PROTEIN AND AN AKAP/CALCINEURI N COMPLE X ...................................................................... 75 Introducti on ..................................................................................................... 75 Materials and Methods .................................................................................... 78 Primary Rat Cortical Neuron Prepar ation .................................................... 78 Calcium Imaging Me asurements ................................................................. 78 Electrophysiology Recordi ngs ..................................................................... 79 Immunohistochem istry................................................................................. 80 Confocal Micr oscopy ................................................................................... 81 Solutions and Reagent s .............................................................................. 82 Data Anal ysis .............................................................................................. 82

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ii Result s ............................................................................................................ 83 Discussion ..................................................................................................... 107 CHAPTER 4 SIGMA-1 RECEPTOR I NHIBITION OF INTRACELLULAR Ca2+ DYSREGULATION DUE TO SYNERGISTIC INTERACTION BETWEEN ACIDOSIS AND ISCHEM IA ........................................................... 113 Introducti on ................................................................................................... 113 Materials and Methods .................................................................................. 117 Primary Rat Cortical Neuron Prepar ation .................................................. 117 Calcium Imaging Me asurement s ............................................................... 117 Electrophysiology Recordi ngs ................................................................... 118 Solutions and Reagent s ............................................................................ 118 Data Anal ysis ............................................................................................ 119 Result s .......................................................................................................... 120 Discussion ..................................................................................................... 150 CHAPTER 5 DIS CUSSION .............................................................................. 159 Conclusi ons .................................................................................................. 159 Overall Sign ificanc e ...................................................................................... 176 LIST OF RE FERENCES .................................................................................. 184 APPENDICE S .................................................................................................. 204 Appendix A – Pharmaco logical Co mpounds ................................................. 205 ABOUT THE AUTH OR ........................................................................... E nd Page

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iii LIST OF FIGURES Figure 1.1 Pathobiology of ischemic stroke.………………………………………7 Figure 2.1 ASIC1a blockers inhibi t proton-evoked increases in [Ca2+]i in cultured cortical neurons from embryonic (E18) rats………………46 Figure 2.2 Pan-selective sigma agonist s inhibit proton-evoked transient increases in [Ca2+]i…………………………………………………….48 Figure 2.3 Activation of -1 receptors inhibits ASIC1a-induced increases in [Ca2+]i……………………………………...…………………………....50 Figure 2.4 Inhibition of -1 receptors blocks CBP-mediated suppression of proton-evoked increases in [Ca2+]i……………………………….…..52 Figure 2.5 Sigma-2 receptor ligands i nhibit ASIC1a-mediated elevations in [Ca2+]i at concentrations inconsistent with -2 mediated effects and in a metaphit-insensitive manner……………………………….54 Figure 2.6 Intracellular Ca2+ stores are not involved in DTG modulation of ASIC1a-induced increases in [Ca2+]i………………………………...56 Figure 2.7 ASIC1a-mediated [Ca2+]i increases are membrane potential dependent………………………………………………………………58 Figure 2.8 ASIC1a activation prom otes membrane depolarization...…………60

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iv Figure 2.9 Sigma-1 receptor activation inhibits Ca2+ channels downstream of ASIC1a……………………………………………………………….62 Figure 2.10 Multiple plasma membr ane ion channels downst ream of ASIC1a activation contribute to acidosis-evoked [Ca2+]i increases…….…..64 Figure 2.11 ASIC1a blockers inhibit ac idosis-mediated currents in voltageclamped neurons……………………………………………………....66 Figure 2.12 Sigma receptor agonists inhibit ASIC1a-mediated currents in voltage-clamped neurons……………………………………………..68 Figure 3.1 Sigma-1 receptors colo calize with both ASIC1a channels and AKAP150 in cortical neurons…………………………………………91 Figure 3.2 Calcineurin inhibition prevents -1 receptor modulation of ASIC1a………………………………………………………………….94 Figure 3.3 Sigma-1 receptors inhibi t acid-induced elevations in [Ca2+]i via a PTX-sensitive G protein……………………………………………….96 Figure 3.4 AKAP150 disso ciation from the plas ma membrane prevents -1 receptor modulation of acid-induced elevations in [Ca2+]i…….98 Figure 3.5 Disruption of t he actin cytoskeleton prevents -1 receptor inhibition of acid-induced increases in [Ca2+]i……………………..100 Figure 3.6 Latrunculin A pr eincubation disrupts the actin cytoskeleton…….102

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v Figure 3.7 Sigma receptor-mediated i nhibition of ASIC1a currents are blocked by preincubation in pertussis toxin……………………….104 Figure 3.8 Sigma-1 receptors c ouple to ASIC1a channels via an AKAP150/calcineurin complex……………………………………...106 Figure 4.1 ASIC activation contri butes to ischemia-evoked [Ca2+]i increases in cultured rat cortical neurons………………………….130 Figure 4.2 ASIC activation and ischemia interact to produce a synergistic potentiation of elevations in [Ca2+]i…………………………………132 Figure 4.3 Protons potentiate i schemia-induced increases in [Ca2+]i……….134 Figure 4.4 Inhibition of homomeric ASIC1a channels decreases ischemia + acidosis-induced elevations in [Ca2+]i at pH values ranging from 7.4 to 6.0………………………………………...………………136 Figure 4.5 Heat inactivated PcTx1 venom does not inhibit ischemiainduced increases in [Ca2+]i………………………………………....138 Figure 4.6 PcTx1 peptide inhibits i schemia-induced elevations in [Ca2+]i…..140 Figure 4.7 Temporal effects of acidosis on ischemia-induced Ca2+ dysregulation………………………………………………………….143 Figure 4.8 Sigma-1 receptor activation inhibits ischemia-mediated Ca2+ dysregulation at pH values r anging from 7.4 to 6.0………………145 Figure 4.9 Effects of synaptic transmission inhibition are overcome by presynaptic ASIC1a channels ……………………………………...147

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vi Figure 4.10 TTX inhibits Na+ currents at pH 7.4 and 6.0..……………………..149 Figure 5.1 Signaling cascade li nking ASIC1a channels and sigma-1 receptors.…………………………………….............. ............. ........166 Figure 5.2 Presynaptic ASIC1a channels under physiological conditions.....178 Figure 5.3 Role of presynatic ASIC1a channels duri ng pathological conditions…..……………………………………........ .............. .......181

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vii Modulation of ASIC1a Function by Sigm a-1 Receptors: Physiological and Pathophysiological Implications Yelenis Herrera ABSTRACT Acid-sensing ion channels (ASIC) ar e a class of ligand gated plasma membrane ion channels that are activated by low extracellular pH. During ischemia, ASIC1a are activated and cont ribute to the demise of neurons. Pharmacological block of ASIC1a prov ides neuroprotection at delayed time points. However, no endogenous recept ors have been implicated in the modulation of ASIC1a activity. The hypothesis presented is that sigma receptor activation inhibits ASIC 1a function and ASIC1a-induced [Ca2+]i elevations during acidosis and ischem ia, which may be a mechanism by which sigma ligands provide ne uroprotection following stroke. This hypothesis is based on the followi ng observations: First, sigma ( ) receptors regulate multiple ion channels that bec ome activated during ischemia. Second, ASIC1a remain functionally active hours beyond the ischemic insult and receptors have been shown to be neuroprotective at delayed time points following stroke. Ratiometric Ca2+ fluorometry and whole-cell patch clamp experiments showed that -1 receptor activation depresses elevations in [Ca2+]i and

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viii membrane currents mediated by ASIC 1a channels in cortical neurons. Furthermore, most of the elevations in [Ca2+]i triggered by acidos is are the result of Ca2+ channels opening downstream of ASIC 1a activation. Stimulation of -1 receptors effectively s uppressed these secondary Ca2+ fluxes both by inhibiting ASIC1a and the other channels directly. The signaling cascade linking -1 receptors and ASIC1a was determined to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein 150/calcineurin complex, which resulted in a decrease of acid-induced [Ca2+]i elevations and ASIC1a-medi ated currents. Furthermore, immunohistochemical studies confirmed that -1 receptors, ASIC1a and AKAP150 colocalize in the plasma membrane of cortical neuron cell bodies and in the dendr itic processes of these cells. Additionally, concurrent exposure to acidosis and ischemia resulted in synergistic potentiation of [Ca2+]i dysregulation. Although ASIC1a activation does not induce long-lived priming of synaptic vesicles for release, channel activation does have a temporal effect on ischemia-mediated [Ca2+]i increases after ischemia onset. Moreover, presynapt ic ASIC1a channels promote synaptic transmission during ischemia by overco ming block of neurotransmission and thus enhance postsynaptic [Ca2+]i elevations. -1 receptor actvation decreased ischemia-mediated Ca2+ dysregulation at pH values of 7.4 6.0 and prevented the synergistic interaction between ischemia and acidosis.

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1 CHAPTER 1 INTRODUCTION Significance ASIC are proton activated ion channels expressed in the brain and are involved in several physiological f unctions including neurotransmission and excitability. ASIC are also activated during pathological conditions, like stroke, resulting in Ca2+ dysregulation and eventually neurodegeneration. Furthermore, regulation of ASIC function woul d result in neuroprotection. receptors are known to regulate multiple ion channels in neurons, all of which are activated during ischemia. Taken t ogether, these observations demonstrate the importance of studying the regulati on of ASIC function by receptors under physiological as well as during ischemic conditions and to determine the signaling cascade involved. Results from this study will i dentify potential therapeut ic targets for the treatment of stroke. Background Stroke is the third leading cause of death in the industrialized world behind heart disease and cancer, and the major cause of long-term disability. Stroke is a sudden interruption in the blood supply to the brain. Ischemic (embolic) stroke is

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2 the most common type of stroke, accounting for almost 88% of all strokes, and is caused by a clot or other blockage withi n a cerebral artery. Following a stroke, there are two major regions of injury withi n the ischemic cerebrovascular bed: the core ischemic region and the ischemic penumbra region (Dir nagl et al., 1999). Within minutes of the ischemic insult, t he core area forms. The core region is an area characterized by reduced blood flow, le ading to a loss of adequate supply of oxygen and glucose resulting in rapid depletion of cellular energy stores. Ischemia results in neuronal death and supporti ng cellular elements such as glial cells by necrosis (characteri zed by cell swelling, cell rupture and activation of inflammatory response) wit hin the severely ischemic core area (Dirnagl et al., 1999). The penumbra region is an area that is generally defined to be ischemic but still containing viable cerebral tissue. Brain cells within t he penumbra, a rim of compromised ischemic tissue lying betwe en tissue that is normally perfused and the area in which infarction is evolving, may remain viable for several days (Dirnagl et al., 1999; Lo et al., 2004). The penumbral region is supplied with blood by collateral arteries. However, even cells in this region will die if reperfusion is not established in a timely manner since collateral circulation is inadequate to maintain the neuronal deman d for oxygen and glucose indefinitely. As time goes by, hours and days after t he insult, apoptosis in the penumbra results in cell death and exp ansion of the core (Dirnagl et al., 1999; Lo et al., 2005). Most of research taking place t oday focuses on the prevention of the apoptotic death that occurs within the penum bra region. This may be achieved by

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3 restoring blood flow to the penumbral zone and/or by disrupting several pathways that are activated resulting in neuronal death. The thrombolytic agent, tissue plas minogen activator (tPA), is the only drug approved for clinical treatment of ac ute stroke. tPA cleav es the precursor molecule plasminogen producing the acti ve enzyme plasmin, which in turn dissolves blood clots. Thus, tPA is not a neuroprotective drug. The limitations of tPA arise from the high risk of intracr anial hemorrhage associated with its use, and the short therapeutic window in which it must be administered ( 3 hours) (Dirnagl et al., 1999). Additi onally, tPA can not be admin istered to hemorrhagic stroke patients or patients with either hypertension or diabetes. Therefore, there is only a very small percentage of people (~ 3%) that are candidates for this drug. The significant inadequacy of current ac ute stroke therapy has increased the demand for the development of alternativ e drugs to improve outcome following stroke injury. A major component of current stroke research focuses on excitotoxicity and less is known about acidotoxicity or the interaction of ASIC1a channel activation and ischemia. Currently, ther e are no drugs that directly enhance neuronal survival approved for clinical use. Furthermore, drugs that have targeted a single component in the pathobiology cascade of stroke (e.g. NMDA receptors, glutamate re ceptors and voltage-gated Ca2+ channels) have failed in clinical trials.

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4 Pathobiology of Stroke To better understand neurodegeneration duri ng ischemia, it is important to analyze the pathobiology of stroke. Ischemia is a restriction of blood flow to the brain triggering a series of events that lead to nec rotic neuronal death in the affected infarct area (core region) and delayed apoptotic neuronal death in the penumbra. Brain ischemia is associat ed with glucose and oxygen deprivation, and a cellular switch to anaerobic glycolysis that results in energy failure (Figure 1.1). The accumulation of lactic acid produced by anaerobic glycolysis leads to acidosis in the ischemic region, and aci dotoxicity eventually results in cell death (Xiong et al., 2004; Yermolaie va et al., 2004). Dysregul ation of intracellular calcium (Ca2+) triggers various events that also result in cell death (Figure 1.1). Loss of adenosine triphosphate (ATP) production results in membrane dysfunction and a rapid depolariz ation of neurons promotes Ca2+ entry, which in turn evokes the release of neurotransmi tters, such as glutamate (Figure 1.1). Glutamate, acting via postsynaptic receptor s, elicits depolarization of the neurons and further elevations in intracellular Ca2+ (Figure 1.1). At the same time, the depletion of ATP blocks the reuptake of glutamate, which leads to an accumulation of glutamate at synaptic and extrasynaptic sites. Accumulation of glutamate in the extracellular space l eads to over-stimulation of N-methyl-Daspartate (NMDA) receptors resulting in Ca2+ overload. This becomes a vicious cycle that leads to ion fluxes into the cell via the activation of Cl-, Na+, Ca2+ and K+ channels (Tanaka et al., 1997). The a ccumulation of extracellular K+ allows surrounding neurons to depolarize, whereas the buildup of intracellular Na+

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5 results in cell swelling, or oedem a (Figure 1.1). Intracellular Ca2+ dysregulation promotes neuronal death by disrupting plasma membrane function via activation of Ca2+-sensitive ion channels (Murai et al ., 1997), and by triggering biochemical cascades that lead to proteolysis, lipolys is, and the production of reactive oxygen species (Mattson, 2000) (Figure 1.1). Ca2+ overload and energy depletion leads to activation of secondary events includi ng release of inflammatory mediators, expression of pro-apoptotic genes, all of which result in cell death and subsequent expansion of the core (Figure 1.1). Though not depicted in Figure 1.1, ce ll death and expansion of the core region is also enhanced by inflammati on caused by the immune response. Activated microglia and reactive astro cytes are capable of entering the central nervous system since the blood brain ba rrier is degraded (Stoll et al., 1998). These cells then exacerbate damage by ac tivating proinflammatory mediators, releasing nitric oxide and glutamate (Heales et al., 1999; Hertz et al., 2001; BalPrice et al., 2002; Trendel enburg and Dirnagl, 2005). All these events are also detrimental to the neurons and result in subsequent expansion of the core.

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6 Figure 1.1 Pathobiology of ischemic stroke Intracellular calcium plays a critical role during ischemia. Ischemia leads to ac idosis and acidotoxicity in the ischemic region, which results in further increases in [Ca2+]i. Calcium dysregulation ultimately leads to the activation of all these events that result in cell death and subsequent expansion of the core region.

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7

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8 Acid-Sensing Ion Channels (ASIC) Recently, the acid-sensing ion channel has been shown to be a major contributor to ischemic injury in th e central nervous system. Physiological extracellular and intracellular pH is main tained at ~ 7.3 and 7.0 through various proton transporting mechanisms. Under pathol ogical conditions, like stroke, the metabolic switch to anaerobic glycolysis produc es lactic acid leading to a drop in pH and acidotoxicity in the ischemic regi on. During ischemia, the pH can drop to as low as 6.0 (Nedergaard et al., 1991). More recent studies imaging brains in rats post-MCAO and patients with cortical ischemic stroke symptoms show that lactate accumulations remain elevated for 3-5 days (Weinstein et al., 2004; Munoz Maniega et al., 2008). The decrease in pH is sufficient to trigger the opening of ASIC and consequently, permitting calcium influx into the neurons (Xiong et al., 2004). Both in vitro and in vivo studies have shown that acidotoxicity aggravates ischemic neuronal injury and a direct correlation has been shown between infarct size and brai n acidosis (Xiong et al., 2004; Pignataro et al., 2007). Thus, activa tion of ASIC has now been unequivocally linked to brain injury resulting from i schemic stroke. It has been suggested that low tissue pH may lead to protein and nuc leic acid denaturation, trigger cell swelling via activation of pr oton exchangers, lead to exci totoxicity by inhibiting astrocyte glutamate reuptak e, and damage glial cells (see review (Xiong et al., 2006)). In contrast, mild acidosis has been implicated to be beneficial due to NMDA receptor inhibition by extracellu lar low pH. Therefore, during ischemia,

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9 other calcium influx pathways may ex ist independently of NMDA receptor function. ASIC are a class of ligand-gated ion c hannels that are members of the degenerin/epithelial sodium channel (Deg/ENac) superfamily (Benos and Stanton, 1999). ASIC are expressed in both peripheral and central nervous system neurons and become activated as a resu lt of low extracellular pH caused by ischemia (Xiong et al., 2004; Yermolaie va et al., 2004). Four genes (ASIC1 – ASIC4) encoding for six ASIC subunits have been cloned, and four of those subunits can form functional homomultime ric or heteromultimeric channels that are activated by extracellular protons. Recent stoichiometric studies of the chicken ASIC1a subtype suggests that 3 subunits are required to form a functional channel (Jasti et al., 2007). T he pH of half-maximal activation (pH0.5) of these channels and the tissue expression pattern differs between each subtype. Pertinent to ischemia, ASIC1a has a pH0.5 = 6.2, and is mainly expressed in the brain (Waldmann et al., 1997b; Alva rez de la Rosa et al., 2003). ASIC1a is a nonselective cation channel that is sodium and calcium permeable and generates fast activating and inactivati ng membrane currents (Waldmann et al., 1997b; Chu et al., 2002; Xiong et al., 2004; Yermolaieva et al., 2004). ASIC1b, a splice variant of ASIC1a with a unique N-terminal, has a pH0.5 = 5.9 (Chen et al., 1998) and is only expressed in sensory neurons (Chen et al., 1998; Bassler et al., 2001). ASIC2a is widely expressed in peripheral and central nervous system with a pH0.5 = 4.4 (Garcia-Anoveros et al., 1997; Lingueglia et al., 1997). ASIC2a channels generate slower activating and in activating currents compared to the

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10 ASIC1a subtype. ASIC3 has a pH0.5 = 6.5, and it is predom inantly expressed in neurons of the dorsal root ganglia (Wal dmann et al., 1997a), while ASIC4 is highly expressed in the pituitary gland but does not form functional channels on their own (Akopian et al., 2000; Grunder et al., 2000). It is been established that neither ASIC2b nor ASIC4 can form functi onal homomeric channels, but ASIC2b can associate with other subunits and modulate their activity, and thus biophysical properties of the channels. Fo r example, studies of heteromeric ASIC1a/2a channels have show n that ASIC1a establishe s the current amplitude, while ASIC2a influences desensitization, re covery from desensitization, and pH sensitivity of the channel (Askwith et al., 2004). ASIC contain two transmembrane domai ns flanked by a large cysteinerich extracellular loop and short intracellu lar Nand Ctermini (Waldmann et al., 1997b; Alvarez de la Rosa et al., 2000; S augstad et al., 2004). In the peripheral nervous system, ASIC localize to neur ons innervating skin, heart, gut, and muscle, and have also been detected in t he eye, ear, taste buds, and bone (see review (Wemmie et al., 2006)). These channels are known to be involved in various processes such as mechanor eception (Price et al., 2000), taste transduction (Ugawa et al., 2003), maintenance of retinal integrity (Ettaiche et al., 2004), and nociception (Allen and Attwell, 20 02), particularly in the ischemic myocardium where ASIC are believed to transduce anginal pain (Benson et al., 1999). ASIC1a have a wide spread distri bution in the brain including hippocampus, cerebral cortex, cerebellu m, striatum, habenul a, amygdale, and

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11 olfactory (Wemmie et al., 2002; Alvare z de la Rosa et al., 2003). The ASIC1a subtype have been localized to dendrites and dendritic spines, axons, and throughout neurons with no preferential dist ribution to synapses (Wemmie et al., 2003). In the central nervous system, ASIC1a is involved in synaptic plasticity, fear conditioning, and learning and me mory (Wemmie et al., 2002; Wemmie et al., 2003). It has been shown that lo ss of ASIC1a disrupted hippocampaldependent long term potentiation and spat ial memory, impaired cerebellumdependent learning, and reduc ed fear response in the amygdala (freezing behavior) (Wemmie et al., 2003). Conversely overexpression of ASIC1a in the amygdala and different regions of the br ain leads to increased ASIC1a currents and enhanced context of fear conditioning (anxiety) (Wemmie et al., 2004). ASIC1 and ASIC1 gene knockout studies hav e shown that this ion channel is calcium permeable and is involved in acidosis-mediated ischemic brain injury and neuronal death (Xiong et al., 2004; Yermola ieva et al., 2004; Pignataro et al., 2007). ASIC also belong to the amiloride-s ensitive superfamily and studies have shown that inhibition of ASIC by this nonspecific blocker leads to diminished [Ca2+]i elevations during acidosis. It has been shown that amiloride dosedependently blocked ASIC currents in mouse cortical neurons with an IC50 of 16.4 M (Xiong et al., 2004). It has also been demonstrated that amiloride and its derivatives benzamil and et hylisopropylamiloride (EIPA) reversibly blocked proton-activated inward ASIC currents in sensory neurons (Kd = 10 M) (Waldmann et al., 1997b). Mutation studies have suggested that residues within

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12 the pore and extracellular domain are crit ical for amiloride binding (Kleyman et al., 1999). This observation suggests that the sites that inte ract with amiloride within the channel’s extracellular domain ma y be in close proximity to residues within the channel’s pore r egion. Supporting the notion that amiloride blocks the outer pore of ASIC and Deg/ENac incl ude the voltage dependence of block, kon increases, and koff decreases linearly at hyperpolarizing membrane voltages, and, therefore, indicates amiloride senses 20% of the membrane electrical field (Alvarez de la Rosa et al., 2000). Amiloride affinity for binding the mouth of the pore also decreases by increasing extracel lular sodium concentrations, indicating a competitive mechanism for the por e binding between amiloride and its permeable cation (Alvarez de la Rosa et al., 2000). Studies with the venom of t he South American tarantula Psalmopoeus cambridgei (psalmotoxin 1, PcTx1) have provi ded great insight about the role of ASIC1a during ischemia. PcTx1 is the firs t potent and specific blocker of ASIC1a (Escoubas et al., 2000; Pidoplichko and Dani, 2006; Diochot et al., 2007). It has been shown that PcTx1 blocks ASIC1a cu rrent in heterologous expression systems (IC50=0.9 nM) at pH 6.0 and also at pH 4 or 5 (10 nM PcTX1). In subpopulations of dorsal root ganglia neurons, t he toxin is also effective at nM concentrations (Escoubas et al., 2000) as well as in mice cortical neurons (Xiong et al., 2004). In whole animal studies, administration of the venom (500 ng/ml) following middle cerebral artery occl usion (MCAO) produced a significant reduction in infarct size and volume (Xiong et al., 2004). It has been suggested that the mechanism by which PcTx1 in hibits ASIC1a channels is via chronic

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13 desensitization of the channe l caused by increased affinity of the channel for protons (Chen et al., 2005). Interestingly, this PcTx1-induced shift of the pHdependent inactivation of ASIC1a is Ca2+-dependent, where increasing extracellular Ca2+ results in a decrease of the PcTx1 inhibition. Acidosis occurs within minutes of stro ke onset (mainly affecting the core region but also the inner penumbral zone) (Back et al., 2000) and ASIC1a remain functionally active beyond 4 hr s following the ischemic insult (Pignataro et al., 2007). Additionally, pharmacological block ade of ASIC1a by amiloride or PcTx1 and administration of PcTx1 even 5 hrs af ter middle cerebral artery occlusion (MCAO) diminishes stroke injury (Xi ong et al., 2004; Pignataro et al., 2007). These observations are consistent with st udies showing the pr esence of lactate 72 hours following transient MCAO in rats (W einstein et al., 200 4). It remains to be elucidated whether endogenous recept ors could regulate ASIC1a channel function. ASIC1a Channels, Calcineurin and Scaffolding Proteins Several extracellular and intracellular modulators of ASIC have been identified. It has been established t hat various divalent cations (Zn2+, Pb2+, Ca2+) (Baron et al., 2001; Chu et al., 2004; Gao et al., 2004; Wang et al., 2006), lactate (Immke and McCleskey, 2001), serine proteas es (Poirot et al., 2004) and redox reagents (Andrey et al., 2005; Chu et al., 2006) may interact with the extracellular domain of ASIC and influence the function of the channels. Other investigators have shown that there is a conserved phosphorylation site within the intracellular

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14 C-terminal domain of ASIC1a for calcium/ calmodulin protein kinase II (CaMKII) (Gao et al., 2005), protein kinase C (B aron et al., 2002) and protein kinase A (PKA) (Leonard et al., 2003) to bind. Ho wever, phosphorylation of ASIC1a has been shown to potentiate ASIC1a function in neurons (Xiong et al., 2004; Gao et al., 2005). Calcineurin-dependent dephosph orylation of ASIC1a channels is the only modulator of ASIC1a that results in dow nregulation of channel function (Chai et al., 2007). Calcineurin, a second mess enger, is a serine/threonine phosphatase that is activated by calcium-calmodul in, and a heterodimer consisting of 2 subunits, A and B (Shibasaki et al., 2002; Dodge and Scott, 2003). The calcineurin A subunit has 3 domains consis ting of a catalytic domain, calmodulinbinding domain, and an autoinhibitor y domain. The regulatory subunit, calcineurin B subunit, encodes t he calcium-binding domain. The immunosuppressive drugs, cyclospori ne A and FK-506, are well known calcineurin inhibitors. Cyclosporine A-cyclophilin A and FK-506-FKBP form drugimmunophilin complexes that bind to the ca lcineurin active site heterodimer to inhibit calcineurin activity competitiv ely and thus, covering the catalytic groove (see review (Dumont, 2000; Shibasaki et al., 2002; Martinez-Martinez and Redondo, 2004)). Calcineurin, also known as PPB2, may exist as free calcineurin in the cytosol but some is also found bound to scaffolding protein A-kinase anchoring protein (AKAP). AKAP is a diverse prot ein family with more than 50 members which are abundantly expresse d in the brain (Feliciello et al., 2001). Neuronal

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15 AKAP150 (rat) and AKAP79 (human) s hare a high degree of sequence homology, differing primarily in a 9 amino acid repeat sequence insert found only in rodents which has no known functi on (Dell'Acqua et al., 2006). AKAP150 anchors both kinases (cAMP-dependent Prot ein Kinase A and Protein Kinase C) and phosphotases (calcineurin) that are inhibited when bound. AKAP150 targets these proteins to specific subcellula r sites through various targeting motifs (Dell'Acqua et al., 1998). AKAP150 has 2 domains: (1) a PKA-anchoring domain to interact with hydrophobic residues in the N-terminal of the RII dimmer, forming an amphipathic helix of residues, and (2) a targeting domain that anchors to the plasma membrane via phospholipids (phos photidylinositol-4,5-biphosphate, PIP2) (see reviews (Dell'Acqua et al., 1998; Diviani and Scott, 2001)). Moreover, AKAP is regulated by calcium-calmodulin. AKAP150 has been shown to be involved in the regulation of receptor activity, localization, and synaptic st ructure during synapse formation during development, synaptic plasticity in learning and memory, and neuronal dysfunction and cell death under pathophysiol ogical conditions (Dell'Acqua et al., 2006). In addition to ASIC1a regulation, AKAP150 modulates in ternalization of AMPA and NMDA receptors during l ong term potentiation and depression (Rosenmund et al., 1994; Westphal et al., 1999; Colledge et al., 2000; Gomez et al., 2002; Smith et al., 2006) and voltage-gated Ca2+ channels function (Oliveria et al., 2007). All these proteins are possi ble constituents of the signaling cascade involving ASIC1a channels and receptors.

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16 Sigma Receptors Sigma receptors have been shown to regulate multiple ion channel function in neurons. This observati on raises the possibility that receptors could modulate ASIC1a function. receptors were discovered by Martin et al. in 1976, and were thought to be an opioid receptor (Martin et al., 1976). Further studies proved that opioid receptors have a high affinity for the (-) enantiomer of benzomorphans while receptors prefer to bind t he (+) enantiomer. Moreover, in vitro and in vivo studies suggested that the opioid antagoni sts, naloxone or naltrexone, do not affect receptor-mediated events (Iwamoto, 1981; Brady et al., 1982; Slifer and Balster, 1983; Vaupel 1983; Katz et al., 1985). Thus, receptors were then classified as their own unique receptor type. Two receptor subtypes have been identified on the basis of pharmacology. receptors have been shown to bind a range of substances including opiates, antipsychotics, ant idepressants, phencyclidine (PCP)-related compounds, and neurosteroids (Walker et al., 1990; Bowen, 2000). -1 receptors have a higher affinity for the positiv e isomer of benzomor phans such as (+)pentazocine and (+)-SKF-10,047, and the -2 receptors have high affinity for ibogaine and its congeners (Bowen, 2000; Vilner and Bowen, 2000). ligands like 1,3-di-o-tolyguanidine (DTG) and hal operidol have been shown to be panselective and bind to both receptor subtypes with high affinity. Evidence suggest that progesterone, neuropeptide Y, peptide YY, and zinc are possible endogenous ligands (Su et al., 1988; Roman et al., 1989; Connor and Chavkin, 1992).

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17 To date, only the -1 receptor has been cloned (Hanner et al., 1996). The structure of the -1 receptor subtype is thought to consist of 2 transmembranespanning domains with internal Cand N-terminals (Aydar et al., 2002). While 1 receptors do not posesses sequence homol ogy to other mammalian proteins, it does have similar homology to fungal st erol isomerases (Hanner et al., 1996; Kekuda et al., 1996; Seth et al., 1998). The cDNA of -1 receptors encodes a protein of 223 amino acids with a molecula r weight of 25 kDa. In humans, the -1 receptor gene is located on chromosome 9p13, a region commonly associated with psychiatric disorders (Prasad et al., 1998). -1 receptors are found in the plasma membrane as well as in intr acellular membranes like the endoplasmic reticulum (Hanner et al., 1996; Kekuda et al., 1996; Aydar et al., 2002). In contrast to -1 receptors, the mo lecular identity of -2 receptors remains to be determined. Reports suggest that a splice variant of -1 receptors displays -2 receptor binding activiti es (Monassier and Bousquet, 2002). However, studies with -1 receptor knockout mouse showed that -1 and -2 receptors were not isoforms of the same protein because -2 effects were still seen in the absence of the -1 receptor subtype (Langa et al., 2003). Similar to -1 receptors, pharmacologica l experiments suggest that -2 receptors are located in the intracellular membr ane of the endoplasmic reticulum and the mitochondria (Bowen, 2000). receptors are ubiquitously express ed in the immune system, the kidney and liver, as well as throughout the cent ral nervous system (Wolfe and De Souza, 1993; Hellewell et al., 1994). Micr oglia activation is one of the main

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18 contributors of the immune re sponse in the brain. Under physiological conditions, microglia respond to substances released by degenerating neurons by migrating to the site of injury, phagocytosing debris and releasing proinflammatory mediators (Streit et al., 2004). However, under pathological conditions, activated microglia are thought to be detrimental sinc e these cells contribute to the demise of compromised neurons, and thus, exac erbate neurodegeneration (Streit et al., 2004). Therefore, the immune response plays a critical role in pathological conditions like stroke. Sigma receptor activation has been shown to inhibit multiple aspects of microglial activation in vitro, including the ability to rearrange the actin cytoskeleton, migrate, an d release cytokine (Hall et al., 2008). Furthermore, in vivo studies also s uggest that sigma receptor activation decreases reactive gliosis follo wing stroke (Ajmo et al., 2006). In the central nervous system, receptors are found in brainstem areas that regulate motor functi on, limbic structures, so me predominantly sensory areas, and areas associated with endocri ne function (della Puppa and London, 1989; Matsumoto et al., 1989; Matsumoto et al., 1990b; Walker et al., 1990; Elsinga et al., 2004). receptors have been implicat ed in numerous physiological and pathophysiological processes such as learning and memory (Senda et al., 1996; Maurice and Privat, 1997), movement disorders (Mats umoto et al., 1990a), and drug addiction (McCracken et al., 1999a; McCracken et al., 1999b). These receptors are emerging as therapeutic targets for various diseases such neuropsychiatric disorders and cancer (C asellas et al., 2004; Hayashi and Su, 2004).

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19 -2 receptors have been shown to regulate sodium and calcium channels. receptor activation blocks seve ral types of calcium channels including N-, T-, P/Qand R-type in neonatal intracardiac (parasympathetic neurons) and superior cervical ganglia ( sympathetic neurons) neurons, and also inhibits voltage-gated sodium channels (Zhang and Cuevas, 2002; Katnik et al., 2006). Studies of mouse and rat hippocam pal neurons have demonstrated that -2 ligands inhibit voltage-gated calcium channels while -1 selective agonists failed to show the same effect, indi cating the inhibition is mediated via -2 receptors (Fletcher et al., 1995). In contrast, -1 receptors regulate inos itol 1,4,5-triphosphate (IP3) receptors and calcium signaling at the endoplasmic reticulum, mobilization of cytoskeletal adaptor proteins modulation of nerve gr owth factor-induced neurite sprouting, modulation of neurotransmi tter release and neuronal firing, and modulation of potassium channels as regulatory subunits (see review, (Su and Hayashi, 2003)). -1 agonists are known to c ause amplification of signal transduction incurred upon activation of the glutamatergic, dopaminergic, IP3related metabotropic, or nerve growth factor-related systems (Su and Hayashi, 2003). Immunohistochemistry studies have shown that -1 receptor, IP3 type-3 receptor, and ankyrin B are colocalized in NG-108 cells in perinuclear areas and regions of cell-to-cell co mmunication, suggesting that -1 ligands may play a role in cells by controlling the function of cy toskeleton proteins and regulating calcium signaling may represent a site of action for neurosteroids and cocaine (Hayashi and Su, 2001).

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20 -1 receptors have been implicated in the regulation of various ion channels. -1 receptors are directly c oupled to potassium channels in intracardiac neurons and activation of th is receptor subtype depresses the excitability of these neurons, blocking par asympathetic input to the heart (Zhang and Cuevas, 2005). In cultured frog melanot rope cells, DTG and (+)-pentazocine have been shown to modulate electrical activity by reducing both a tonic K+ current and a voltage-dependent K+ conductance through activation of a cholera toxin-sensitive G protein (Soriani et al., 1998; Soriani et al., 1999). In Xenopus oocytes, the signal transduction cascade between Kv1.4 and receptors has been shown to be dependent on protein-protei n interaction (Aydar et al., 2002). The ligand, (+)-3-(3-hydroxyphenyl)-N-(1 -propyl)piperidine ((+)3-PPP), has been shown to depolarize sympathetic neurons of the mouse isolated hypogastric ganglion by inhibiting the M-current A-current and calciumdependent K+ current (Kennedy and Henderson, 1990). Activation of sigma receptors regulate multiple ion channels subtypes in neurons (Aydar et al., 2002; Zhan g and Cuevas, 2002; Zhang and Cuevas, 2005). This regulation of ion channels ma y result in neuroprotection during ischemia. It has been suggested that the neuroprotective properties of ligands depend in part on their ability to depress elevations in [Ca2+]i associated with glutamate receptor-mediated excitotoxicity (Klette et al., 1995; Kl ette et al., 1997; Katnik et al., 2006), neurotransmitter re lease regulation (Matsuno et al., 1993; Couture and Debonnel, 1998), an d modulation of multiple ion channel subtypes. The studies of receptor modulation of gl utamate evoked changes in

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21 intracellular calcium have resulted in cons iderable controversy in the literature. There are conflicting re ports as to whether receptor ligands exert their effects as a result of receptor activation (Hayashi et al., 1995) or through non-specific interaction with other targets, in particul ar, NMDA receptors (Kume et al., 2002). receptors and NMDA receptors both bind PCP and related compounds like MK-801 with high affinity (Sircar et al ., 1987), and thus, such drugs cannot be used to discriminate between direct and indirect effects. Activation of receptors has been shown to be neuroprotective at delayed time points in a rat model of ischemic stroke (Ajmo et al., 2006) and blocks ischemia-induced increases in [Ca2+]i (Katnik et al., 2006). -1 receptor activation has also been shown to prevent neuronal death associated with gl utamate excitotoxicity in cultured neurons (Takahashi et al., 1996; Kume et al., 2002), diminish infarct size by blocking nitric oxide production in vivo (Goyagi et al., 2001), inhibit proinflammatory factors (TNF ) and activate the expressi on of anti-inflammatory agents like IL-10 (Bourrie et al., 1995; Bou rrie et al., 1996; Bourrie et al., 2002). Stimulation of receptors have been found to block voltage-gated calcium and potassium channels (Zhang and Cuevas 2002; Zhang and Cuevas, 2005) in intracardiac neurons and ionotropic glutam ate receptors (Monnet et al., 2003). All of these channel types are involved in t he dysregulation of intracellular calcium homeostasis accompanying ischemia, and blocking the function of these channels provides neuroprotection (Schurr, 2004). Thus, receptor ligands have been targeted for the treatment of stroke due to the neur oprotective properties

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22 observed in both in vivo and in vitro model s of ischemia (Lockhart et al., 1995; Takahashi et al., 1996). In Vitro Chemical Ischemia Models The role of acidotoxicity, ASIC1a channels and acid-induced Ca2+ dysregulation during ischemia remains to be established. Furthermore, the effects of receptor activation on acidotoxicity remain to be determined. It is well known that in response to hypoxia-ischemia an in vivo model of stroke, the brain relies on anaerobic glycolysis as the sour ce of energy (Vannucci et al., 1994). This is energetically unfavorable and resu lts in rapid depletion of ATP along with subsequent activation of deliterious bioc hemical cascades. All of these events are similar to those observed in cultured neurons (in vitro): (1) Ca2+ dysregulation due to glutamate accumulation and activa tion of NMDA receptors (Shalak and Perlman, 2004; Vannucci and Hagberg, 2004), (2) production of free oxygen radicals (Shalak and Perlman, 2004) (3) damage to proteins and lipids (Shalak and Perlman, 2004), (4) mitochondrial dysf unction leading to activation of apoptotic pathways (Northington et al ., 2001), and (5) activation of secondary mechanisms including release of proinfla mmatory mediators (Silver and Miller, 2004; Chew et al., 2006; Fukui et al ., 2006; Van Lint and Libert, 2007). There are several models of in vitro chemical ischemia that are used to mimic the injury profile seen in animal models of stroke and conditions associated with cerebral i schemia in vivo. These models include: (1) oxygen glucose deprivation (Monyer et al., 1989; Laake et al., 1999) and the addition of

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23 either (2) sodium azide (Dawson et al., 1991; Dawson et al., 2002; Katnik et al., 2006; Marino et al., 2007), (3) sodium cyanide (Vornov et al., 1994; Vornov, 1995; Zhang et al., 2000), or (4) 2,4-dinitrophenol (Riepe et al., 1996; Sullivan et al., 2003) in glucose-free solutions. Thes e ischemic models are associated with Ca2+ overload triggered by the exce ss influx of extracellular Ca2+ through Ca2+sensitive ion channels and re ceptors (Monyer et al., 1989; Goldberg and Choi, 1993; Katnik et al., 2006). All of these mode ls affect the electron transport chain in the mitochondria, which is the site of oxidative phosphorylation in eukaryotes. Oxidative phosphorylation is the main pathw ay that uses energy released by the oxidation of nutrients to drive the production of adenos ine triphosphate (ATP), which supplies energy for metabolism. I nhibition of enzymes, like cytochrome c, during this metabolic process results in energy failure due to the lack of ATP production. Sodium-azide and sodium-cyanide are both compounds that inhibit oxidative phosphorylation. Azide and cyani de bind to the iron-copper center (when the iron is in the ferric state) of cytochrome c with greater affinity than oxygen. This prevents the reduction of oxygen and stops the electron transfer preventing translocation of protons ac ross the membrane, which disrupts the electrochemical gradient that powers ATP synthase. Therefore, these compounds are inhibitors of the enzyme cytochrome c oxidase in the fourth complex of the electron tr ansport chain and both prevent the aerobic production of ATP as the source of cellular ener gy. However, the main disadvantage of cyanide is that it is a very toxic an d lethal compound to humans since cyanide

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24 interacts with pH and produces hydrogen cyanide, thus making difficult to control its concentration. Thus, azide is a safer compound to handle. In contrast to azide and cyanide, 2,4-dinitrophenol (DNP) uncou ples the electron transport from oxidative phosphorylation, which disr upts the proton gradient by transporting protons back across the inner mitochondrial membrane. There are several disadvantages to using oxygen depletion: (1) pre and early post natal rat tissue is resistant to oxygen depleti on, (2) difficult to comple tely remove oxygen and thus requires long incubation to produce signifi cant injury (Rothman, 1984), and (3) requires using a closed-chamber, making pat ch-clamp experim ents difficult. The major advantage of the sodium azide/ glucose deprivation model over the oxygen/glucose deprivation mode l is that it elicits neurochemical responses that are significantly more rapid and robust, t hus facilitating the recording of changes in [Ca2+]i increases (Katnik et al., 2006). Disad vantages of this model are that azide has multiple affects incl uding inhibition of cytochrome aa3, superoxide dismutase and DNA synthesis (Varming et al., 1996). Azide breaks down to nitric oxide and thus enhances excitatory neurot ransmission (Varming et al., 1996). Moreover, because of the s hort duration of the applicat ion of azide, this only models acute ischemia. This azide/gluc ose deprivation model has previously been used in rat cortical neurons to study receptor inhibition of ischemiainduced [Ca2+]i elevations (Katnik et al., 2006) Moreover, calciu m influx through the plasma membrane and not release from the mitochondria or endoplasmic reticulum, accounts for most of the ischemia-mediated [Ca2+]i increases in cortical neurons using this azide model of ischemia (Katnik et al., 2006).

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25 CHAPTER 2 SIGMA-1 RECEPTOR MODULATION OF ASIC1a CHANNELS AND ASIC1aINDUCED Ca2+ INFLUX IN RAT CORTICAL NEURONS Introduction Acid-sensing ion channels are a cla ss of ligand-gated channels that are members of the degenerin/epithelial s odium channel superfamily and are expressed in both peripher al and central nervous syst em neurons (Waldmann et al., 1997b). Thus far, four genes (ASIC1 – ASIC4) and two splice variants of ASIC1 and ASIC2 (a and b) have been cloned (Wemmie et al., 2006) which encode protein subunits that form func tional proton-gated homomultimeric or heteromultimeric channels (Wemmie et al., 2006).The pH of half-maximal activation and the tissue expression patterns differ between each channel subtype. One of the most common ASIC subtypes in the central nervous system (CNS) contains the ASIC1a subunit, which can form homomultimeric or heteromultimeric channels with ASIC2a (A skwith et al., 2004). These channels are activated by pH 7 and have a pH of half-maxima l activation of ~ 6.0 6.5 (Waldmann et al., 1997b; Hesselager et al., 2004). ASIC1a homomultimeric channels differ from other ASIC subtypes in that they are highly permeable to both Na+ and Ca2+ ions (Waldmann et al., 1997b; Ye rmolaieva et al., 2004). This

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26 ASIC subtype has been implicated in a num ber of physiological processes such as synaptic plasticity, fear conditioning, and learning and memory (Wemmie et al., 2002; Wemmie et al., 2003; Wemmie et al., 2004). ASIC1a has also been shown to be activated following cerebral ischemia, and has been unequivocally linked to neuronal cell death (Xiong et al ., 2004; Gao et al., 2005; Pignataro et al., 2007). Transgenic mice deficient in ASIC1a have reduced infarct size in response to middle cerebral artery occl usion (MCAO) relative to wild-type mice (Xiong et al., 2004). Moreover, pharmacologi cal inhibition of ASIC1a with either amiloride or psalmotoxin1, which is selective for homomultimeric ASIC1a channels (Diochot et al., 2007), diminishes ischemic br ain injury (Xiong et al., 2004). Several studies have suggested that Ca2+ influx through these channels is a key mechanism leading to neurodegenerati on (Xiong et al., 2004; Yermolaieva et al., 2004). Despite efforts to determine the functi on of ASIC1a and the role of these channels in ischemia, little is known about endogenous mechanisms which control ASIC1a activity. Thus far, onl y the NMDA receptor, acting via a Ca2+calmodulin kinase II cascade (CaMKII) has been shown to modulate ASIC1a (Gao et al., 2005). Activation of NMDA receptors enhances ASIC1a-mediated currents, which consequently exacerbates acidotoxicity during ischemia (Gao et al., 2005). receptor activation has been shown to modulate multiple cell membrane ion channels in neurons. -1 receptors regulate ionot ropic glutamate receptors and voltage-gated K+ channels, whereas -2 receptors modulate voltage-gated

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27 Ca2+ channels (Hayashi et al., 1995; Aydar et al., 2002; Zhang and Cuevas, 2002; Zhang and Cuevas, 2005). The inhibi tion of ionotropic glutamatergic receptors by receptors prevents elevations in [Ca2+]i associated with glutamateinduced excitotoxicity (Klette et al., 1995) All of these voltagegated ion channels and NMDA receptors have been shown to contribute to the demise of neurons during an ischemic insult. Our labor atory has recently shown that -1 receptors inhibit Ca2+ dysregulation evoked by isc hemia and that activation of receptors is neuroprotective at delayed time points in a rat model of ischemic stroke (Ajmo et al., 2006; Katnik et al., 2006). Interstiti al pH in the brain remains low several hours after an ischemic event (Nedergaar d et al., 1991), and pharmacological blockade of ASIC1a by amiloride or psalmotoxin1 administered even 5 hours after MCAO has been shown to diminish stroke injury (Simon, 2006). These observations raise the possibility that receptors may regul ate ASIC1a function and ASIC1a-induced intracellular calcium tr ansients, and provide neuroprotection when stimulated at delayed time points after an ischemic insult. Experiments were conducted to determine the effects of receptors on ASIC-mediated membrane cu rrents and transient [Ca2+]i elevations. It was determined that receptor agonists inhibit aci dosis-induced increases in [Ca2+]i and peak membrane currents in cells ex pressing homomeric ASIC1a channels. Pharmacological studies demonstrated that the -1 receptor subtype was responsible for these effects. Moreover, acidosis was also shown to activate downstream Ca2+ influx pathways (e.g. NMDA and AMPA/kainate receptors and voltage-gated Ca2+ channels, VGCC), and activation of -1 receptors also

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28 diminished Ca2+ entry via these c hannels. Therefore, -1 receptors couple to ASIC1a channels to inhibit channel f unction and also block ASIC1a-induced [Ca2+]i dysregulation. Our findings suggest that -1 receptors represent potential targets for improving outcome of stro ke injury and expanding the therapeutic window for ischemic stroke treatment. Materials and Methods Primary Rat Cortical Neuron Preparation Primary cortical neurons from embr yonic (E18) rats were cultured as previously described by our laboratory (K atnik et al., 2006). All procedures were done in accordance with the regulations of the University of South Florida Institutional Animal Care and Use Committe e. Briefly, dams were sacrificed, uterus removed, and embryos dissected out and placed in isotonic buffer consisting of (in mM): 137 NaCl, 5 KCl, 0.2 NaH2PO4, 0.2 KH2PO4, 5.5 glucose, and 6.0 sucrose, titrated to pH 7.4 wit h NaOH. Cortices were excised and minced, followed by digestion in isotoni c buffer containing 0.25% trypsin/EDTA for 10 minutes at 37C and added to 3X vo lume of Dulbecco’s modified Eagle’s Medium (DMEM) (Invitrogen Inc, Carl sbad, CA) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were counted using a hemocytom eter, plated (0.5 X 106 cells/well) on 18-mm pretreated poly-L-lysine coverslips, and incubated at 37C under 95% air, 5% CO2 atmosphere. After 24 hours, the media was replaced with Neurobasal medium supplemented with B27 and 0. 5 mM L-glutamine (Invitrogen Inc, Carlsbad, CA)

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29 to minimize astrocyte pro liferation in the cultures. Cells were used between 10-21 days in culture. Calcium Imaging Measurements The effects of acidosis on intracellular Ca2+ concentrations were examined in isolated cultured cortical neurons using fluorescent imaging techniques. Cytosolic free-Ca2+ was measured using the Ca2+ sensitive dye, fura-2, as previously described (Katnik et al., 2006). Cells, plated on poly-L-lysine coated coverslips, were incubated for 1 hour at room temperature in Neurobasal (Invitrogen) medium supplemented wit h B27 (Invitroge n) and 0.5 mM Lglutamine, or in physiological saline so lution (PSS) consisting of (in mM): 140 NaCl, 5.4 KCl, 1.3 CaCl2, 1.0 MgCl2, 20 glucose, and 25 HEPES (pH to 7.4 with NaOH), 330 10 mOsm. Both solutions contained 3 M acetoxymethyl ester fura-2 and 0.3 % dimethyl sulfoxide. T he coverslips were washed in PSS (fura-2 free) prior to the experiments being carri ed out. All solutions were applied via a rapid application system identical to that previously described (Cuevas and Berg, 1998). A DG-4 high-speed wavelength switcher (Sutter Instrum ents Co., Novato, CA) was used to apply alternating exci tation light. Fluorescent emission was captured using a Sensicam digital CC D camera (Cooke Corporation, Auburn Hills, MI) and recorded with Slidebook 4.02 software (Intelligent Imaging Innovations, Denver, CO). Slidebook 4.02 software was used to calculate changes in [Ca2+]i from the intensity of the emitted fluor escence following excitation with 340 nm and 380 nm light, re spectively, using the Grynkiewicz

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30 equation: [Ca2+]i = Kd *Q(R-Rmin)/(Rmax-R), where R represents the fluorescence intensity ratio (F340/F380) as determined during experiments, Q is the ratio of Fmin to Fmax at 380 nm, and Kd is the Ca2+ dissociation constant for fura-2 (224 M). The system was calibrated using a Fura-2 Calcium Imaging Calibration Kit (Molecular Probes; Eugen e, OR) and values for Fmin/Fmax, Rmin, and Rmax were determined. Electrophysiology Recordings Neurons plated on glass coverslips (a s described above) were transferred to a recording chamber mounted on a Zei ss Axiovert 200 and visualized at 400x. Membrane currents were amplified using an Axopatch 200B (A xon Instruments), filtered at 1 kHz, digitized at 5 kHz with a Digidata 1322A (Axon), and acquired using Clampex 8 (Axon). Electrical a ccess was achieved using the amphotericin B perforated-patch method to preserve intr acellular integrity of neurons (Rae et al., 1991). An amphotericin B stock solu tion (60 mg/ml in DMSO) was made daily, kept on ice, light protected, and diluted to 240 g/ml (0.4% DMSO) in control pipette solution immediately prior to patch clamp experiments. The pipette solution consisted of (in mM): 75 K2SO4, 55 KCl, 5 MgSO4, and 25 HEPES (titrated to pH 7.2 with N-methyl-d-glucamine, 300 5 mOsm). Patch electrodes were pulled from thin-walled borosilicate gla ss (World Precision Instruments Inc., Sarasota, FL) using a Sutter Instrument s P-87 pipette puller (Novato, CA) and had resistances of 1.0–1.5 M Access resistance (Rs) were monitored throughout experiments for stable values 20 M and were always compensated at 40% (lag, 10 s). All ce lls were voltage-clamped at -70 mV.

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31 Solutions and Reagents The control bath solution for all experiments was PSS. In one series of experiments requiring high extracellular K+, an additional 34.6 mM of KCl was isosmotically substituted for NaCl. All drugs were applied in PSS (or high K+ PSS) using a rapid application system ident ical to that previously described (Cuevas and Berg, 1998). ASIC activation was induced by applying PSS with a pH of 6.0 (+/drug) to specifically tar get ASIC1a (Askwith et al., 2004). Individual cells were exposed to no more than four low pH applications, and no rundown of the responses was observed with this protocol. All chemicals used in this investigation were of analytic grad e. The following drugs were used: DTG, opipramol, ibogaine, metaphit, nifedipi ne, AP5 and PB28 (Sigma-Aldrich, St. Louis, MO); carbetapentane, BD1063, CNQX and PRE-084 (Tocris Bioscience, Ellisville, MO); dextromethorphan (MP Biomedicals, Inc, Solon, OH); psalmotoxin1 (Spider Pharm, Yarnelle AZ); tetrodotoxin and thapsigargin (Alomone Labs, Jerusalem, Israel); amiloride (Alexis Biochemicals, Lausen, Switzerland); cadmium (Fischer Sci entific, Fair Lawn, NJ); and fura-2 acetoxymethyl ester (Molecul ar Probes, Eugene, OR). Data Analysis Analyses of measured intracellular Ca2+ and membrane cu rrent responses were conducted using Clampfit 9 (Axon inst ruments). Fluorescenc e intensities were recorded from fura-2 -loaded neuronal cell bodies. Time-lapse imaging data files collected with SlideBook 4.02 (Intelligent Imaging Innovati ons, Inc.) were converted to a text format and imported into Cla mpfit for subs equent analysis. Statistical

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32 analysis was conducted us ing SigmaPlot 9 and SigmaS tat 3 software (Systat Software, Inc.). Statistical differences were determined usin g paired and unpaired t-tests for within group an d between group experiments, respectively, and were considered significant if p < 0.05. For multip le group comparisons either a 1-way or a 2-way ANOVA, with or without repeat measures, were used, as appropriate. When significant difference s were determined with an ANOVA, post-hoc analysis was conducted using a Tukey Test to determine differ ences between individual groups. For the generation of concentration response curves, data were best fit using a single-site Langmuir-Hill equation. Results ASIC subtypes are distinguishable by pH sensitivity and ion selectivity, with homomultimeric ASIC1a being the only subtype that is Ca2+ permeable (Yermolaieva et al., 2004). Experiments were carried out to determine the effects of ASIC activation on intracellular Ca2+ transients and to identify the specific ASIC subtype(s) affecting [Ca2+]i in our rat cortical neuron model. Figure 2.1A shows representative traces of [Ca2+]i as a function of time recorded from a single neuron during acidosis (pH 6.0) in the absence (Control) and presence of amiloride (100 M), and following a 10 minute washout of drug (Wash). The general ASIC inhibitor, amiloride, reversib ly blocked ASIC-mediated increases in [Ca2+]i by 88 1 % (Figure 2.1B). Psalmoto xin1 (PcTx1) from the venom of the tarantula Psalmopoeus cambridgei has been shown to be a selective blocker of homomultimeric ASIC1a channels (Dioc hot et al., 2007). Figure 2.1C shows

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33 representative traces of [Ca2+]i as a function of time recorded from a neuron prior to (Control), following a 10 20 min prei ncubation in bath applied PcTx1 (500 ng/ml venom protein), and after washout of the toxin (Wash). In identical experiments, PcTx1 produced a statistically significant reversible decrease in ASIC1a-mediated elevations in [Ca2+]i (57 2 %) (Figure 2.1D). The effects produced by this concentration of PcTx1 ar e consistent with results obtained for ASIC1a responses in mouse cortical neurons (Xiong et al., 2004). These data indicate that, in cultured cortical neurons from embryonic rats, acidosis results in elevations in [Ca2+]i that are mediated via the ac tivation of homomultimeric ASIC1a channels, as reported for cultured cortical neurons from embryonic mice (Xiong et al., 2004). Activation of receptors has been shown to inhibit numerous plasma membrane ion channels in neurons (Hay ashi et al., 1995; Zhang and Cuevas, 2002; Zhang and Cuevas, 2005). Therefore, experiments were carried out to study the effects of receptor activation on ASI C1a function using the panselective receptor agonist, DTG. Figure 2.2A shows representative traces of [Ca2+]i recorded from a single neuron during acidosis in the absence (Control) and presence of 100 M DTG, a concentration previ ously shown to effectively regulate other ion channels (Zhang and Cuevas, 2002; Zhang and Cuevas, 2005), and following 10 minute washout of the drug (Wash). DTG rapidly and reversibly inhibited the low pH-i nduced transient increases in [Ca2+]i. In identical experiments, 100 M DTG produced a statistically si gnificant decrease (46 3 %) in ASIC1a-mediated elevations in [Ca2+]i (Figure 2.2B). Opipramol (10 M),

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34 another pan-selective -1/ -2 agonist, also inhibited ASIC1a-mediated increases in [Ca2+]i by 57 1 % (Figure 2.2B). The c oncentration-response relationship for DTG inhibition of ASIC1a was determined to confirm that the actions of DTG on ASIC1a are consistent with receptor activation. Incr easing DTG concentrations resulted in further depression of ASIC1a-mediated increases in [Ca2+]i (Figure 2.2C). A plot of the concentrati on-response relationship obtained from measurements made in multip le cells is shown in Figure 2.2D. The data were best fit using a single-site Langmuir-Hill equation with an IC50 value of 109 M and a Hill coefficient of 0.9. These values are consistent with the effects of DTG being mediated via activation of receptors (Zhang and Cuevas, 2002; Zhang and Cuevas, 2005; Katnik et al., 2006). Taken together these results suggest that receptors depress acid-induced increases in [Ca2+]i and modulate ASIC1a function. receptor subtype-selective agonists were used to identify the specific receptor subtype(s) medi ating these effects. Repr esentative traces of [Ca2+]i as a function of time recorded fr om two cells during acidosis in the absence (Control) and presence of the -1 selective agonists carbetapentane (CBP) and dextromethorphan (DEX) at t he indicated concentrations are shown in Figures 2.3A and 2.3B, respectively. -1 selective agonists blocked the low pH-induced elevations in [Ca2+]i in a concentration dependent and reversible manner. Concentration-response plots for mean changes in peak [Ca2+]i recorded in identical experiments using the -1 selective ligands CBP, DEX, and PRE-084, are shown in Figure 2.3C. The data were best fit using the Langmuir-Hill equation

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35 and values obtained for IC50 and Hill coefficients were 13.8 M and 0.7 (CBP), 22 M and 0.8 (DEX), and 13.7 M and 0.6 (PRE-084), respectively. Presented for comparison is the best fit to the da ta obtained for CBP inhibition, via -1 receptors, of chemical isc hemia-induced increases in [Ca2+]i in cortical neurons (dotted line, IC50 =18.7 M; Hill coefficient, 0.8) (K atnik et al., 2006). This curve superimposes on the responses to CBP observed in the current study. To provide further evidence that -1 receptors modulate ASIC1a-induced Ca2+ elevations experiments were conducted using the irreversible antagonist, metaphit, and a selective -1 antagonist, BD1063, in combination with the -1 agonist, CBP. Cells were exposed to acidosis in the absence and presence of CBP (30 M), with or without preinc ubation in metaphit (50 M; 30 min -1hr, 23 C). CBP decreased the acid-induced elevations in [Ca2+]i in control cells, and this inhibition was lessened by preincubatio n with metaphit (Figure 2.4A). Figure 2.4B shows results from several si milar experiments determining percent inhibition of acid-induced increases in [Ca2+]i observed in the presence of CBP in control neurons (Control) and neurons preincubated with metaphit (MET). Whereas CBP decreased the elevations in [Ca2+]i evoked by acidosis in control cells by 52 2 %, the -1 receptor agonist only reduced t he response by 30 3 % in cells preincubated in the irreversible receptor antagonist. This >40% decrease in the effects of CBP following metaphit preincubation was statistically significant (p < 0.001). The selective -1 antagonist, BD1063, showed more pronounced effects. Figure 2.4C s hows representative traces of [Ca2+]i as a function of time recorded fr om two neurons during acidos is in absence (Control)

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36 and presence of CBP (30 M), without ( PSS) and with co-application in BD1063 (BD1063, 10 nM). In ident ical experiments, the agonist CBP completely loses its ability to block the low pH-induced elevations in [Ca2+]i in the presence of the antagonist BD1063 when compared to 40% block in control cells (Figure 2.4D). Inhibition of -1 receptors by BD1063 significantly blocked the effects of CBP on ASIC1a-mediated [Ca2+]i elevations when compared to control cells (p <0.001). These data confirm the effects of -1 agonists on ASIC1a mediated increases in [Ca2+]i are the result of these compounds acting on -1 receptors. Further experiments were carried out using the -2 selective ligands ibogaine and PB28 to determine if the -2 receptor subtype also affects ASIC1a function. Figure 2.5A shows r epresentative traces of [Ca2+]i as a function of time recorded from two cells during acidosis in the absence (Control) and presence of ibogaine (IBO, left traces) and PB28 (right traces) at the indicated concentrations. The -2 ligands inhibited acidosis-evoked increases in mean peak changes in [Ca2+]i in a concentration dependent m anner (Figure 2.5B). Best fits to the data demonstrated that applic ation of ibogaine and PB28 resulted in inhibition of ASIC1a-mediated increases in [Ca2+]i with IC50 values of 69 M and 11 M, and Hill coefficients of 0.86 and 0.85, respectively. For comparison, the best fit to the data obtained fo r ibogaine inhibition of ICa-induced increases in [Ca2+]i is presented, which has been shown to be mediated by -2 receptors (dashed line; IC50 = 31 M; Hill coefficient, 1. 1) (Zhang and Cuevas, 2002). Unlike the similar concentration-res ponse relationship observed for CBP inhibition of ASIC1a and ischemia res ponses, there is a discrepancy between the

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37 ibogaine block of responses mediat ed by ASIC1a and by voltage-gated Ca2+ channels (VGCC). To determine if the effects of these -2 ligands were mediated by activation of -2 receptors, experiments usi ng metaphit were carried out. PB28 (20 M) produced an inhibition of ASIC1a-mediated increases in [Ca2+]i in both control (PSS) and metaphit (+MET) treated cells (Figure 2.5C). PB28 blocked ASIC1a-mediated increases in [Ca2+]i by 67 1% and 60 2 % in the absence and presence of metaphit preincubat ion, respectively (Figure 2.5D). The ~10% reduction in the effects of PB28 pr oduced by metaphit was not statistically significant (p = 0.64). Furthermore, BD1063 (10 nM) failed to block the effects of PB28 (20 M) on ASIC1a-induced elevations in [Ca2+]i (Figure 2.5D). These results demonstrate that the effects of PB28 on ASIC1a-medited increases in [Ca2+]i are not mediated by -2 receptors since the inhi bition of these responses to acidosis by PB-28 is metaphit-ins ensitive and occurs at concentrations inconsistent with -2 receptor activation. The activation of receptors has previously been shown to directly affect Ca2+ release from intracellular stores (C assano et al., 2006). Thus, experiments were conducted to resolve if receptor activation reduces acid-induced increases in [Ca2+]i in part via the inhibition of calcium-induced-calcium release from the endoplasmic reticulum, triggered by Ca2+ influx through the plasma membrane. For these experiments, thapsigargin was used to block the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which results in depletion of both ryanodineand IP3-sensitive stores. Figure 2. 6A shows representative traces of [Ca2+]i as a function of time recorded from a neuron during acidosis in

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38 the absence (Control) and pres ence of 100 M DTG (DTG), while Figure 2.6B shows traces in the absence and presence of DTG following pre-incubation (1 hr, 23 C) in 10 M thapsigargin (THAP and THAP + DTG, respectively). Thapsigargin alone did not decr ease the elevations in [Ca2+]i produced by acidosis, and DTG depressed the increases in [Ca2+]i under both conditions ( thapsigargin preincubati on). Analysis of the data collected in identical experiments indicates that preincubation with thapsigargin does not significantly alter the effects of DTG on acid-mediated increases in [Ca2+]i (Figure 2.6C). Thus, DTG does not decrease the low pH-induced elevations in [Ca2+]i by affecting release of calcium from intrace llular stores. The fact that depletion of intracellular stores fails to depress increases in [Ca2+]i evoked by acidosis suggests that Ca2+ influx through the plasma memb rane accounts for most, if not all, of the increases in [Ca2+]i evoked by ASIC1a activation. Simultaneous Ca2+ fluorometry and whole-ce ll patch clamp recordings were performed to study ASIC1a-mediat ed membrane currents and to determine how much of the observed Ca2+ influx is due to Ca2+ entry through ASIC1a channels. Cells were voltage-clamped at -70 mV to minimize NMDA receptor and VGCC activation, and thus, isolate ASIC1a currents. Figure 2.7A shows that ASIC1a stimulation by low pH solution (pH 6.0), resulted in a small intracellular Ca2+ transient measured in a patched cell ([Ca2+]i V-Clamp). In contrast, a second cell ([Ca2+]i Control) in the same field of view, which was not voltage clamped, had a significantly larger increase in [Ca2+]i. Acidosis also resulted in a large inward current in the patched cell (Figure 2.7A, inset). The cumulative

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39 results from several similar experiment s demonstrate that ASIC1a activation of neurons voltage clamped at -70 mV resulted in elevations in [Ca2+]i which were an order of magnitude sma ller than changes evoked in cells that were not voltage-clamped (Figure 2.7B). These data confirm ASIC1a activation results in minimal Ca2+ influx through the ASIC1a channel itse lf and that the majority of the acid-induced [Ca2+]i increases are mediated by downstream Ca2+ influx pathways. To test the possibility that ASIC1a channels prom ote cell depolarization, cortical neurons were held under curr ent-clamp mode. Figur es 2.8A and 2.8C show representative traces of membrane pot ential as a function of time recorded from a single cell in absence (Control ) and presence of amiloride (100 M) and from a different neuron in the absence (C ontrol) and presence of PcTx1 peptide (50 nM). A summary of the changes in membrane potential recorded from several cells in the absence (Control) and presence of ASIC blockers, amiloride and PcTx1 peptide, are shown in Figures 2.8C and 2.8D. Inhibition of ASIC1a channels by amiloride, but not PcTx1 peptid e, produced a significant reduction in changes in membrane potential (Figure 2. 8C). Thus, these data suggest that activation of ASIC1a channels depolarizes cortical neurons to potentials capable of activating other Ca2+ channels. Application of protons evoked a rapi d depolarization of neurons held under current-clamp mode which, unlike the case in voltage-clamped neurons, was associated with pronounced elevations in [Ca2+]i. Thus, ASIC1a channels are likely promoting significant Ca2+ influx into the neurons by depolarizing the cells.

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40 To mimic this change in membrane pot ential evoked upon ASI C1a activation, cells were exposed to high K+ (40 mM) extracellular solu tion. Figure 2.9A shows representative [Ca2+]i traces recorded in response to high K+ o application in the absence (Control) and presence of CBP (100 M, +CBP). Depolarizing the neurons in this manner evoked robust [Ca2+]i elevations which were blocked by addition of CBP. In identical experiments CBP reduced the high K+ o-evoked increases in [Ca2+]i by 83 1% and this decrease was statistically significant (p < 0.001) (Figure 2.9B). Thus, receptor activation inhibits Ca2+ channels downstream of ASIC1a in addition to t he proton-gated channels themselves. To determine the specific ion channels contributing to the ASIC1a-induced [Ca2+]i influxes, several inhibitors of plas ma membrane ion channels were used. Activation of ASIC1a depolarizes these neurons which could stimulate action potential firing and, consequently, synaptic transmission, both of which may elevate [Ca2+]i. Thus, tetrodotoxin (TTX, 500 n M) was used to inhibit voltagegated Na+ channels to prevent the genesis of action potentials. Figure 2.10A shows that application of TTX inhibited ~ 10% of t he acid-induced increases in [Ca2+]i, but did not significantly affect CBP (50 M) modulation of the Ca2+ responses, when compared to the PSS group. Because TTX had no effect on the -1/ASIC1a interaction, all subsequent experiments included 500 nM TTX in the bath solutions to prevent spontaneous action potentials from contributing to the measured increases in [Ca2+]i. Inhibition of NMDA receptors by AP5 (100 M) significantly reduced the acid-induced elevations in [Ca2+]i, and co-application with CBP (50 M) resulted in a further decrease in [Ca2+]i which was statistically

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41 significant (Figure 2.10A). These resu lts show that ~ 30% of ASIC1a-induced [Ca2+]i increases are dependent on NMDA re ceptor activation. Furthermore, since the effects of AP5 and CBP are less than additive, activation of -1 receptors depresses ASIC1a-evoked increases in [Ca2+]i in part by blocking this NMDA receptor-dependent component. Block ade of L-type VGCC by nifedipine (10 M) significantly inhibited (>75%) acid-induced [Ca2+]i elevations, and CBP (50 M) continued to provide further blo ckade of the residual increases in [Ca2+]i (Figure 2.10A). Cadmium (100 M), a broad-spectrum blocker of plasma membrane calcium channels, inhibited ~ 90% of the acid-induced increases in [Ca2+]i and CBP (50 M) had no effe ct on the remaining Ca2+ influx (Figure 2.10A). Thus, voltage-gated Ca2+ channels either directly (influx through the channel) or indirectly (facilitating glut amate release) account for most of the [Ca2+]i increases resulting from ASIC1a activation, and -1 receptor activation provides an inhibition sim ilar to that observed with Cd2+. Further experiments were conduct ed to determine if AMPA/kainate receptors are involved in the [Ca2+]i elevations elicited upon ASIC1a activation. Figure 2.10B shows relative changes in [Ca2+]i in the absence (Control) and presence of the AMPA/kainate receptor blocker CNQX (10 M), and the effects of CBP at two different concentrations ( 50 M and 300 M). Maximal blockade of AMPA/kainate receptors alone produced ~ 40% reduction of the elevations of [Ca2+]i and 50 M CBP did not prov ide additional, statistica lly significant block. Increasing the CBP concentration to 300 M, however, did produce an additional block of the Ca2+ response on top of the effect s of CNQX. Figure 2.10B shows

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42 that co-application of 300 M CBP and CNQX blocked >80% of the acid-induced increases in [Ca2+]i and this was statistically significant when compared to CNQX alone. Thus, [Ca2+]i elevations elicited upon ASIC1a activation involve Ca2+ influx through both ASIC1a and other Ca2+ permeable plasma membrane ion channels expressed in cortical neurons. Importantly, it is also evident that activation of -1 receptors results in depression of Ca2+ influx through all of these sources, either by inhibiting ASIC1a or the dow nstream channels themselves. Two ASIC blockers, amiloride and PcTx1, were used to confirm that the acid-activated inward currents observed in our cortical neuron model were mediated by ASIC1 channels. Figure 2. 11A shows representative traces of membrane currents as a function of time recorded from a single cell in absence (Control) and presence of am iloride (100 M, top traces, i ), and from a different neuron in the absence (Control) and pres ence of PcTx1 (500 ng/ml venom, bottom traces, ii ). Figure 2.11B summarizes the normalized peak proton-gated whole-cell currents recorded from several cells in the absence (Control) and presence of amiloride and PcTx1. Amilori de produced a 79 6 % inhibition of acid-activated currents while 500 ng/ml of PcTx1 containing venom produced a 42 11 % reduction in this current. Both r eductions were significantly different from control (p<0.01). Experiments were also carried out to confirm that ASIC1a currents were not affected by the channel blockers used in the imaging studies described above. Figure 2.11C shows relative peak proton-gated currents, normalized to control (PSS), during acidos is in the absence (PSS) and presence of the indicated drugs. These data demons trate that neither TTX, AP5, CNQX,

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43 nifedipine, nor cadmium have any di rect effects on ASIC1a channels. receptor activation has been shown to modulate multiple cell membrane ion channels in neurons that are activated following ASI C1a stimulation (Hayashi et al., 1995; Zhang and Cuevas, 2002). This fact raises the possibility that -1 receptors only couple to the secondary even ts that result fr om ASIC1a activation and not to ASIC1a itsel f. To investigate if receptors affect ASIC1a function, whole-cell patch clamp recordings were performed in the presence of agonists. Figure 2.12A shows representative membra ne current traces as a function of time recorded from a cell in the ab sence (Control) and presence of the panselective agonist DTG (100 M). Results fr om several similar experiments show that activation of receptors with the DTG inhibited ASIC1a-mediated currents by 53 9 %, and this decrease was statistically significant manner (Figure 2.12B). To determine if the e ffects of DTG on ASIC1a currents were mediated by -1 or -2 receptors, the effects of the -1 selective agonist, CBP, on whole-cell currents were measured. Figure 2.12C shows representative membrane current traces as a function of time recorded in the absence (Control) and presence of CBP (50 M). After mu ltiple experiments, analysis of the measured current densities showed that cont rol cells had statistically significant larger current densities than CBP treated ce lls (Figure 2.12D). In the presence of 50 M CBP, ASIC1a-mediated currents were decreased by 30 5 % relative to control. In contrast, the -2 selective agonist, PB28 (20 M), failed to inhibit ASIC1a-mediated membrane currents, suggesting -2 receptors do not regulate ASIC1a function (Figure 2.12D) Therefore, these resu lts demonstrate that -1

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44 receptors functionally couple to ASIC1a cha nnels in cortical neurons as well as to targets downstream of ASIC1a activation.

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45 Figure 2.1 ASIC1a blockers inhibi t proton-evoked increases in [Ca2+]i in cultured cortical neurons from embryonic (E18) rats. A, Representative traces of [Ca2+]i as a function of time recor ded from a single cell duri ng acidosis in the absence (Control, Wash) and presence of 100 M Amiloride (Amiloride). B, Mean change in peak [Ca2+]i ( SEM) measured in response to low pH solution (pH 6.0) under the indicated conditions (n = 69). C, Representative traces of [Ca2+]i as a function of time recorded from a neuron during acidosis (Control, Wash) and following 20 minutes preincubation in 500 ng/ml Psal motoxin1 venom (PcTx1). Psalmotoxin1 venom was only present in the pH 7.4 conditioning solution. D, Mean change in peak [Ca2+]i ( SEM) measured during acidosis under the indicated conditions (n = 158). Asterisks denote significant differ ence from Control and Wash groups in (B) and (D) ( p <0.001).

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46

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47 Figure 2.2 Pan-selective sigma agonist s inhibit proton-evoked transient increases in [Ca2+]i. A, Representative traces of [Ca2+]i as a function of time recorded from a single neuron during acidos is in the absence (Control, Wash) and presence of 100 M DTG (DTG). B, Me an change in peak [Ca2+]i ( SEM) measured during acidosis in the absence (Control, n = 119) and presence of 100 M DTG (DTG; n = 99) or 10 M opipramol (OPI, n = 119). Asterisks denote significant difference from Control group ( p < 0.001), and pound symbol indicates significant difference from DTG group ( p < 0.05). C, Representative traces of [Ca2+]i as a function of time recorded from a neuron during acidosis in the absence (Control) and presence of 100 M and 300 M DTG. D, Concentrationresponse relationship for DTG inhi bition of mean change in peak [Ca2+]i ( SEM). Values obtained for each cell were normalized to their respective controls (no DTG) (n = 117-124). Line represents a bes t fit to the data using a single-site Langmuir-Hill equation.

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48

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49 Figure 2.3 Activation of -1 receptors inhibits ASIC1a-induced increases in [Ca2+]i. Representative traces of [Ca2+]i as a function of time recorded from two cells during acidosis in the absence (Control) and presence of 10 M and 100 M carbetapentane (A, CBP), or in t he absence (Control) and presence of 10 M and 100 M dextromethorphan (B, DEX). C, Concentration-response relationships for mean change in peak [Ca2+]i ( SEM) measured during acidosis in the presence of the indicated -1 selective ligands D EX, PRE-084, and CBP. Values were normalized to control (absence of ligand). Solid and dashed lines represent best fits to the data using single-site Langmuir-Hill equations. Dotted line represents best fit to the data obt ained for CBP inhibition of chemical ischemia-induced (4 mM azide in glucose-free PSS) increases in [Ca2+]i, and is shown for comparison (Katnik et al., 2006). For each condition n > 73.

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50

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51 Figure 2.4 Inhibition of -1 receptors blocks CBP-mediated suppression of proton-evoked increases in [Ca2+]i. A, Representative traces of [Ca2+]i as a function of time recorded from 2 different neurons during acidosis in the absence (Control) and presence of 30 M CBP (CBP ), without (PSS, left traces) and with 1hr preincubation in 50 M metaphit (Metaph it, right traces). B, Percent inhibition of [Ca2+]i increases ( SEM) by CBP measured during acidosis under control conditions (Control, n = 135) and follo wing metaphit preincubation (MET, n = 113). C, Representative traces of acid-induced increases in [Ca2+]i as a function of time recorded from 2 di fferent neurons in the absenc e (Control) and presence of 30 M CBP (CBP), without (PSS, left tr aces) and with co-application of 10 nM BD1063 (BD1063, right traces). D, Percent inhibition of [Ca2+]i increases ( SEM) by CBP under control conditions (Cont rol, n = 178) and in the presence of BD1063 (BD1063, n = 116). Asterisks in (B ) and (D) denote significant difference from Control groups ( p < 0.001).

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53 Figure 2.5 Sigma-2 receptor ligands in hibit ASIC1a-mediated elevations in [Ca2+]i at concentrations inconsistent with -2 mediated effects and in a metaphitinsensitive manner. A, Repr esentative traces of [Ca2+]i as a function of time recorded from two neurons during acidosis in the absence (Control) and presence of 10 M and 100 M ibogaine (IB O, left traces), or in the absence (Control) and presence of 1 M and 10 M PB28 (right traces). B, Mean change in peak [Ca2+]i ( SEM) measured during acidosis in the presence of the -2 selective ligands PB28 and IBO. Solid lines represent best fits to the data using single-site Langmuir-Hill equatio ns. Dashed line represent s best fit to the data obtained for IBO inhibition of ICa, and is shown for comparison (Zhang and Cuevas, 2002). For each condi tion, n > 108 cells. C, [Ca2+]i as a function of time recorded from 2 different neurons during acidosis in the absence (Control) and presence of 20 M PB28 (PB28), without (PSS) and with (+MET) 1hr preincubation in 50 M metaphit. D, Percent inhibition of [Ca2+]i ( SEM) by PB28 measured during acidosis under control c onditions (Control, n = 324), following metaphit preincubation (+MET, n = 276) and co-applied with 10 nM BD1063 (BD1063, n = 139). There is no signific ant difference between Control and +MET groups, ( p = 0.64) or Contro l and BD1063 groups ( p = 0.70).

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55 Figure 2.6 Intracellular Ca2+ stores are not involved in DTG modulation of ASIC1a-induced increases in [Ca2+]i. A, Representative traces of [Ca2+]i as a function of time recorded from a neuron dur ing acidosis in the absence (Control) and presence of 100 M DTG (DTG). B, Representative traces of [Ca2+]i as a function of time recorded from a neuron dur ing acidosis followin g preincubation in 10 M thapsigargin (1 hr, 23 C) in the absence (THAP) and presence of 100 M DTG (THAP + DTG). C, Mean change in peak [Ca2+]i ( SEM) measured in response to acidosis with (THAP, n = 346) or without (PSS, n = 216) thapsigargin preincubation in the absence (Control) and presence of 100 M DTG (DTG). Asterisks denote significant diffe rence from Control group ( p < 0.001). There was no significant difference between PSS and THAP groups ( p = 0.48).

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57 Figure 2.7 ASIC1a-mediated [Ca2+]i increases are membrane potential dependent. A, Representative traces of [Ca2+]i recorded in response to ASIC1a activation from a neuron electrically a ccessed using the perforated patch wholecell configuration and held at -70 mV ([Ca2+]i V-Clamp), and from a second neuron in the same field of view which was not electrically accessed ([Ca2+]i Control). Inset, whole-cell current tr ace recorded simultaneously from the voltage-clamped neuron. Lines above traces indicate application of pH 6.0 solution. Scale bars: 500 pA, 5 sec. B, Mean change in peak [Ca2+]i ( SEM) measured during acidosis in non voltage-clamped neurons (Control, n = 37) and in voltage-clamped neurons (V-Clamp, n = 4). Asterisks denote significant difference between groups ( p < 0.001).

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59 Figure 2.8 ASIC1a activation prom otes membrane depolarization. A, Representative traces of ASIC1a-m ediated membrane potential changes as a function of time recorded from a neur on held under current-clamp mode in the absence (Control) and presence of 100 M amiloride (Amiloride). B, Changes in peak membrane potential ( SEM) reco rded from neurons in normal PSS (Control) or in PSS containing 100 M amiloride (Amiloride, n = 3). Asterisk denotes significant differ ence from Control group ( p < 0.05). C, Representative traces of ASIC1a-mediated membrane potent ial changes as a function of time recorded from a different cell held under current-clamp mode in the absence (Control) and presence of 50 nM PcTx1 pept ide (PcTx1). D, Changes in peak membrane potential ( SEM) recorded from neurons in the absence (Control) or presence of 50 nM PcTx1 peptide (PcTx1 n = 3). No significant difference between Control and PcTx1 groups were noted ( p = 0.11).

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61 Figure 2.9 Sigma-1 recept or activation inhibits Ca2+ channels downstream of ASIC1a. A, Representat ive traces of [Ca2+]i recorded in response to application of extracellular high K+ (40 mM) solution in the abs ence (Control) and presence of 100 M CBP. B, Bar graph of mean change in peak [Ca2+]i ( SEM) measured after multiple experiments (n = 59). Asterisks denote significant difference between groups ( p < 0.001).

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63 Figure 2.10 Multiple plasma memb rane ion channels downstream of ASIC1a activation contribute to acidosis-evoked [Ca2+]i increases. A, Relative changes in [Ca2+]i ( SEM) during acidosis in the absence (PSS) and presence of 500 nM tetrodotoxin (TTX) alone or TTX (500 nM) co-applied with 100 M AP5, 10 M nifedipine or 100 M cadmium. All combinations were done without (Control) and with 50 M CBP (CBP). For each condition n > 101. Asterisks denote significant differences from respective control groups ( p < 0.001), pound symbol from PSS group ( p < 0.01 for TTX vs. PSS, p < 0.001 for all others), and dagger symbols from TTX group ( p < 0.001). B, Relative changes in [Ca2+]i ( SEM) during acidosis in the absence (C ontrol) and presence of 10 M CNQX (CNQX) without CBP (PSS) or with CBP at the indicat ed concentrations. For each condition n > 95. Asterisks denote significant differenc es from PSS group within Control or CNQX groups ( p < 0.001), pound symbols indicate significance differences between 50 M and 300 M CBP groups within Control or CNQX groups ( p < 0.001), and daggers denote significant di fferences between Control and CNQX within PSS ( p < 0.001) and 300 M CBP ( p < 0.05) groups.

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65 Figure 2.11 ASIC1a blockers inhibit acidosis-mediated currents in voltageclamped neurons. A, Repres entative traces of ASIC1a-mediated currents as a function of time record ed from two neurons held at -70 mV in the absence (Control) and presence of 100 M amiloride (Amiloride) ( i .) or 500 ng/ml Psalmotoxin1 venom (PcTx1) ( ii .). B, Relative mean peak proton-gated currents ( SEM) recorded from neurons in norma l PSS (Control) or in PSS containing either 100 M amiloride (Amiloride, n = 6) or 500 ng/ml Psalmotoxin1 venom (PcTx1, n = 4). Values were normalized to the maximum proton-evoked response recorded for control conditions (no drug) in each cell (I/Imax). Asterisks denote significant difference from Control group (p < 0.01). C, Relative peak protongated currents (I/Imax, SEM) recorded in the absence (PSS) and presence of 500 nM tetrodotoxin (TTX, n = 4) alone and TTX with 100 M AP-5 (n = 3), 10 M CNQX (n = 4), 10 M nifedipine (Nif, n = 6) or 100 M cadmium (Cd, n = 3) inthe bath solution. Cells were volt age-clamped at -70 mV. No significant differences between the groups were noted ( p = 0.826).

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67 Figure 2.12 Sigma receptor agonists inhibit ASIC1a-mediated currents in voltage-clamped neurons. A, Representativ e traces of ASIC1a currents as a function of time recorded from a single perforated-patched neuron held at -70 mV in the absence (Control) and presence of 100 M DTG (DTG). B, Mean peak proton-gated current densities ( SEM) m easured from neurons held at -70 mV without (Control) or with 100 M DTG (DTG) in the bath solution (n = 6). C, Representative traces of ASIC1a-medi ated currents as a function of time recorded from a voltage-clamped neuron (-70 mV) in the absence (Control) and presence of 50 M CBP (CBP). D, Mean peak prot on-gated current densities ( SEM) recorded from neurons held at 70 mV without (Contro l) or with 50 M CBP (CBP, n = 6) or 20 M PB28 (PB28, n = 4). Asterisks in (B) and (C) denote significant differences from respective Control groups ( p < 0.001).

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69 Discussion Activation of receptors depresses membrane currents and elevations in [Ca2+]i mediated by ASIC1a channels in cortical neurons. The pharmacological properties of the receptor involved are consistent with the effects being specifically mediated by the -1 receptor subtype. Fu rthermore, most of the elevations in [Ca2+]i triggered by acidosis are the result of Ca2+ channels opening downstream of ASIC1a acti vation. Stimulation of -1 receptors effectively suppressed these secondary Ca2+ fluxes both by inhibi ting ASIC1a and the other channels directly. ASIC are regulated by various factor s such as pH, membrane distention and arachidonic acid, and therefore, functi on as signal integrators in the CNS (Allen and Attwell, 2002; Lopez, 2002). All of these factors elic it or potentiate ASIC-mediated responses. Information on endogenous mechanisms that inhibit ASIC function is lacking. It has been s hown that NMDA re ceptors modulate ASIC1a function via the activation of a CaMKII signaling cascade, but activation of this pathway results in an increase in currents through ASIC1a (Gao et al., 2005). Thus, our finding that activation of receptors depresses ASIC1amediated responses is novel. Our conclusion that the responses observed are mediated specifically by ASIC1a is supported by the inhibition produced with the selective ASIC1a channel blocker, PcTx1 (Diochot et al., 2007), and that cultured cortical neurons from embryonic mice defic ient in the ASIC1a subunit fail to show increases in [Ca2+]i or membrane currents at the proton concentrations used here (Xiong et al., 2004). ASIC2a and ASIC2b su bunits are also expressed in the

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70 CNS, but homomeric ASIC2a channels are activated below pH 5.5, and ASIC2b does not generate currents in response to low pH (Lingueglia et al., 1997). Furthermore, neither homomeric ASIC 2a nor heteromultimeric ASIC1a/ASIC2a channels conduct Ca2+, and thus could not account for the changes in [Ca2+]i observed here (Yermolaieva et al., 2004). Results from Ca2+ imaging experiments suggest that it is specifically the -1 receptor subtype that modulates neur onal responses to ASIC1a activation. Studies have shown that the affinity of carbetapentane for -1 receptors is >50-fold greater than for -2 receptors (Rothman et al ., 1991; Vilner and Bowen, 2000). The calculated IC50 for carbetapentane inhibi tion of ischemia-evoked increases in [Ca2+]i via -1 receptor activation is 18. 7 M (Katnik et al., 2006), which is comparable to the 13.8 M IC50 for CBP inhibition of ASIC1a-induced [Ca2+]i increases. Carbetapentane also inhibi ts epileptiform activity in rat hippocampal slices via -1 receptors with an IC50 value of 38 M (Thurgur and Church, 1998). Similarly, we show that the -1 agonists dextromethorphan (IC50 = 22 M) and PRE-084 (IC50 = 13.7 M), both of which have >100-fold greater affinities for -1 than -2 receptors, block ASIC 1a-mediated responses at concentrations consistent with thos e reported in the literature. Dextromethorphan inhibits spreading depre ssion in rat neocortical brain slices with an IC50 ~ 30 M (Anderson and Andrew, 2002), whereas PRE-084 protects human retinal cells against oxidative stress with an IC50 ~ 10 M (Bucolo et al., 2006). The fact that IC50 values determined here for carbetapentane, dextromethorphan and PRE084 are in the low M range suggests that it is

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71 unlikely these agonists are affecting ASIC1a activity via -2 receptors, since high M to mM concentrations of these co mpounds are required to stimulate -2 receptors. Moreover, -2-selective agonists failed to inhibit ASIC1a-mediated responses at concentrations consistent with -2 specific effects. The strongest evidence that -1 receptor activation modulates ASIC1a comes from experiments using the antagonists, metaphit and BD1063. Metaphit has been shown to bind irreversibly to -1 receptors with an IC50 value of 50 M (Wu, 2003). Preincubation in metaphit blocks -1 receptor mediated modulation of voltage-gated K+ channels in intracardiac neurons and depression of ischemia-induced elevations in [Ca2+]i in cortical neurons (Zhang and Cuevas, 2005; Katnik et al., 2006). Preincubation of cortical neurons in 50 M metaphit antagonized CBP inhibition of ASIC1a by ~ 40%. BD1063 has been shown to have a higher affinity for -1 than -2 receptors and attenuates the dystonia produced by DTG in rats in a dose-dependent manner, suggesting this ligand acts as an antagonist at sites (Matsumoto et al., 1995). Here we show that CBP is unable to block acid-induced increases in [Ca2+]i when co-applied with BD1063. In addition, we found that metaphi t fails to inhibit the effects of the -2 agonist, PB28, on ASIC1a-mediated res ponses. Taken together, these data show that increases in [Ca2+]i in response to ASIC1a activation are modulated only by -1 receptors. Several studies have suggested that Ca2+ influx through ASIC1a channels is a key mechanism leading to cell death (Xiong et al., 2004; Yermolaieva et al., 2004). Depletion of Ca2+ from intracellular stores indica tes that most, if not all, of

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72 the acid-induced increases in [Ca2+]i is due to plasma memb rane influx. However, our results show that multiple ion channels downstream of ASIC1a activation contribute to acidosis-induced elevations in [Ca2+]i, including NMDA and AMPA/kainate receptors and VGCC. The activation of NMDA and AMPA/kainate receptors following ASIC1a stimulation was observed even when neuronal conduction was inhibited with tetrodot oxin. This observation suggests a presynaptic localization of ASIC1a, whereby activation of the channel by protons results in synaptic transmission and s ubsequent activation of postsynaptic glutamatergic receptors. C onsistent with this hypothesis, ASIC1a has been found to regulate neurotransmitter release probability in mouse hippocampal neurons (Cho and Askwith, 2008). receptors have been identified in both presynaptic and postsynaptic sites (Gonzalez-Alvear and Werling, 1995; Alonso et al., 2000), and thus may modulate channels in both regions. In t he presence of specific inhibitors of ionotropic glutamate rec eptors, activation of -1 receptors with CBP further decreased proton-evoked increases in [Ca2+]i, but the effects of CBP and the glutamate channel inhibitors were less than additive. Thus, -1 receptors also inhibit Ca2+ entry via NMDA and AMPA/kainate re ceptors directly by inhibiting these channels and indirectly by depressing ASIC1a activation. Application of the L-type VGCC inhibitor, nifedi pine, and the broad-spectrum Ca2+ channel inhibitor, cadmium, blocked ASIC1a-induced increases in [Ca2+]i by >70% and >90%, respectively. This observation indicates that most of the increases in [Ca2+]i produced upon ASIC1a activa tion is dependent on Ca2+ influx through VGCC.

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73 Co-application of CBP with nifedipine, but not with Cd2+, resulted in further reduction in the proton-evoked increases in [Ca2+]i. The conclusion that Ca2+ influx through ASIC1a channels itself cont ributed only a small fraction to the total observed [Ca2+]i increases was confirmed with simultaneous Ca2+ fluorometry and whole-cell patch clamp recordings. Cells voltage-clamped at –70 mV, which prevents NMDA receptor and VGCC acti vation, demonstrated minimal acidevoked elevations in [Ca2+]i. Taken together, our results show that the increases in [Ca2+]i evoked by ASIC1a activation are the result of synaptic transmission and subsequent opening of multiple Ca2+ channels, and that stimulation of -1 receptors downregulates all of these events. However, the fact that activation of -1 receptors depressed ASIC1a -mediated currents in cells voltage-clamped at -70 mV indicate that -1 receptors are functionally coupled to ASIC1a, and that the depression in acid-evoked increases in [Ca2+]i is not exclusively the result of -1 receptors blocking c hannels downstream of ASIC1a. The finding that -1 receptors can inhibit AS IC1a channels has significant physiological and pathophysiological imp lications. It has been proposed that ASIC1a activation may facilitate neurot ransmission by compensating for the decrease in excitatory neurotransmission caused by direct inhibition of postsynaptic Na+ and Ca2+ channels by protons which are released during exocytosis (Krishtal et al., 1987; Zha et al., 2006). Furthermore, the expression levels of ASIC1a have direct effects on the densit y of dendritic spines in hippocampal neurons (Zha et al., 2006). Thus, -1 receptors may influence cell-to-cell signaling in the CNS by a ffecting ASIC1a activity. One of the consequences of

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74 ASIC1a overexpression in mice is enhanc ed fear conditioning (Wemmie et al., 2004), whereas stimulation of -1 receptors is known to ameliorate conditioned fear stress (Kamei et al ., 1997). These observations, coupled with our current report, suggest that -1 receptor activation may pro duce anxiolytic effects via the inhibition of ASIC1a channels. The inhibition of ASIC1a by -1 receptors is a pot ential component of the neuroprotective properties of receptors, since activa tion of ASIC1a has been shown to contribute to stroke injury (Xi ong et al., 2004). Important ly, inhibition of ASIC1a has been shown to be neuroprotective at delayed time points following ischemic stroke (Simon, 2006). Thus, -1 receptor-mediated inhibition of ASIC1a may contribute to the enhanced neuronal survival following receptor activation 24 hr post-stroke in rats (Ajmo et al., 2006). Furthermore our data suggests that activation of ASIC1a stimulates the activity of NMDA and AMPA/kainate receptors and VGCC, all of which have been linked to ischemia-induced brain injury. Thus, -1 receptor activation may provide further neuroprotection by reducing the activity of these channel s which occurs subsequent to ASIC1a stimulation. Consistent with this pleiotropic effect of -1 receptors is our observation that -1 receptor activation suppressed extracellular high K+-induced increases in [Ca2+]i, which would also activate th ese downstream effectors. In conclusion, -1 receptors inhibit ASIC1a channel function and blunt acidosisevoked ionic fluxes and increases in [Ca2+]i. Thus, -1 receptors can be targeted for therapeutic intervention in pathophysi ological conditions involving ASIC1a activation.

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75 CHAPTER 3 SIGMA-1 RECEPTOR ACTIVATION I NHIBITS ASIC1a CHANNELS VIA A PERTUSSIS TOXIN SENSITIVE G PROTEIN AND AN AKAP/CALCINEURIN COMPLEX Introduction Acid-sensing ion channels (ASIC) are a class of ligand-gated ion channels that are members of th e degenerin/epithelial sodi um channel (Deg/ENac) superfamily (Waldmann et al., 1997b; Benos and Stanton, 1999). ASIC are expressed in both peripheral and cent ral nervous system neurons. Several extracellular and intracellu lar modulators of ASIC hav e been identified. Divalent cations (Zn2+, Pb2+, Ca2+) (Baron et al., 2001; Chu et al., 2004; Gao et al., 2004; Wang et al., 2006), lactate (Immke and McCleskey, 2001), serine proteases (Poirot et al., 2004) and redox reagents (A ndrey et al., 2005; Chu et al., 2006) have been shown to interact with the extr acellular domain of ASIC and influence the function of these channels. Moreover there is a conserved phosphorylation site within the intracellular C-terminal domain of ASIC1a that is also the calcium/calmodulin protein kinase II (CaM KII) (Gao et al., 2005), protein kinace C (PKC) (Baron et al., 2002) and protei n kinase A (PKA) (Leonard et al., 2003) binding site. Phosphorylation of ASIC 1a has been shown to potentiate ASIC1a function in neurons (Xiong et al., 2004; G ao et al., 2005). In co ntrast, the second

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76 messenger, calcineurin, dephosphorylates ASIC1a channels and results in downregulation of channel function (Chai et al., 2007). Dephosphorylation of ASIC1a is m ediated through the interaction with cytoskeletal anchoring protein, A-kinase anc horing protein (AKAP). Interestingly, AKAP150 and calcineurin have been implic ated in the downregulation of both ASIC1a and ASIC2a channels (Chai et al ., 2007). AKAP is a diverse protein family with more than 50 members which are abundantly expressed in the brain (Feliciello et al., 2001). Neuronal AKAP150 (rat) and AKAP79 (human) share a high degree of sequence homology, differing pr imarily in a 9 amino acid repeat sequence insert found only in rodents which has no known function (Dell'Acqua et al., 2006). AKAP150 anchors both kinases (cAMP-dependent Protein Kinase A and Protein Kinase C) and phosphatases (cal cineurin) that ar e inhibited when bound. AKAP150 targets these proteins, through a unique targeting motif, to specific subcellular sites and plasma me mbrane via association with structural proteins, membranes, or cellular organelles (Dell'Acqua et al., 1998; Diviani and Scott, 2001). AKAP150 has been shown to modulate the internalization of AMPA receptors, NMDA receptors duri ng long term potentiation and depression (Rosenmund et al., 1994; Westphal et al., 1999; Colledge et al., 2000; Gomez et al., 2002; Smith et al., 2006) and voltage-gated Ca2+ channel function (Oliveria et al., 2007). All of these ion channel s have shown to be regulated by receptor activation in neurons via various signaling cascade mechanisms. receptor activation has been shown to depress both membrane currents and elevations in

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77 [Ca2+]i mediated by ASIC1a channels in cort ical neurons. Furthermore, most of the elevations in [Ca2+]i triggered by acidosis were determined to be the result of Ca2+ channels opening downstream of ASIC 1a activation, and activation of -1 receptors effectively s uppressed these secondary Ca2+ fluxes by both inhibiting ASIC1a and/or the Ca2+ channels directly. But the signaling cascade mechanism by which -1 receptors regulate ASIC1a channels remains to be elucidated. -1 receptors are directly coupled to potassium channels in intracardiac neurons and activation of this receptor subtype depresses the excitability of these neurons, blocking parasympathetic input to the heart (Zhang and Cuevas, 2005). In cultured frog melanotrope ce lls, DTG and (+)-pentazocine have been shown to modulate electrical activity by reducing both a tonic K+ current and a voltage-dependent K+ conductance through activation of a cholera toxin-sensitive G protein (Soriani et al., 1998; Soriani et al., 1999). In Xenopus oocytes, the signal transduction cascade between Kv1.4 and receptors has been shown to be dependent on protein-protei n interactions (Aydar et al., 2002). In this study, experiments were conducted to determine the signaling cascade linking -1 receptors to ASIC1a channels and downstream Ca2+ channels. -1 receptors, ASIC1a and AKAP150 were shown to colocalize not only in the plasma membrane of cortical neuron cell bodies but also in the dendritic processes of these cells. Calc ineurin inhibitors, cyclosporin A and FK506, and the G protein inhibitor pertu ssis toxin (PTX) diminished the downregulation of ASIC1a by -1 receptors, suggesting that -1 receptors exert their effect via calcineurin-dependent dephosphorylation of ASIC1a and a PTX-

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78 sensitive G protein. Furthermore, disr uption of the acti n cytoskeleton or dissociation AKAP150 from the plasma membrane were shown to diminish -1 receptor mediated inhibiti on of ASIC1a channels. Mo reover, whole-cell patch clamp experiments confirmed that preinc ubation in PTX or disruption of the AKAP/calcineurin interact ion with VIVIT, prevented -1 receptor modulation of ASIC1a-mediated membrane currents, suggesting -1 receptors couple to ASIC1a channels via a PTX-sensitive G protein and an AKAP150/calcineurin complex. This is the second repo rt of receptor-mediated functional downregulation of ASIC1a channels. Thus far, -1 receptors are the only receptors identified to inhibi t ASIC1a channel function. Materials and Methods Primary Rat Cortical Neuron Preparation Primary cortical neurons from embr yonic (E18) rats were cultured as previously described by our laboratory (Kat nik et al., 2006). All procedures were done in accordance with the regulations of the University of South Florida Institutional Animal Care and Use Committe e. Cells were used after 10-21 days in culture. Calcium Imaging Measurements The effects of acidosis on intracellular Ca2+ concentrations were examined in isolated cortical neurons using rati ometric calcium imaging. Cytosolic free-Ca2+ was measured using the Ca2+ sensitive dye, fura-2. The membrane permeable ester form of fura-2, fura-2 AM, acetoxymethyl ester (AM), was loaded and

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79 imaged as we have previously described (DeHaven and Cuevas, 2004; Katnik et al., 2006). Briefly, cells plated on coverslip s were incubated for 1 hour at room temperature in Neurobasal (Invit rogen) medium supplemented with B27 (Invitrogen) and 0.5 mM L-gl utamine, or in physiological saline solution (PSS) consisting of (in mM): 140 NaCl, 5.4 KCl, 1.3 CaCl2, 1.0 MgCl2, 20 glucose, and 25 HEPES (pH to 7.4 with NaOH). Both solutions contained 4 M of fura-2, acetoxymethylester (fura2 AM) and 0.4 % dimethyl sulfoxide. The coverslips were washed in PSS (fura-2-AM free) prior to the exper iments being carried out. Electrophysiology Recordings ASIC1a-mediated membrane currents we re recorded using the protocol previously described by our laborato ry. Briefly, neurons plated on glass coverslips were transferred to a record ing chamber and membrane currents were amplified, filtered at 1 kHz, digitized at 5 kH z, and acquired using Clampex 8 (Axon). Electrical access was achiev ed using the amphotericin B perforatedpatch method to preserve intracellular in tegrity of neurons (Rae et al., 1991). An amphotericin B stock solution (60 mg/m l in DMSO) was made fresh everyday, kept on ice, light protected, and dilut ed to 240 g/ml (0.4% DMSO) in control pipette solution immediately prior to patching. The control pipette solution consisted of (in mM): 75 K2SO4, 55 KCl, 5 MgSO4, and 10 HEPES (titrated to pH 7.2 with N-methyl-d-glucamine). Patch el ectrodes were pulled from thin-walled borosilicate glass (World Precision Instru ments Inc., Sarasota, FL) using a Sutter Instruments P-87 pipette puller (Novat o, CA) and had resist ances of 1.0–1.5 M Access resistances (Rs) were monitored throughout experiments for stable

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80 values 20 M and were always compensated at 40% (lag, 10 s). All cells were voltage-clamped at -70 mV. Immunohistochemistry Cortical neurons plated on covers lips were first rinsed with phosphate buffer saline (PBS) to remove excess medi a. Cells were fixed by incubating in 95% ethanol and 5% acetic acid at -20C for 20 minutes, followed by four 2minute incubations at room temperatur e in 95%, 75%, 50% and 0% ethanol. Neurons were then permeabilized with PBS so lution containing 0.1% Triton X for 15 minutes and rehydrated with 3 washes in PBS and 5 washes in PBS with 0.5% bovine serum albumin (BSA). The ce lls were then blocked with 2% BSA in PBS for 45 minutes at room temperature. Neurons were incubated in the primary antibodies (ASIC1 guinea-pi g polyclonal IgG at 1: 500 dilution, AKAP150 goat polyclonal IgG at 1:50 dilution and -1 rabbit polyclonal Ig G at 1:500 dilution) in 0.5% BSA in PBS solution overnight at 4C and in the secondary antibodies (Alexa-Fluor 633 goat anti-guinea pig, Alexa-Fluor 555 donkey anti-goat and Alexa-Fluor 488 goat anti-rabbit, all at 1:1000 fold dilutions) for an hour at room temperature. Primary ant ibodies were purchased fr om: AKAP150 (Santa Cruz Biotechnology, Santa Cruz CA) and ASIC1a (Lifespan Biosciences, Seattle, WA). -1 receptor antibody was kindly prov ided by Drs. T. P. Su and Teruo Hayashi (Baltimore, Maryland). Secondar y antibodies were purchased from Invitrogen (Eugene, OR): Alexa Fluor 488 anti-rabbit, Alexa Fluor 555 anti-goat, and Alexa Fluor 633 anti-guinea-pig. Cove rslips with labeled cells were then

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81 mounted onto a microscope slide using Ve ctaShield Hardset Media with DAPI purchased from Vector Laborat ories (Burlingame, CA). Confocal Microscopy Confocal images of triple labeled neurons were collected using a Leica DMI6000 inverted microscope, TCS SP5 c onfocal scanner and a 100x/1.4 N.A. Plan Apochromat oil immersion objective (Leica Microsystems, Germany). Laser lines (405 Diode, Argon, HeNe 543, and HeNe 633) were applied to excite stained cells and tunable filters were us ed to minimize crosstalk between fluorchromes. Image sections at 0.4 m were acquired with photomultiplier detectors, processed and analyzed using Le ica LAS AF software version 1.8.2 (Leica Microsystems, Germ any). 2-D images of fluore scence emission for each fluorophore (AF488, green; AF555, red; AF6 33, purple) were collected for 20-30 optical sections along the z-axis for each field of view. Areas of colocalization for pairs of fluorophores in a single z-secti on are depicted as white pixels in the merged images and represent pixels within the boundaries drawn in the scatter plot (Figure 3.1). The area underneath the arc represents background pixels which have been eliminated, while pixels out side the two straight lines are pixels with intensities greater than background in only one of the images. The Overlap Coefficient (R), a co rrelation coefficient relating tw o images or regions within an image, is defined as ii 2 i 2 i i i i2 S 1 S 2 S 1 S R (1)

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82 where S1i is the intensity of pixel i in image 1 and S2i its intensity in image 2 (Manders, 1993). R ranges from 1, complete colocalizati on, to 0, complete noncolocalization and was calcul ated for entire images and wit hin regions of interest drawn around individual cells. Solutions and Reagents The control bath solution for all experiments was PSS. All drugs were applied in this solution using a rapid application system identical to that previously described (Cuevas and Berg 1998). ASIC activation was induced by applying PSS with a pH of 6.0 (+ /drug) to specifically target ASIC1a. Individual cells were exposed to 3 low pH applications. No rundown of responses was observed using this protocol. All chemical s used in this investigation were of analytic grade. The following drugs were used: DTG and pertussis toxin (SigmaAldrich, St. Louis, MO); carbetapentane and NMDA (Tocris Bioscience, Ellisville, MO); latrunculin-A, cyclospor ineA and VIVIT (Calbioche m, San Diego, CA); FK506 (LC Laboratories, Wobur n, MA); tetrodotoxin (Alomone Labs, Jerusalem, Israel); and fura-2 AM (Mo lecular Probes, Eugene, OR). Data Analysis Analysis of measured intracellular Ca2+ and membrane current responses was conducted using Clampfit 9 (Axon inst ruments). Imaging dat a files collected with SlideBook 4.02 (Intelli gent Imaging Innovations, Inc. ) were converted to a text format and imported into Clampfit for subsequent analysis. Statistical analysis was conducted using SigmaPlot 9 and SigmaStat 3 software (Systat Software, Inc.). Statistical differe nces were determined using paired and

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83 unpaired t-tests for within group and betwe en group experiments, respectively, and were considered significant if p < 0.05. For multiple group comparisons either a 1-way or a 2-way ANOVA was used, as appropriate. When significant differences were determined with an AN OVA, post-hoc analysis was conducted using a Tukey’s Test to determine in teraction between individual groups. Results To answer whether the signaling cascade linking receptors to ASIC channels might involve protein-protei n interactions, immunohistochemical fluorescent staining experiments were performed on cortical neurons to determine if -1 receptors, ASIC1a channels and AKAP150 colocalize. Figure 3.1A shows fluorescent images of neurons triple labeled for -1 receptors(i), AKAP150 (ii) and ASIC1a channels (iii) from the same field of view. The white pixels represent areas in the image where -1 receptor and AKAP150 (Figure 3.1B, i) labeling appear together as selected in the scatt er plot (Figure 3.1B, ii). The scatter plot maps pixe l intensities from the -1 receptor labeling versus intensities from the AKAP150 labelin g. The overlap coefficient for -1 receptor and AKAP150 colocalization calculated us ing Eq. 1 for the entire image was calculated to be 0.74. Figure 3.1C, i, shows the merged image of -1 receptor and ASIC1a labeling with co localized pixels represented in white and the colocalization scatter plot (Figure 3. 1C, ii). The overlap coefficient for -1 receptors and ASIC1a channels for the entire image was calculated to be 0.78. After multiple experiments (3 stains wit h a total of 59 cell bodies and 45 neuronal

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84 processes), the mean ov erlap coefficient for -1 receptor colocalization with AKAP150 and ASIC1a channels for individual cell bodies was calculated to be 0.723 .007 and 0.790 .008, and for defi ned dendritic processes to be 0.696 .013 and 0.751 .009, respectively (Figure 3.1D). These images suggest that 1 receptors colocalize with both AKAP150 and ASIC1a in the cell body and in the dendritic processes. The similarity of t he pixel patterns in both sets of images (Figure 3.1A, ii and iii) is consistent with previous results showing colocalization of AKAP150 and ASIC1a (Chai et al., 2007). Reports suggest that calcineurin may be involved in the functional downregulation of ASIC1a and ASIC2a (Cha i et al., 2007). T hus, experiments were carried out to determine if calcineur in is a constituent of the signaling cascade linking -1 receptors to ASIC1a ch annels. Figure 3.2A show characteristic traces of [Ca2+]i as a function of time recorded from three cells exposed to normal PSS solution (PSS, i) or PSS containing the calcineurin inhibitors FK-506 (1 M, ii) and cyclosporin A (1 M, cyclo A, iii), all in the absence and presence of CBP (10 M). Both calcineurin in hibitors reversed the effects of -1 receptor activation on ASIC 1a function. A summary of data collected from identical experiments is s hown in Figure 3.2B. Bath application of FK-506 or cyclosporin A alone had no effe cts on control responses. However, following inhibition of calc ineurin with either FK506 or cyclosporin A, CBP blocked ASIC1a-mediated responses si gnificantly less than under control conditions (Figure 3.2B). The percent-i nhibition of ASIC1a-mediated responses by CBP was significantly decreased from 45 1% in control cells to 31 1% and

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85 4 2% in the presence of FK-506 and cyclos porin A, respectively (Figure 3.2C). These results suggest -1 receptors are functionally linked to ASIC1a via a signaling cascade involving calcineurin. Th ese observations are consistent with reports in the literature which sugges t that dephosphorylat ion of ASIC1a and ASIC2a channels by this phosphatase ma y be involved in the functional downregulation of ASIC (C hai et al., 2007). Calcineurin has been shown to be activa ted by a pertussis toxin-sensitive G protein, specifically Gi2 (Gromada et al., 2001). Prev ious reports suggest that -1 receptors may couple to ion channels, such as voltage-sensitive K+ channels, via G proteins (Soriani et al., 1998; Soriani et al., 1999). Experiments were performed to determine whether G pr oteins were also involved in -1 receptor regulation of ASIC1a channe ls. Figures 3.3A and 3.3B show representative traces of [Ca2+]i as a function of time recorded during acidosis in the absence (Control) and presence of CBP (C BP, 50uM) without (A) and with PTX preincubation (200 ng/ml for 24 hrs at 37C, B). Results from multiple experiments suggest that PTX alone has some effect on ASIC1a-mediated [Ca2+]i increases but it does not change the kinetics of the responses (Figure 3.3B). Preincubation in PTX significantly reduced -1 receptor mediated inhibition of ASIC1a-induced [Ca2+]i increases (Figure 3.3C). While CBP inhibited 41 1% of acid-induced increases in [Ca2+]i in control cells, the -1 ligand only blocked 26 1% of the increases follo wing PTX treatment. This inhibition was statistically significant (Figure 3. 3D). These results suggest that -1 receptor

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86 activation inhibits ASIC1a-induced increases in [Ca2+]i via a PTX-sensitive G protein. Having identified calcineurin and a PTX-sensitive G protein in the signaling pathway linking ASIC1a and -1 receptors, it was of interest to determine other constituents of the si gnaling cascade. The scaffolding protein AKAP150 has been shown to anchor kinases (PKA and PKC) and the phosphatase calcineurin to regulate ion channels and receptors (Dell'Acqua et al., 1998; Dodge and Scott, 2000; Feliciello et al., 2001; Moita et al., 2002; Wong and Scott, 2004; Beene and Scott, 2007). Activation of NMDA due to Ca2+ influx through the receptor has been shown to cause rearrangement of the cytoskeleton resulting in dissociation of AKAP150 from the plasma membrane (Gomez et al., 2002). Figures 3.4A and 3.4B show representative traces of [Ca2+]i as a function of time recorded during acidosis in the absence (Control) and presence of CBP (CBP, 50 M) without (PSS, A) and with NMDA (10 M, B) preincubation. Pre-incubation in NMDA significantly reduced ASIC1a-mediated elevations in [Ca2+]i (Figure 3.4C). In addition, pre-incubation in NMDA also significantly reduced -1 receptor mediated inhibi tion of the remaining ASIC1ainduced [Ca2+ ]i increase (Figure 3.4C). While CBP inhibited 44 1% of acidinduced increases in [Ca2+]i in control cells, it only inhibited 40 1% in NMDA treated cells and this inhibition was statis tically significant (Figure 3.4D). The small but significant block by NMDA of -1 receptor modulat ion of ASIC1a can probably be attributed to additional effects of Ca2+ overload following preincubation in NMDA which led to increases in basal calcium levels. These

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87 results are consistent wit h the theory that the sca ffolding protein AKAP150 is involved in -1 receptors modulation of ASI C1a-induced increases in [Ca2+]i, assuming NMDA receptor activation results in AKAP150 dissociation. AKAP150 has been shown to associate with the plasma membrane in neurons by binding to the cytoskeleton via f-actin (Dell'Acqua et al., 1998; Dodge and Scott, 2000; Diviani and Scott, 2001; Felic iello et al., 2001; Wong and Scott, 2004). Latrunculin-A, a bioactive 2-thiazoli dinone macrolide, reversibly disrupts the actin cytoskeleton resulting in AKAP dissociation away from the membrane (Allison et al., 1998; Sattler et al., 2000; Zhou et al., 2001; Popp and Dertien, 2008). Figures 3.5A and 3.5B show representative traces of [Ca2+]i as a function of time recorded during acidosis in t he absence (Control) and presence of CBP (CBP, 50 M) without (DMSO, A) and with latrunculin-A (5 M, B) preincubation for 4 hours at 37C. While latrunculin-A alone did not significantly reduce ASIC1a-mediated [Ca2+]i elevations, disruption of th e neuronal cytoskeleton more substantially affected -1 receptor mediated inhibition of ASIC1a Ca2+ dysregulation (Figure 3.5C). In control cells CBP inhibited 47 1% of acidinduced increases in [Ca2+]i but only inhibited 29 1% of these increases in latrunculin-A treated cells (Figure 3.5D ). This inhibition was statistically significant. Disruption of the cytoskelet on by latrunculin-A was confirmed using phalloidin stain, which detects actin filaments cytochemically (Figure 3.6). Results showed that control cells had pres erved cytoskeleton staining around the cell body and along the dendritic processe s, while latrunculin-A treated cells

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88 showed staining patterns consistent with retraction of the cyt oskeleton from the processes and synapses (Figure 3.6). Previous studies have demonstrated that the major component of acidinduced increases in neuronal [Ca2+]i is from influx through Ca2+ channels activated by ASIC1a-induced membrane depolar ization. To isolate what direct effects disruption of the -1/G-protein/calcineurin/AKA P150 protein structure has on ASIC1a function, whole-cell patch cl amp experiments using the perforated patch method were performed. Cells were voltage-clamped at -70 mV to prevent NMDA receptor and voltage-gated Ca2+ channel activation, thus isolating ASIC1a channels. In these experiments the pan-selective agonist DTG was used instead of the -1 selective agonist CBP. Figures 3.7A and 3.7B show superimposed ASIC1a currents evoked by application of lo w pH solution (pH = 6) in the absence (Control) and presence (DTG) of 100 M DTG (DTG) without (A) and with (B) pertussis toxin (PTX, 24 hr preincubation at 37 C, 200 ng/ml). Figure 3.7C shows a bar graph of mean percent inhibition ( SEM) of ASIC1amediated currents elicited by bath application of 100 M DTG in neurons under control conditions and following preincubat ion in pertussis toxin (PTX, n = 7). Preincubation in PTX significantly decr eased the effects of DTG on acid-evoked currents, 10% versus 30% inhibition, res pectively (Figure 3.7B). Therefore, in cortical neurons, the effects of -1 receptors on ASIC 1a currents are dependent on activation of a PTX-sensitive G protein. VIVIT is peptide that selectively and potently inhibits calcineurin/AKAP interaction by disrupting calcineurin binding to a PxIxIT-docking motif in AKAP

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89 (Oliveria et al., 2007). Mor eover, VIVIT does not affect phosphatase activity (Hogan et al., 2003; Im and Rao, 2004). To el ucidate the effects of VIVIT on the signaling cascade linking ASIC1a channels to -1 receptors, cortical neurons were voltage-clamped at -70 mV to isolate ASIC1a currents. Figure 3.8A shows representative ASIC1a-medi ated currents evoked by low pH solution in the absence (Control) and presence of CBP (CBP, 100 M). In contrast, Figure 3.8B shows traces of ASIC1a-mediated current during acidosis from a different neuron in the absence (Control), presence of pr eincubation in the ce ll permeable peptide VIVIT (VIVIT, 10 nM, 5 minutes) and c oapplication of VIVIT and CBP (VIVIT/CBP 100 M). Analysis of the currents densities shows that while CBP significantly inhibited ASIC1a-mediated currents compar ed to control cells (Figure 3.8C), VIVIT alone or in combination with CBP had no affect on ASIC1a-mediated currents (Figure 3.8D). Taken toget her, these data, consistent with Ca2+ imaging results, suggest that -1 receptors couple to ASIC1a channels via a calcineurin/AKAP complex in addition to a PTX-sensitive G protein.

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90 Figure 3.1 Sigma-1 receptors co localize with both ASIC1a channels and AKAP150 in cortical neurons. A, Represent ative fluorescent images of neurons triple labeled for -1 receptors( i ), AKAP150 ( ii ) and ASIC1a channels ( iii ) in the same field of view. B, From A, mer ged image of -1 receptor and AKAP150. The white pixels represent areas in the image where -1 receptor and AKAP150 labeling appear together (B, i ) as selected in the scatter plot (B, ii ). The scatter plot maps pixel in tensities from the -1 receptor labeling versus intensities from the AKAP150 labeling. The overlap coefficient for -1 receptor and AKAP150 colocalization for the entire image was ca lculated to be 0.74. C, From A, merged image of -1 receptor and ASIC1a l abeling with colocalized pi xels represented in white (C, i ) and the colocalization scatter plot (C, ii ). The overlap coefficient for 1 receptor and AKAP150 colocalization for the entire image was calculated to be 0.78. All scale bars (A, B, C) are 25 m. D, Mean overlap coefficient for -1 receptor colocalization with AKAP150 and ASIC1a channels for individual cell bodies was calculated to be 0.723 .007 and 0.790 .008, and for defined dendritic processes 0.696 .013 and 0.751 .009, respectively.

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93 Figure 3.2 Calcineurin inhibition prevents -1 receptor modulation of ASIC1a. A, Characteristic traces of [Ca2+]i as a function of time recorded from three neurons in the absence (Cont rol) and presence of 10 M carbetapentane (CBP) during acidosis in normal PSS (i), or PSS containing 1 M FK-506 (ii, FK-506) or 1 M cyclosporin A (iii, Cyclo A). B, M ean peak proton-evoked changes in [Ca2+]i recorded from neurons in the absenc e (Control) and presence of 10 M carbetapentane (CBP) in normal PSS (n = 184), or PSS containing either 1 M FK-506 (n = 216) or 1 M cyclosporin A (n = 155). Asterisks denote significant differences from respective control groups ( p < 0.05), and pound symbols indicate significant differences from the PSS gr oup within CBP ( p < 0.05). C, Bar graph of mean CBP-induced inhibitions of peak proton-evoked changes in [Ca2+]i from the same experiments as B. Cont rol group represents cells exposed to acidosis in PSS alone. Asterisks denote sign ificant differences from control group ( p < 0.05).

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95 Figure 3.3 Sigma-1 receptors inhibi t acid-induced elevations in [Ca2+]i via a PTX-sensitive G protein. A, Representative traces of [Ca2+]i as a function of time recorded from a vehicle treated (PSS) neuron during acidosis in the absence (Control) and presence of 50 M CBP (CBP). B, Representative traces of [Ca2+]i as a function of time recorded from a different neuron during acidosis following preincubation in 200 ng/ml PTX (PTX, 24 hrs at 37 C) in the absence (Control) and presence of 50 M CBP (CBP). C, Mean changes in peak [Ca2+]i ( SEM) measured in response to acidosis with (P TX, n = 246) or without (PSS, n = 188) PTX preincubation in the absence (Control) and presence of 50 M CBP (CBP). Asterisks denote significant differences from respective Control groups ( p < 0.05), pound symbol indicates signific ant difference between PSS and PTX groups within CBP ( p < 0.05), and dagger denotes sign ificant difference between PSS and PTX groups within Control ( p < 0.05). D, Percent inhibitions of [Ca2+]i increases ( SEM) by CBP under cont rol conditions (Control) and in the presence of PTX (PTX). Asterisk denot es significant difference between the groups ( p < 0.05).

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97 Figure 3.4 AKAP150 dissociation from the plasma membrane prevents -1 receptor modulation of acid-induced elevations in [Ca2+]i. A, Representative traces of [Ca2+]i as a function of time recorded from a neuron during acidosis (PSS) in the absence (Control) and pr esence of 50 M CBP (CBP). B, Representative traces of [Ca2+]i as a function of time recorded from a different neuron during acidosis follo wing preincubation in 10 M NMDA (NMDA, 5 min -1 hr) in the absence (Control) and presence of 50 M CBP (CBP). C, Mean changes in peak [Ca2+]i ( SEM) measured in respons e to acidosis with (NMDA, n = 398) or without (PSS, n = 198) NMDA preincubation in the absence (Control) and presence of 50 M CBP (CBP). Asterisks denote significant differences from respective Control groups ( p < 0.05), pound symbol indicates significant difference between PSS and NMDA groups within CBP ( p < 0.05), and dagger denotes significant difference between PSS and NMDA groups within Control ( p < 0.05). D, Percent inhibition of [Ca2+]i increases ( SEM) by CBP under control conditions (Control) and in the presence of NMDA (NMDA). Asterisk denotes significant difference between the groups ( p < 0.05).

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99 Figure 3.5 Disruption of t he actin cytoskeleton prevents -1 receptor inhibition of acid-induced increases in [Ca2+]i. A, Representative traces of [Ca2+]i as a function of time recorded from a vehi cle (DMSO) treated neuron during acidosis in the absence (Control) and presence of 50 M CBP (CBP). B, Representative traces of [Ca2+]i as a function of time recorded from a different neuron during acidosis following preincubation in 5 M latrunculin A (Latrunc ulin A, 4 hrs at 37 C) in the absence (Control) and presence of 50 M CBP (CBP). C, Mean changes in peak [Ca2+]i ( SEM) measured in response to acidosis with (Latrunculin A, n = 222) or without (DMS O, n = 276) latruncu lin A preincubation in the absence (Control) and presence of 50 M CBP (CBP). Asterisks denote significant differences from respective Control groups ( p < 0.05), pound symbol indicates significant difference betw een DMSO and latrunculin A groups within CBP ( p < 0.05), and dagger denotes signific ant difference between DMSO and latrunculin A groups within Control ( p < 0.05). D, Percent inhibition of [Ca2+]i increases ( SEM) by CBP under cont rol conditions (Control) and in the presence of latrunculin A (Latrunculin A). Asterisk denotes significant difference between the groups ( p < 0.05).

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101 Figure 3.6 Latrunculin A preincubation disrupts the actin cytoskeleton. Cells were treated with either vehicle (A, DMSO) or latrunculin A (B, 5 M) for 4 hours at 37C. Cells were then stained with phalloidin to detect actin filaments cytochemically (A and B, right images). Fo r comparison, bright field pictures are also shown (A and B, left images). All scale bars are 25 m.

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103 Figure 3.7 Sigma receptor-mediated inhi bition of ASIC1a currents are blocked by preincubation in pertussis toxi n. A and B, Superim posed ASIC1a currents evoked by application of low pH solution (pH = 6) in the absence (Control) and presence of 100 M DTG (DTG) from cortical neurons without (A) and with preincubation for 24 hrs (37 C) in 200 ng/ml pertussis toxin (B, PTX). Cells were held at -70 mV. C, Bar graph of mean per cent inhibitions ( SEM) of ASIC1amediated currents elicited by 100 M DTG under control conditions (Control) or following preincubation in pertussis toxin (PTX), n = 7. Asterisk denotes significant difference from Control group ( p < 0.05).

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105 Figure 3.8 Sigma-1 receptors couple to ASIC1a channels via an AKAP150/calcineurin comple x. ASIC1a currents were elicited in whole-cell perforated patched, voltage-clamped (-70 mV) neurons by a 10 second application of pH 6.0 PSS. A, Representative traces of ASIC1a currents as a function of time recorded from a neuron in the absence (Control) and presence of 100 M CBP (CBP). B, Representative traces of ASIC1a-mediated currents as a function of time recorded from a diffe rent cell in the absence (Control) and presence of 10 nM VIVIT (VIVIT) and during coapplication of VIVIT with 100 M CBP (VIVIT/CBP). C, Mean peak prot on-gated current densities ( SEM) measured from neurons without (Control) or with 100 M CBP (CBP) in the bath solution (n = 6). Asterisks denote significant difference from respective Control group ( p < 0.01). D, Mean peak proton-gated current densities ( SEM) recorded under control conditions, in the presence of VIVIT alone, and in the presence of VIVIT plus CBP (n = 5). There were no significant differences between Control, VIVIT and VIVIT/CBP groups, ( p = 0.972).

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107 Discussion Results from this study sugges t the signaling cascade linking -1 receptors to ASIC1a channels in rat cort ical neurons involves direct proteinprotein interactions involving PTXsensitive G proteins, calcineurin and AKAP150. -1 receptors are shown to colocalize with both ASIC1a channels and the scaffolding protein AKAP 150, not only in regions of the cell body but also along the dendritic processes of these cells. Like previous studies in our laboratory and others, which showed that receptors affect ion channel function via mechanisms consistent with protein-protein interactions, -1 receptors modulate ASIC1a channels via a pertussi s toxin-sensitive G protein-coupled signal transduction cascade. Furthermore, -1 receptors are also shown here to couple to ASIC1a channels via the second messenger calcineurin anchored to AKAP150. This coupling results in decr eases in both ASIC1a-mediated currents and concomitant elevations in cytosolic Ca2+ following -1 receptor activation. receptors are the only reported instance of receptor mediated downregulation of ASIC activity. Thus far, only the NMDA receptor, acting via a Ca2+-calmodulin kinase II cascade (CaM KII), has been shown to modulate ASIC1a channels (Gao et al., 2005). Acti vation of NMDA receptors enhances ASIC1a-mediated currents, which consequent ly exacerbates acidotoxicity during ischemia (Gao et al., 2005). ASIC channe ls are also regulated in a similar manner by C kinase-1 (PICK-1), which bi nds to the C-terminus of several ASIC isoforms (Duggan et al., 2002). PICK1 has been shown to promote the stimulation of homomeric ASIC2a and heteromultimeric ASIC3/ASIC2b channels

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108 by protein kinase C (PKC ) (Baron et al., 2002; Deval et al., 2004), while several protein kinase C isoforms (PKC I or PKC II) have also been shown to inhibit ASIC1 (Berdiev et al., 2002). Protein ki nase A phosphorylation of ASIC interferes with the binding of PICK-1 to thes e channels, and disrupts PICK1-ASIC1 colocalization (Leonard et al., 2003). Fu rthermore, various signaling molecules have been linked to the regulat ion of ASIC. Three dist inct kinases (CaMKII, protein kinase C and protein kinase A) have all been shown to modulate the function of ASIC, by direct phosphorylat ion of the channel (Baron et al., 2002; Leonard et al., 2003; Gao et al ., 2005). Kinase anchoring pr oteins such as PICK1 and AKAP150 have also been shown to bind to ASIC subtypes, and appear to increase currents through these channels by facilitating protein kinase C and protein kinase A phosphorylation of t he channels, respectively (Baron et al., 2002; Deval et al., 2004; Chai et al., 2007). Conversely, inhibition of calcineurin has been shown to potentiate current s through ASIC1a and ASIC2a channels, and thus dephosphorylation of the c hannels by this phosphatase may be involved in downregulation of ASIC (Chai et al., 2007). Our results with the calcineurin inhibitors, cyclospor in A and FK-506, demonstrate that -1 receptormediated block of ASIC1a is dependent on activation of this phosphatase. Interestingly, reports have shown t hat calcineurin may be activated by a pertussis toxin-sensitive G protein (Gro mada et al., 2001). Consistent with this observation, our study shows that a pertuss is toxin-sensitive G protein is involved in the signaling cascade coupling -1 receptors to ASIC1a. It has been proposed that receptors regulate ion channel function via protein-protein interactions

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109 (Aydar et al., 2002). Moreover, inhibition of G proteins either by cell dialysis with GDP-S or preincubation in pertussis toxin failed to affect -1 and -2 receptor modulation of voltage-gated K+ and Ca2+ channels in intracardiac neurons (Zhang and Cuevas, 2002; Zhang and Cuevas 2005). To date, nearly all reports of receptor modulation of ion channels suggest that the effects involve a membrane-delimited signaling pathway which likely involves a protein-protein interaction. However, receptor activation has been shown to stimulate GTPase activity in mouse prefrontal membr anes (Tokuyama et al., 1997). A pertussis toxin-sensitive G protein has been im plicated in the modulation of NMDA receptors by receptors in rat CA3 dorsal hippocampus neurons (Monnet et al., 1994), whereas a cholera toxin-sensitive G protein has been suggested to couple -1 receptors to the channels mediati ng the A-current in frog pituitary melanotopes (Soriani et al., 1999) However, others have argued that receptors do not couple to G proteins (Hopf et al., 1996). Our findings demonstrate that -1 receptors can modulate ion channel functi on via activation of a PTX-sensitive G protein, and these results are the first ev idence of a receptor coupling to ASIC1a through this mechanism. AKAP150 has been shown to be involved in the regulation of receptor activity and localization, and in the r egulation of synaptic structure during developmental synapse formation, in synapt ic plasticity in learning and memory, and neuronal dysfunction and cell death dur ing pathophysiological conditions (Dell'Acqua et al., 2006). Additionally a calcineurin/AKAP150 complex has been shown to modulate both ASIC1a and ASIC 2a function (Chai et al., 2007). Our

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110 immunohistochemical studies suggest that -1 receptors colocalize with both ASIC1a channels and AKAP150 in the plasma membrane of the cell body and along the neuronal processes of the cells Furthermore, the si milarity of the distributions of receptors colocalized with ASIC1a channels and with AKAP150 is consistent with previous studies sho wing colocalization of ASIC channels and AKAP (Chai et al., 2007). Disr uption of the actin cytoskele ton by chemical agents or by NMDA activation has been shown to result in the redistribution of AKAP150 away from the plasma membrane (Gomez et al., 2002). Our data shows that both of these manipulations significantly prevents -1 receptor modulation of ASIC1ainduced [Ca2+]i elevations. The small but significant block by NMDA of -1 receptor modulation of ASI C1a can probably be attributed to additional effects of Ca2+ overload following preincubation in NM DA which led to increases in basal calcium levels. Previous results in our laboratory have shown that most of the elevations in [Ca2+]i triggered by acidosis are the result of Ca2+ channels (NMDA and AMPA receptors, voltage-gated Ca2+ channels) opening down stream of ASIC1a activation, and that activation of -1 receptors effectively suppresses these secondary Ca2+ fluxes. AKAP150 has been shown to modulate glutamate receptors like the internalization of AMPA receptors and NMDA receptors during long term potentiation or depression (Ros enmund et al., 1994; Westphal et al., 1999; Colledge et al., 2000; Gomez et al., 2002; Smith et al., 2006). In addition, AKAP also regulates voltage-gated Ca2+ channel function (Oliveria et al., 2007). Data presented here shows that -1 receptors functionally couple to ASIC1a

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111 channels via AKAP150, which is consis tent with all of these observations. Moreover, these results also raise the possibility that AKAP150 is a component in the modulation of other i on channels (voltage-gated Ca2+ channels) and receptors (NMDA and AMPA) by -1 receptors. The strongest evidence that -1 receptors couple to ASIC1a channels via a calcineurin/AKAP150 complex comes from experiments using the VIVIT peptide. Whole-cell patch clamp experim ents with VIVIT suggest that when this peptide competes with calcineurin for bindi ng to the PxIxIT-like motif in AKAP150 and thus prevents calcineurin binding to AKAP150, receptor activation loses its ability to regulate ASIC1a function. The di sruption of this interaction prevents -1 receptors from functionally coupling to ASIC1a channels. Similarly, VIVIT has been implicated in the disrupt ion of calcineurin/AKAP150 modulation of L-type Ca2+ channels in hippocampal neur ons (Oliveria et al., 2007). In conclusion, our data show t hat the signaling cascade between -1 receptors and ASIC1a channe ls involves a PTX-sensitive G protein and an AKAP150/calcineurin comple x in cortical neurons. This coupling results in decreases in both ASIC1a-mediated membrane currents and concomitant elevations in cytosolic Ca2+ following -1 receptor activation. Moreover, calcineurin may be a potent ial component of the neuropr otective properties of receptors. Furthermore, our result s also raise the possibility of -1 receptors coupling to downstream Ca2+ channels (voltage-gated Ca2+ channels) and receptors (NMDA and AMPA) via a sim ilar signaling cascade. All of these channels and receptors have been implicated in pathophysiological conditions,

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112 such as stroke (Tanaka et al., 1997; T anaka et al., 1999; Tanaka et al., 2002). 1 receptors are the first receptor shown to downregulate ASIC1a channel function and remain as the only recept or identified thus far to negatively modulate ASIC1a channels.

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113 CHAPTER 4 SIGMA-1 RECEPTOR INHIBIT ION OF INTRACELLULAR Ca2+ DYSREGULATION DUE TO SYNERGISTIC INTERACTION BETWEEN ACIDOSIS AND ISCHEMIA Introduction Glucose and oxygen deprivation associ ated with brain ischemia initiates a switch in metabolism from aerobic to anaerobic glycolysis to produce cellular energy. The accumulation of lactic acid, the end product of anaerobic glycolysis, leads to acidosis in the ischemic regi on, activating Acid-Sensing Ion Channels (ASIC) (Xiong et al., 2004; Gao et al., 2005; Pignataro et al., 2007). Synaptic vesicles contain not only the neurotransmitt er glutamate but also protons that are released along with glutam ate during neurotransmissi on. It remains to be elucidated if protons rel eased during neurotransmission also activate ASIC. ASIC subtypes are distinguishable by the pH of half-maximal activation, Ca2+ permeability and tissue expression pa ttern. The predominant ASIC subtype in the central nervous system (CNS) c ontains the ASIC1a subunit, which can form homomultimeric or heteromultimeri c channels with ASIC2a (Askwith et al., 2004). ASIC1a channels are activated by pH 7 and have a pH of half-maximal activation of ~ 6.0 6.5 (Waldmann et al., 1997b; Xiong et al., 2004), which is similar to the pH seen during an ischemic insult (Nedergaard et al., 1991; Back et

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114 al., 2000). The homomeric ASIC1a channel is the only ASIC subtype that is highly permeable to both Na+ and Ca2+ ions (Xiong et al., 2004; Yermolaieva et al., 2004). ASIC1a has been implicated in various neuronal physiological processes such as synaptic plasticity, fear condi tioning, and learning and memory (Wemmie et al., 2002; Wemmie et al., 2003; Wemm ie et al., 2004). ASIC1a has also been shown to be activated following cerebral ischemia, and has been linked to neuronal cell death (Xiong et al., 2004; Gao et al., 2005; Pignataro et al., 2007). Compared to wild-type mice, transgenic mice deficient in ASIC1a have reduced infarct size in response to middle cerebr al artery occlusion (MCAO) (Xiong et al., 2004). Furthermore, pharmacological inhi bition of ASIC1a with either the nonselective Na+ channel blocker amiloride or t he homomultimeric ASIC1a selective inhibitor psalmotoxin1 (Diochot et al., 2007), diminishes ischemic brain injury (Xiong et al., 2004). Thus, there is a dire ct correlation between infarct size, brain acidosis and ASIC activation, suggesting t he acidotoxicity occurring during stroke is in part mediated by ASIC1a channels (Xiong et al., 2004). The demise of neurons during ischemia and acidosis is predicated by intracellular calcium overload (Xiong et al ., 2004; Yermolaieva et al., 2004). Our laboratory has shown that -1 receptors inhibit Ca2+ dysregulation evoked by ischemia (Katnik et al., 2006) as well as acidosis, and that activation of receptors is neuroprotective at delayed ti me points in a rat model of ischemic stroke (Ajmo et al., 2006). Moreover, -1 receptors regulate ionotropic glutamate receptors, voltage-gated K+ channels and voltage-gated Ca2+ channels (Hayashi

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115 et al., 1995; Aydar et al., 2002; Zhang and Cuevas, 2002; Zhang and Cuevas, 2005). The inhibition of ionotropi c glutamatergic receptors by receptors prevents elevations in [Ca2+]i associated with glutamate-induced excitotoxicity (Klette et al., 1995; Klette et al., 1997). All of these voltage-gated ion channels as well as NMDA receptors have been shown to contribute to the demise of neurons during ischemia (Tanaka et al., 2002). C onsistent with this observation, our laboratory has also shown that mo st of the elevations in [Ca2+]i triggered by acidosis are the result of Ca2+ channels (NMDA and AMPA receptors and voltage-gated Ca2+ channels) opening following ASIC1a activation, and stimulation of -1 receptors effectively suppressed these secondary Ca2+ fluxes by inhibiting both ASIC1a and these Ca2+ channels directly. Previous reports have indicated t hat the glucose-oxygen deprivation model of ischemia potentiates ASIC1a -mediated currents in neurons (Xiong et al., 2004). Moreover, it has also been shown that ischemia enhances ASIC1a currents through phosphorylation of the channel by CaMKII as a result of activation of NMDA receptor (Gao et al., 2005). But how ASIC1a activation affects the responses to ischemia and whet her acidosis has any temporal effects during ischemia remains to be determined. Furthermore, it is unknown whether acidosis and ischemia interact in terms of the Ca2+ dysregulation produced. Interestingly, ASIC have been shown to re main active for greater than 4 hours following an ischemic insult (Pignataro et al., 2007). Pharmacological blockade of ASIC1a by amiloride or PcTx1 administ ered even 5 hours after MCAO has been shown to diminish stroke injury (S imon and Xiong, 2006; Pignataro et al., 2007).

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116 These observations raise the possibility that receptors may provide neuroprotection when stimulated se veral hours after an ischemic insult by directly inhibiting ASIC1a channels as well as Ca2+ channels opening in response to membrane depolarizations produc ed by ASIC1a activation. Experiments were undertaken to dete rmine whether ASIC1a activation and ischemia interact to produce potentiated elevations in [Ca2+]i and to determine if activation of -1 receptors is able to block this synergistic potentiation. Ratiometric Ca2+ fluorometry experimen ts demonstrated that acidification of the extr acellular solution from pH 7.4 to 6.0 produced a potentiation of the ischemia -induced elevations in [Ca2+]i. Inhibition of ASIC1a channels with either amiloride or PcTx 1, significantly decreased ischemiainduced elevations in [Ca2+]i at pH values ranging from 7.4 to 6.0, suggesting that homomeric ASIC1a channels ar e activated during ischem ia alone and that these channels contribute to the pH dependence of these [Ca2+]i increases. Furthermore, inhibition of synaptic transmission with tetrodotoxin prevented ischemia-evoked increases in [Ca2+]i but the effect of te trodotoxin was overcome by acidification of t he extracellular solution (pH 6.0). The selective -1 receptor agonist, carbetapentane, significantly decreased ischemia-mediated Ca2+ dysregulation at all pH values tested (7 .4-6.0), suggesting that activation of -1 receptors modulate ischemic and ASIC1a-activated [Ca2+]i increases individually while also effecting the mechanism mediat ing the synergistic interaction between these two initiators of Ca2+ influx pathways in co rtical neurons.

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117 Materials and Methods Primary Rat Cortical Neuron Preparation Primary cortical neurons from embr yonic (E18) rats were cultured as previously described by our laboratory (K atnik et al., 2006). All procedures were done in accordance with the regulations of the University of South Florida Institutional Animal Care and Use Commi ttee. Cells were used after 10-21 days in culture. Calcium Imaging Measurements The effects of acidosis and chemic al ischemia on intracellular Ca2+ concentrations were examined in isolat ed cortical neurons using fluorescent imaging techniques. Cytosolic free-Ca2+ was measured using the Ca2+ sensitive dye, fura-2. The membrane permeable ester form of fura -2, fura-2 AM, acetoxymethyl ester (AM), was loaded and imaged as we have previously described (DeHaven and Cuevas, 2004). Cells plated on coverslips were incubated for 1 hour at room temperature in Neurobas al (Invitrogen) medium supplemented with B27 (Invit rogen) and 0.5 mM L-glutami ne, or in physiological saline solution (PSS) consisting of (in mM): 140 NaCl, 5.4 KCl, 1.3 CaCl2, 1.0 MgCl2, 20 glucose, and 25 HEPES (pH to 7.4 with NaOH). Both solutions contained 3 M fura-2, acetoxymethylester (f ura-2 AM) and 0.3 % dimethyl sulfoxide. The coverslips were was hed in PSS (fura-2-AM free) prior to experiments being performed.

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118 Electrophysiology Recordings Na+-mediated membrane currents were recorded using a protocol previously described by our laboratory (Dr. Hongling Zhang, unpublished data). Briefly, neurons plated on glass covers lips were transferred to a recording chamber and membrane currents were amplifi ed, filtered at 1 kH z, digitized at 5 kHz, and acquired using Clampex 8 (Axon). Cells were patch-clamped using the conventional dialysis whole-cell confi guration and voltage-cl amped at -90 mV. Na+ currents were activated by stepping cells to -10 mV for 250 msec. The control pipette solution consisted of (in mM): 130 CsCl, 10 NaCl and 10 HEPES (titrated to pH 7.2 with CsOH ). The extracellular solution consisted of (in mM): 72 NaCl, 79 TEA-Cl, 5 KCl, 1.4 CaCl2, 1 MgCl2, 10 glucose, 5 BaCl2, 0.1 CdCl2, and 10 HEPES (titrated to pH 7. 4 or 6.0 with TEA-OH). Both the external and internal solutions were modified from other pr otocols (Dr. Hongli ng Zhang, unpublished data, and (Mike et al., 2003)). Patch elec trodes were pulled from thin-walled borosilicate glass (World Precision Instru ments Inc., Sarasota, FL) using a Sutter Instruments P-87 pipette puller (Novato, CA) and had resistances of 2.0 – 5.0 M Access resistances (Rs) were monitored throughout experiments for stable values 20 M and were always compensated at 40% (lag, 10 s). All cells were voltage-clamped at -90 mV. Solutions and Reagents The control bath solution for all experiments was PSS. All drugs were applied in this solution using a rapid application system identical to that previously described (Cuevas and Berg 1998). ASIC activation was induced by

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119 applying PSS buffered to pH values of 7.4, 7.0, 6.5 and 6.0 (+ /drug). Chemical ischemia was induced by applying glucos e-free PSS containing 4mM azide (+/drug) and titrated to pH 7.4, 7.0, 6.5, and 6.0. Indivi dual cells were exposed to 3 ischemic or acidic+ischemic insults to prevent rundown of responses (Katnik et al., 2006). All chemicals used in this invest igation were of analytical grade. The following drugs were used: carbetapentane (T ocris Bioscience, Ellisville, MO); psalmotoxin1 venom (Spider Pharm, Ya rnelle, AZ); psalmotoxin1 peptide (Peptide International, Louisville, KY); te trodotoxin (Alomone Labs, Jerusalem, Israel); amiloride (Alexis Biochemic als, Lausen, Switzerland); sodium-azide (Sigma-Aldrich, St. Louis, MO); and fura-2 AM (Molecular Pr obes, Eugene, OR). Data Analysis Analysis of measured intracellular Ca2+ response s and Na+-current activation was performed us ing Clampfit 9 (Axon instrume nts). Imaging data files collected with SlideBook 4.02 (Intelligent Imaging Innovati ons, Inc.) were converted to a text format and imported into Cla mpfit for subs equent analysis. Statistical analysis was conducted us ing SigmaPlot 9 and SigmaS tat 3 software (Systat Software, Inc.). Statistical differences were determined usin g paired and unpaired t-tests for within group an d between group experiments, respectively, and were considered significant if p < 0.05. For multiple group co mparisons either a 1-way or a 2-way ANOVA, with or without repeat measures, were used, as appropriate. When significant difference s were determined with an ANOVA, post-hoc analysis was conducted using a Tukey Test to determine differ ences between individual groups.

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120 Results The low extracellular pH associated with ischemia can activate ASIC (Xiong et al., 2004; Gao et al., 2005; Pi gnataro et al., 2007), and ASIC-mediated currents are potentiated dur ing ischemia (Xiong et al., 2004; Gao et al., 2005). During neurotransmission, protons are released along with glutamate but it remains to be elucidated whether t hese protons can activate ASIC at physiological pH values. Experiments were carried out to determine if ASIC are activated during ischemia at normal ph ysiological pH (7.4) and to determine the effects of pathophysiological pH values (7.0 6.0) during ischemia on [Ca2+]i dysregulation. Figures 4.1A and 4.1B show representative traces of [Ca2+]i as a function of time evoked by ischemia at pH 7.4 (A) and pH 6.0 (B) in the absence (Control) and presence of the ASIC bl ocker amiloride (100 M). Similar experiments demonstrate that amiloride si gnificantly inhibits ischemia-induced peak elevations in [Ca2+]i by 48% in solutions buffer ed to pH 7.4 (Figure 4.1C). These results indicate that proton s released along with glutamate during neurotransmission under physiol ogical conditions activa te ASIC. The application of ischemia + acidosis resulted in more robust peak increases in [Ca2+]i and these elevations were also inhibited by amiloride (Figure 4.1C). These data suggest that ASIC are activated during ischemia and potentiate ischemiainduced Ca2+ dysregulation. The kinetics of the Ca2+ responses to the combination of ischemia + acidosis are noticeably differ ent from those to ischemia or acidosis alone. To compare the kinetics of these responses, a single cell was exposed to acidosis

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121 (pH 6.0), ischemia at pH 7.4, and isc hemia at pH 6.0. Figure 4.2A shows representative traces of [Ca2+]i as a function of time fr om a single neuron during 2 minute applications of acidosis (pH 6.0 PSS) ischemia at pH 7.4 (Ischemia), and ischemia at pH 6.0 (Acidosis + Isc hemia) separated by 10 minute recovery periods. Acidosis alone produced an initial transient increase in [Ca2+]i with a rapid desensitization of the Ca2+ influx resulting in a retu rn to basal levels within seconds of the acid application. In contra st, ischemia alone resulted in smaller initial transient increase in [Ca2+]i that decreased to a sustained elevated level which returned to basal levels only upon washout of the ischemia solution. The combination of acidosis + ischemia pr oduced a significant potentiation in the initial transient peak [Ca2+]i elevations, which rapidly decreased to an elevated concentration which monotonically increased in the continual presence of the acidosis + ischemia solution. Upon washout of this solution, there was a transient rebound increase in [Ca2+]i which then slowly decayed to baseline levels. To quantitate the net elevati ons in the cytosolic Ca2+ concentration during these stimulations, increases in [Ca2+]i were integrated over the time period surrounding the acidosis + ischemia cha llenge yielding the ar ea under the curve. Figure 4.2B shows that there is a synergistic increase in [Ca2+]i in response to the combination of ischemia + acidosis when compared to the responses observed to acidosis and ischemia alone. These results indicate that not only does ischemia enhances ASIC activation, as previously reported (Xiong et al., 2004; Gao et al., 2005), but that acidosis potent iates the response to ischemia. This is

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122 the first report showing the role of aci dosis during ischemia and the effects of ASIC activation on ischemia-induced [Ca2+]i elevations. To better understand this synergistic interaction between acidosis and ischemia, experiments were performed to measure the [Ca2+]i responses to ischemic insults as a function of pH. Four parameters were used to compare these responses: (1) magnitude of the initial [Ca2+]i peak, (2) the steady state level of [Ca2+]i at the end of the ischemia applic ation, (3) the p eak magnitude of the rebound transient [Ca2+]i increase following washout of the ischemia solution, and (4) the net Ca2+ elevations measured in t he cytosol as determined by integrating the area under the [Ca2+]i vs. time curve. Results from multiple experiments show a signific ant increase in initial [Ca2+]i peak (Figure 4.3A), rebound [Ca2+]i peak (Figure 4.3C) and area (Fi gure 4.3D) as the pH of the ischemia solution was reduced from 7.4 to 6.0. Interestingly, the steady state level of [Ca2+]i obtained at the end of azide application increased when the pH was reduced from 7.4 to 7. 0, but then remained constant as the pH was further reduced to 6.0 (Figure 4.3B). Thes e data suggest that ischemia-induced increases in [Ca2+]i are pH dependent. Furthermore, acidosis significantly affects ischemia-induced [Ca2+]i elevations such that greater synergy is observed as the pH decreased from 7.4 to 6.0. Psalmotoxin1 (PcTx1) from t he venom of the tarantula Psalmopoeus cambridgei has been shown to be a selective inhibitor of homomultimeric ASIC1a channels (Diochot et al., 2007). To confirm that ASIC 1a channels were mediating this synergistic potentiation of changes in [Ca2+]i during ischemia at low pH

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123 values, cells were preincubated in the absence (PSS, Control) and presence of PcTx1 venom (PcTx1) in solutions buffe red to the indicated pH values. The venom was present in the conditioning solu tion (PSS pH 7.4) as well as all the ischemia solutions (pH 7.4 6.0). Pc Tx1 venom inhibit ed the initial peak elevations in [Ca2+]i to a statistically significant degree at all pH values tested (Figure 4.4A). The venom also blocked the rebound increases in [Ca2+]i following washout of the ischemia solution at pH values lower than 7.0 (Figure 4.4C). Interestingly, at the lower pH val ues, the PcTx1 venom is unable to block elevated steady state levels of [Ca2+]i or the net [Ca2+]i elevation (area) in response to ischemia + acidosis (Figure 4.4B and 4.4D). This observation could be explained as a result of PcTx1 inducing a shift in the steady-state desensitization of the channe l. In contrast, amiloride which directly blocks the channel, does inhibit steady state [Ca2+]i elevations. These results show that inhibition of ASIC1a channels prevent s acidosis-associated synergistic potentiation of [Ca2+]i dysregulation during ischemia. As a negative control, PcTx1 venom was heat inactivated to denature all proteins. Figures 4.5A and 4.5B s how representative traces of [Ca2+]i as a function of time recorded from a neuron dur ing ischemia at pH 7.0 and from a second cell during ischemia + acidosis in the absence (Control) and presence of heat inactivated PcTx1 venom (h.PcTx1). After multiple experiments, changes in initial peak, steady state and rebound [Ca2+]i, and net [Ca2+]i (area) ( SEM) were measured in response to ischemia at pH 7.0 and ischemia at pH 6.0 (Figure 4.5C

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124 and 4.5D). These results show that heat inactivated PcTx1 venom do not significantly affect ischemia-induced [Ca2+]i elevations at pH 7.0 or 6.0. Because PcTX venom is a cocktail of toxins (Psalmotoxin 1 (PcTx1), Psalmopeotoxin I (PcFK1), Psalmopeotoxin II (PcFK2), Vanillotoxin 1 (VaTx1), Vanillotoxin 2 (VaTx2), Vani llotoxin 3 (VaTx3)) experime nts were performed with pure PcTx1 peptide during acidosis alone (pH 6.0), ischemia at pH 7.0 and ischemia at pH 6.0. Figures 4.6A and 4. 6B show representative traces of [Ca2+]i as a function of time recorded from a neuron during ischemia at pH 7.0 and from a second neuron during ischemia + aci dosis in the absence (Control) and presence of synthetic PcTx1 peptide (s.PcTx1 ). Similar to results from the venom experiments, PcTx1 peptide significantly inhibited chan ges in initial peak, steady state and rebound [Ca2+]i, and net [Ca2+]i (area) ( SEM) m easured in response to ischemia pH 7.0 (Figure 4.6C). Furt hermore, the peptide inhibited initial and rebound [Ca2+]i peaks in response to ischemia at pH 6.0 (Figure 4.6D). These data confirm the results shown in Figure 4.4 were due to the activity of PcTx1 and not the other toxins in the venom. Therefore, ASIC1a c hannels are mediating the synergistic potentiation seen duri ng the combination of ischemia and acidosis. The synergistic interaction between ischemia and acidosis raises the possibility that ASIC1a channels promot e long-lived priming of presynaptic vesicles, and could thus, enhance release of neurotransmitter if stimulated prior to an ischemic event. To test this hypot hesis, cells were exposed to acid alone prior to the ischemia app lication. Figure 4.7A shows representative traces of

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125 [Ca2+]i as a function of time recorded from a neuron exposed to ischemia (black trace) and from a different cell exposed to a 10 second acid application prior to ischemia (gray trace). Analysis of the data comparing the two ischemia applications suggest that ASIC1a activa tion alone does not promote long-lived vesicle priming since the mean changes in peak [Ca2+]i were comparable (Figure 4.7B). These data suggest t hat ASIC1a activation alone prior to ischemia does not result in greater glutamate release. Furthermore, these data are consistent with results presented in this study and conf irm that the synergist ic potentiation in Ca2+ observed is the result of the combinatio n of ischemia + acidosis and that for the potentiation to occur, the events must take place simultaneously. Consistent with this observation, ischemia potent iates ASIC1a-mediated currents following OGD preincubation (Xiong et al., 2004). To further substantiate that the c oncurrent incidence of ischemia and acidosis is required to produce synergistic potentiation of Ca2+ dysregulation, the temporal effects of acidosis on the i schemia responses were studied. Figure 4.7C shows representat ive traces of [Ca2+]i as a function of ti me recorded from a cell exposed to ischemia alone (black tr ace) and from a sec ond neuron exposed to ischemia followed by the combination of ischemia + acidosis (gray trace). Addition of protons resulted in a stat istically significant increases in mean changes in [Ca2+]i in peak, steady state, rebound peak, and net [Ca2+]i (area) (Figure 4.7D). These data confirm that the synergistic potentiation of Ca2+ dysregulation is due to the interaction between ischemia and acidosis. Moreover,

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126 these results indicate that ASIC1a channels can be activated again after the initial ischemic onset, exac erbating ischemia-induced Ca2+ dysregulation. Next we wanted to determine the mechanism responsible for the rebound increase in [Ca2+]i following washout of ischemia and acidosis. Figure 4.7E shows representative traces of [Ca2+]i as a function of time recorded from a neuron exposed to the combination of isc hemia + acidosis (black trace) and from a second cell exposed to the combination of ischemia + acidosis then rapidly switched to ischemia (pH 7.4) alone (gra y trace). In identical experiments, populations of cells exposed to these two treatments did not exhibit significant differences comparing mean changes in peak, steady state and net [Ca2+]i (area) (Figure 4.7F). In contrast, mean changes in rebound were significantly different (Figure 4.7F). Thus, these results sugges t that the relief of proton block of downstream Ca2+ channels allows for the influx of Ca2+ contributing to the rebound [Ca2+]i elevations. receptor activation has been shown to modulate multiple ion channels which are opened following an ischemic insult. Figure 4.8 shows bar graph representation of data from multiple experiments in the absence (Control) and presence of the -1 receptor agonist carbetapent ane (CBP) during ischemia at the indicated pH values. CBP significantly inhibited the initial transient ischemiainduced [Ca2+]i elevations (Figure 4.8A), consis tent with previous results showing -1 receptor inhibition of ASIC1a-activated Ca2+ increases. Similarly, CBP significantly reduced steady state (Figur e 4.8B) and rebound (Figure 4.8C) levels of [Ca2+]i induced by ischemia at all pH values tested, consistent with -1

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127 receptor inhibition of Ca2+ channels activated by ASIC1a-mediated depolarization. Likewise, CBP reduced the net [Ca2+]i elevations (area), suggesting -1 receptor activation not only inhibit Ca2+ influx pathways but may also modulate release from intracellu lar stores and/or extrusion pathways (exchangers and ATPases) (Figure 4. 8D). These results show that -1 receptor activation prevents the synergistic interaction between acidosis and ischemia. Moreover, the -1 selective ligand inhibits ischemia-induced Ca2+ dysregulation at all pH values (7.4 6.0). Tetrodotoxin (TTX) is an i nhibitor of voltage-gated Na+ channels, and thus, subsequent synaptic transmission. The activation of postsynaptic Ca2+ channels and receptors following ASIC1a stimul ation has been observed even when neuronal conduction is inhibited by TTX, s uggesting a presynaptic localization of ASIC1a channels. Therefore, experiments were conducted to determine the effects of presynaptic ASIC1a channels duri ng ischemia in the presence of TTX. Figures 4.9A and 4.9B show r epresentative traces of [Ca2+]i as a function of time evoked by ischemia pH 7.4 (A) and ischem ia pH 6.0 (B) in the absence (Control) and presence of tetrodotoxin (TTX, 500 nM). Similar to earlier reports, addition of TTX prevented ischemia-induced elevations in [Ca2+]i by 69 2% when compared to control responses (Figur e 4.9C and 4.9D) (Katnik et al., 2006). While in response to ischemia + ac idosis, the effects of TTX on [Ca2+]i dysregulation were diminished, reducing only 9 2% of the initial peak increase in [Ca2+]i (Figure 4.9C and 4.9D). These dat a suggest that presynaptic ASIC1a channels promote synaptic transmission during ischemia, and thus, overcome

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128 block of synaptic transmission and enhance postsynaptic [Ca2+]i increases. This observation is consistent with the hypot hesis that ASIC1a channels are located presynapticly and regulate neurotrans mitter release probability (Cho and Askwith, 2008). To confirm that the lack of TTX bl ock of ischemia-induced increases in [Ca2+]i by acidosis was not due to TTX being pH sensitive, whole-cell voltageclamp experiments were perform ed to measure voltage-gated Na+ currents in the absence and presence of TTX at pH 7. 4 and 6.0. Cells were patch-clamped using the conventional dialysis whole-cell configuration and voltage-clamped at -90 mV. Na+ currents were activated by step ping cells to -10 mV for 250 msec. Figure 4.10A show represent ative current traces reco rded from a single cell in the absence (Control) and pres ence of TTX (TTX, 500 nM) at pH 7.4 (i) and pH 6.0 (ii). Analysis of the m easured current densities show s that TTX significantly inhibits Na+ currents at pH 7.4 and pH 6.0 (F igure 4.10 B and 4.10C). Consistent with reports in the literature, our data also suggest that Na+ channels are pH sensitive (Figure 4.10B). Results from recordings of peak inward Na+ currents from 12 cells showed that TTX (500 nM) inhibited INa by 96.1 0.9% at pH 7.4 and 96.6 1.0% at pH 6.0 (Figure 4.10C). No significant difference was noted between TTX-evoked inhibition at pH 7.4 and 6.0 (p = 0.68) (Figure 4.10C). In conclusion, our data indicate that TTX inhibition of voltage gated Na+ currents is not pH sensitive, and thus, confirm t hat presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission by TTX and enhancing postsynaptic [Ca2+]i increases.

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129 Figure 4.1 ASIC activation contri butes to ischemia-evoked [Ca2+]i increases in cultured rat cortical neurons. A, Representative traces of [Ca2+]i as a function of time recorded from a single cell during ischemia at pH 7.4 in the absence (Control) and presence of 100 M amiloride (Amiloride). B, Representative traces of [Ca2+]i as a function of time record ed from a single neuron during ischemia and acidosis (pH 6.0) in t he absence (Control) and presence of 100 M amiloride (Amiloride). C, Mean changes in peak [Ca2+]i ( SEM) measured in response to ischemia at pH 7.4 (n = 182) and pH 6.0 (n = 166) in the absence (Control) and presence of 100 M Amiloride. Asterisks denote significant differences from respective Control groups ( p < 0.001) and pound symbols indicates significant difference between pH 6.0 and pH 7.4 within Control and Amiloride groups ( p < 0.001).

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131 Figure 4.2 ASIC activation and ischemia interact to produce a synergistic potentiation of elevations in [Ca2+]i. A, Representative traces of [Ca2+]i as a function of time recorded from a single ce ll during the indicated conditions. B, Quantification of net [Ca2+]i elevations from multiple experiments identical to that in (A) (n = 84). Net [Ca2+]i elevation is calculated by integrating [Ca2+]i over time. Asterisks denote significant differences from the Acidosis group ( p < 0.001), and pound symbols indicates significant di fference from Ischemia group ( p < 0.001).

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133 Figure 4.3 Protons potentiate i schemia-induced increases in [Ca2+]i. Mean change in initial peak (A), steady state (B) and rebound peak (C) [Ca2+]i, and net increase in [Ca2+]i (area, D) ( SEM) measured in re sponse to ischemia at pH 7.4 (n = 80), pH 7.0 (n = 120), pH 6.5 (n = 120) and pH 6.0 (n = 121). Asterisks denote significant differenc e from pH 7.4 group ( p < 0.05), pound symbols indicate significant differences from pH 7.0 group ( p < 0.05), and daggers denote significant differences from pH 6.5 group ( p < 0.05).

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135 Figure 4.4 Inhibition of homomeric ASIC1a channels decreases ischemia + acidosis-induced elevations in [Ca2+]i at pH values ranging from 7.4 to 6.0. Mean change in initial peak (A), steady state (B) and rebound peak (C) [Ca2+]i, and net [Ca2+]i increase (area, D) ( SEM) measured in response to ischemia at pH 7.4 (n = 72), pH 7.0 (n = 87), pH 6.5 (n = 62) and pH 6.0 (n = 215) in the absence (Control) and presence of 500 ng/ml Pc Tx1 venom (PcTx1). Asterisks denote significant differences from pH 7.4 group ( p < 0.05) and pound symbols indicates significant differences from respective Control groups ( p < 0.05).

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137 Figure 4.5 Heat inactivated PcTx1 v enom does not inhibit ischemia-induced increases in [Ca2+]i. Representative traces of [Ca2+]i as a function of time recorded from a neuron during ischemia at pH 7.0 (A) and from a second cell during ischemia + acidosis (B) in t he absence (Control) and presence of heat inactivated PcTx1 venom (h.PcTx1). Mean changes in initial peak (peak), steady state (SS) and r ebound (Rbd) [Ca2+]i, and net [Ca2+]i elevation (area) ( SEM) measured in response to ischemia pH 7.0 (C, n = 79) and ischemia pH 6.0 (D, n = 89). There is no significant diffe rence between any of the groups.

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139 Figure 4.6 PcTx1 peptide inhibits ischemia-induced elevations in [Ca2+]i. Representative traces of [Ca2+]i as a function of time recorded from a neuron during ischemia at pH 7.0 (A) and from a second cell during ischemia + acidosis (B) in the absence (Control) and presence of synthetic PcTx1 peptide (s.PcTx1). Mean changes in initial peak (peak), steady state (SS) and rebound (Rbd) [Ca2+]i, and net [Ca2+]i elevation (area) ( SEM) measur ed in response to ischemia pH 7.0 (C, n = 165) and ischemia pH 6.0 (D, n = 171). Asterisks denote significant difference between groups ( p <0.001).

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141 Figure 4.7 Temporal effects of acidosis on ischemia-induced Ca2+ dysregulation. A, Representative traces of [Ca2+]i as a function of time recorded from a cell during ischemia pH 7.4 (bla ck trace, protocol A) and from a second neuron during a 10 second acid application followed by ischemia pH 7.4 (gray trace, protocol B). B, M ean changes in initial peak ( peak), steady state (SS) and rebound (Rbd) peak [Ca2+]i, (nM), and net [Ca2+]i elevation (area, nM min) ( SEM) measured in response to ischemia pH 7.4 (protocol A, n = 81) and acid followed by ischemia pH 7.4 (protocol B, n = 108). Area was ca lculated using the two minute application of control prot ocol (A). Asterisks denote significant differences between the groups in peak [Ca2+]i ( p < 0.05), rebound [Ca2+]i peak ( p < 0.001) and area ( p < 0.001). C, Represent ative traces of [Ca2+]i as a function of time recorded from a cell during ischemia pH 7.4 (black trace, protocol A) and from another neuron during ischemia pH 7. 4 followed by ischemia + acidosis (gray trace, protocol C). D, Mean changes in initial ischemia pH 7.4 and ischemia + acidosis peaks (Peak), steady st ate (SS) and rebound (Rbd) [Ca2+]i (nM), and net elevation in [Ca2+]i (area, nM min) ( SE M) measured in response to ischemia pH 7.4 (protocol A, n = 49) and ischemia pH 7.4 followed by ischemia + pH 6.0 (protocol C, n = 149). Area was calculated using the two minute application of control protocol (A). Asterisks denote significant differences between the groups ( p < 0.001). E, Represent ative traces of [Ca2+]i as a function of time recorded from a neur on during ischemia + acidosis (black trace, protocol D) and from a second cell during ischemia + acidosis followed by ischemia pH 7.4 (gray trace, protocol E). F, Mean changes in init ial peak (Peak), steady state

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142 (SS) and rebound (Rbd) [Ca2+]i (nM), and net [Ca2+]i elevation (area, nM min) ( SEM) measured in response to ischemia + acidosis (protocol D, n = 79) and ischemia + pH 6.0 followed by ischemia at pH 7.4 (protocol E, n = 294). Area was calculated using the two minute applicati on of control protocol (D). Asterisks denote significant differences between the groups ( p < 0.001).

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144 Figure 4.8 Sigma-1 receptor activa tion inhibits ischemia-mediated Ca2+ dysregulation at pH values ranging from 7. 4 to 6.0. Mean changes in initial peak (A), steady state (B) and rebound peak (C) [Ca2+]i, and net [Ca2+]i elevation (area) (D) ( SEM) measured in response to ischemia at pH 7.4 (n = 141), pH 7.0 (n = 110), pH 6.5 (n = 196) and pH 6.0 (n = 181) in the absence (Control) and presence of 50 M carbetapentane (CBP). Asterisks denote significant differences from pH 7.4 group ( p < 0.05) and pound symbols indicate significant differences from respective Control groups ( p < 0.05).

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145

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146 Figure 4.9 Effects of synaptic trans mission inhibition are overcome by presynaptic ASIC1a channels. A, Representative traces of [Ca2+]i as a function of time recorded from a neuron during ischem ia (pH 7.4) in the absence (Control) and presence of 500 nM tetrodotoxin (TTX ). B, Representative traces of [Ca2+]i as a function of time recorded from a different cell during the combination of ischemia and acidosis (pH 6.0) in t he absence (Control) and presence of 500 nM tetrodotoxin (TTX). C, Mean changes in peak [Ca2+]i ( SEM) measured in the absence (Control) and presence of TTX (TTX 500 nM) in response to ischemia (pH 7.4, n = 126) and ischemia + acidosis (pH 6.0, n = 240). D, Percent inhibition of changes in peak [Ca2+]i by TTX measured during ischemia at pH 7.4 and pH 6.0. Asterisks denote significant differenc es from respective Control groups ( p < 0.001).

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147

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148 Figure 4.10 TTX inhibits Na+ currents at pH 7.4 and 6. 0. A, Representative Na+ current traces as a function of time recorded from a single cell in the absence (Control) and presence of TTX (TTX, 500 nM) at pH 7.4 ( i ) and pH 6.0 ( ii ). Cells were patch-clamped using the conventional dialysis whole-cell configuration and voltage-clamped at -90 mV. Na+ currents were activated by stepping cells to -10 mV for 250 msec. B, Mean peak voltage-gated Na+ current densities ( SEM) measured from neurons under the indicated conditions (n = 12). Asterisks denote significant difference from respective Control group ( p < 0.01) and pound symbols indicate significant difference bet ween pH 7.4 and pH 6.0 within Control ( p < 0.01). C, Percent inhibition of Na+ currents ( SEM) by TTX at pH 7.4 and pH 6.0. There was no significant diffe rence between pH 7.4 and pH 6.0 groups, ( p = 0.683).

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149

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150 Discussion The results from this study demonstr ate that in rat cortical neurons, acidosis and ischemia synergistically inte ract to produce potentiated elevations in [Ca2+]i that are inhibited by -1 receptor activation. Acidification of the ischemiainducing extracellular solution to pathophysiological pH values (pH 7.0) produced elevations in [Ca2+]i that were greater than the sum of the changes evoked by acidosis and ischemia al one. Moreover, the kinetics of these responses were significantly different. Inhibition of ASIC1a channels with either amiloride or PcTx1, significantly decr eased ischemia-induced increases in [Ca2+]i at pH values ranging from 7.4 to 6.0, suggesting that homomeric ASIC1a channels are activated during ischemia and that these channels contribute to the pH dependency of these [Ca2+]i increases. While our data indicate that activation of ASIC1a channels does not induce long-li ved priming of synaptic vesicles for release, channel activation does have a te mporal effect on ischemia-mediated [Ca2+]i increases after ischemia onset. More over, relief of proton block of Ca2+ influx pathways mediates the rebound of [Ca2+]i following washout of the acidic ischemia solution. The selective -1 receptor agonist, carbetapentane, decreased ischemia-mediated Ca2+ dysregulation at all pH values tested (7.46.0). TTX was shown to block ~70% of the initial ischemia-induced [Ca2+]i increases when the external solution was buffered to 7.4, but only produced ~10% block when the solution was acid ified to pH 6.0, suggesting that presynaptic ASIC1a channels promote sy naptic transmission which leads to postsynaptic [Ca2+ ]i increases.

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151 Previous studies have shown that ischemia and the acidosis that accompanies ischemia, produces marked increases in [Ca2+]i (Katnik et al., 2006), which is one of the main mechani sms leading to cell death (Tombaugh and Sapolsky, 1993; Murai et al., 1997; Mattson, 2000; Xiong et al., 2004; Yermolaieva et al., 2004). ASIC1a-mediat ed currents in neurons have also been shown to be potentiated by oxygen-glucos e deprivation (Xiong et al., 2004; Gao et al., 2005). Though the effects of ASIC 1a currents during ischemia have been established, this study is the first r eport showing how ASIC1a activation affects the responses to ischemia and how acidosis and ischemia interact with each other to produce a synergistic dysregulation in [Ca2+]i. Our in vitro model of ischemia using the combination of az ide in glucose-free PSS and acidosis (pH 6.0), resembles in vivo m odels of stroke since simila r pH values have been observed during ischemia in whole ani mal studies (Nedergaard et al., 1991). Together, ischemia and acidosis produced elevations in [Ca2+]i that were greater than the sum of the changes evoked by ac idosis and ischemia alone. There was also a noticeable difference in t he kinetics of the observed [Ca2+]i transients resulting from acidosis, ischemia, and ischemia + acidosis. These differences cannot be explained by ischemia potent iating ASIC currents because ASIC1a channels desensitize within seconds. T hus, we conclude the effects seen minutes after the initial insult (steady state, rebound and net Ca2+ elevations) following ischemia + acidosis are the re sult of ASIC1a activation potentiating ischemia-mediated [Ca2+]i increases. Therefore, isc hemia and ASIC1a activation interact to produce potent iated elevations in [Ca2+]i dysregulation.

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152 The fact that ASIC1a inhibitors am iloride and PcTx1 reduce the ischemiainduced increases in [Ca2+]i when the external solution was buffered to pH 7.4 suggests that the acidosis produced by meta bolic inhibition is not responsible for ASIC1a activation. Instead, it is reasoned that protons released from glutamate containing synaptic vesicles during neurot ransmission (DeVries, 2001; Traynelis and Chesler, 2001) are the source of ASIC1a channel activation. Therefore, there are two sources of protons associated with isch emia. First, an internal source as glucose and oxygen deprivat ion initiates a metabolic switch to anaerobic glycolysis to produce cellular ene rgy leading to the accumulation of lactic acid, the end product of anaerobic glycolysis. Second, protons released along with glutamate during synaptic tr ansmission. In our model, ischemia produces increased action potential firing an d thus acidification of the synaptic cleft and the accumulation of lactic acid is mimicked by buffering external solutions to pH 6.0. Increased neurotr ansmission would activate more ASIC1a channels, inducing a feed-forward mechani sm which promotes further synaptic transmission and greater Ca2+ accumulation. These increases in [Ca2+]i, if of sufficient duration, would produce Ca2+ overload and eventually lead to cell death. Consistent with these conclusions, the interaction between ischemia and acidosis leads to the syner gistic potentiation of Ca2+ dysregulaton, which is what is expected to be seen in vivo follo wing an ischemic stroke (Tombaugh and Sapolsky, 1993; Murai et al., 1997; Mattson, 2000; Xiong et al., 2004; Yermolaieva et al., 2004).

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153 The [Ca2+]i responses to ischemia + acidosis were characterized by four parameters, the initial transient peak amplit ude, the steady state level just prior to washout of the ischemia + acid solu tion, the transient rebound amplitude following washout and the net [Ca2+]i elevations, calculated by integrating [Ca2+]i for the duration of the response. The st rongest evidence that the initial peak in ischemia-induced [Ca2+]i elevations reported here is due to activation of homomeric ASIC1a channels is the inhibi tion produced by both amiloride and the selective ASIC1a channel blocker PcTx1 (D iochot et al., 2007). The steady state [Ca2+]i is a function of the activated Ca2+ influx and efflux pathw ays. Interestingly, the steady state level of [Ca2+]i obtained at the end of ischemia applications increased as the pH was reduced from 7.4 to 7.0, but then remained constant as the pH was reduced to 6.0. Previous studi es suggest most of the elevations in [Ca2+]i triggered by acidosis are the result of Ca2+ channels (NMDA and AMPA receptors and voltage-gated Ca2+ channels) opening in response to ASIC1a mediated membrane depolarizati on. These results suggest that acidosis results in increased ASIC1a channel opening but t he effects of pH are countered by increased H+ block of other ion channels. The antioxidant MnTMPyP (25 nM) did not significantly affect the transient rebound increase in [Ca2+]i, suggesting that reactive oxygen species production do not account for the rebound mechanism. In contrast, the transi ent rebound increase in [Ca2+]i observed during washout of acidosis + ischemia solution is du e to the removal of a block of Ca2+ influx pathways by protons as the proton concent ration is reduced to pH 7.4. Since this feature of the [Ca2+]i response is not observed followi ng applications of ischemia

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154 at pH 7.4, it is unlikely that when observ ed at lower pH values it is due to the washout of the ischemia solution. The tr ansient rebound is expected to be more robust at the lower pH values due to a greater proton block of these Ca2+ channels being relieved. Conditions which pr oduce large net [Ca2+]i elevations, or Ca2+ overload, are likely to lead to apoptosis. Acidosis alone, while producing a large [Ca2+]i increase, has a very small net Ca2+ elevation because the transient increase is short lived. Likewise, while ischemia produces a maintained elevated [Ca2+]i level in the presence of the ischemia solution, it is only a moderately high level which returns quickly to baseline upon washout, and thus results in a small net elevation. The synergistic interaction of acidosis and ischemia, on the other hand, produces potentiated increases at all times during the agonist-evoked application as well as an extenuated r ebound increase and a slower decay back to baseline. All these contribute to produce net elevations in [Ca2+]i which are ~400% times greater than that for acidos is or ischemia alone. This pronounced Ca2+ overload would be more lik ely to trigger apoptosis. PcTx1 from the venom of the tarantula Psalmopoeus cambridgei has been shown to be a selective blocker of homomultimeric ASIC1a channels with no effect on other ASIC subtypes (1b, 2a or 3) or voltagegated channels (Na+, K+, Ca2+) (Diochot et al., 2007). PcTx1 induces a shift in the steady-state desensitization of the c hannel. At normal pH (7.4) this shift puts ASIC1a channels in the inactive state (Ch en et al., 2005). When studying ASIC1a activation, it is sufficient to include the toxin only in the conditioning media

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155 because the on and off rates are slow compared to the rate of channel desensitization (Chen et al., 2005; Chen et al., 2006). This also avoids the problem of the peptide being degraded in th e low pH solutions. For this study, however, the venom was included in the c onditioning media as well as the test solutions because the cellular response to ischemia lasted for the duration of the ischemia application. PcTx1 venom bl ocks the initial ischemia + acidosismediated [Ca2+]i increases as well as rebound and the elevated steady state levels at pH values > 6.5. Thus, blocking ASIC1a channels significantly prevented activation of the downstream Ca2+ channels responsible for the initial [Ca2+]i peak as well as disrupting the synergi stic interaction between acidosis and ischemia. Moreover, experiments per formed with a synthetic PcTx1 peptide provided similar results to those obt ained with the venom. Therefore, ASIC1a channels are mediating the synergistic pot entiation seen during the combination of ischemia and acidosis. Similar to earlier reports, blockade of voltage-gated Na+ channels with TTX and subsequent synaptic transmission i nhibition, prevents ischemia-induced elevations in [Ca2+]i at physiological pH (Katnik et al., 2006). Inte restingly, with the more acidic conditions present during the combination of ischemia and acidosis at pH 6.0, the inhibition of [Ca2+]i increases by TTX were overcome, most likely by the additional activation of ASIC1a channels. This observation suggests a presynaptic localization of ASI C1a, whereby activation of the channel by protons results in synaptic tr ansmission and subsequent activation of postsynaptic receptors. Consistent with th is theory, the activation of postsynaptic

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156 Ca2+ channels and receptors following ASIC1a stimulation during acidosis alone (pH 6.0) are observed even when neuronal c onduction is inhibited by TTX. It has been proposed that ASIC1a activation ma y facilitate neurotransmission by compensating for the decrease in excita tory neurotransmission caused by direct inhibition of postsynaptic Na+ and Ca2+ channels by protons which are released during exocytosis (Krishtal et al., 1987; Zha et al., 2006). Thus, action potential inhibition alone is not su fficient to prevent Ca2+ overload, as ASIC1a channel activation is able to overcome this block of glutamate release by depolarizing the presynaptic membrane to potentials capable of activating voltage-gated Ca2+ channels and thus, evoking synaptic transmi ssion. In addition, these results also raise the possibility of ASIC1a activation resulting in enough Ca2+ influx to promote synaptic transmission and glutamat e release. Similarly, heteromeric complexes of alpha 5 and/or alpha 7 subuni ts of nicotinic receptors have been shown to conduct Ca2+ and thus, resulting in nicotine-induced presynaptic facilitation (Girod et al., 1999). receptors have been identified in both pre and postsynaptic membranes (Gonzalez-Alvear and Werling, 1995; Alonso et al., 2000), modulating ion channels in both regions. receptor activation has been shown to modulate the function of multiple ion channels, seve ral of which have been linked to neuronal death following an ischemic insult. Studies have shown that the affinity of carbetapentane for -1 receptors is >50-fo ld greater than for -2 receptors (Rothman et al., 1991; Vilner and Bow en, 2000). The concentrations of CBP used in this study are cons istent with CBP acting as a -1 receptor agonist

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157 (Thurgur and Church, 1998). Moreover, similar concentrations of CBP have previously been shown to inhibit i schemia-evoked increases in [Ca2+]i (Katnik et al., 2006) as well as ASIC1a-induced [Ca2+]i increases and membrane currents. Unlike the results seen with inhibi tion of ASIC1a by PcTx1 venom, CBP activation of -1 receptors is pH insensitiv e and significantly inhibited the synergistic increases in [Ca2+]i produced by ischemia and acidosis as measured by initial peak amplitud e, steady state, rebound and net elevations of [Ca2+]i. In conclusion, -1 receptors inhibit [Ca2+]i increases following ASIC1a channel activation, ischemia-induced increases in [Ca2+]i as well as the synergistic potentiation during ischemia + acidosis. Because acidic conditions exist during ischemic episodes, -1 receptors should be targeted for development of treatments for stroke and other pathophysiological conditions where ASIC1a channel activation are involved. -1 receptor mediated inhibition of Ca2+ channels activated by membrane depolarizations produced by ASIC1a channel activation or metabolic inhibiti on and the ASIC1a channels themselves, may contribute to the enhanced neur onal survival observed following administration of receptor agonists 24 hr post-stroke in rats (Ajmo et al., 2006). For these reasons, -1 receptors should be targeted for therapeutic intervention during ischemia, expandi ng the therapeutic window of stroke treatment. The finding that ASIC1a activation at any point during an ischemic insult results in synergistic [Ca2+]i increases has significant physiological and pathophysiological implications. Our dat a indicate that though ASIC1a channels rapidly desensitize, they are able to be re-activated minutes into the ischemic

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158 insult. This suggests that during pathophysiol ogical conditions it is possible to get repeated activation of ASIC1a channels if the extracellular media is buffered back to normal pH for even a short time. Even more dramatic though, is that under ischemic conditions these repetit ive activations of ASIC1a channels synergistically interact with ischemia to produce potentiated elevations in peak, steady state, and rebound [Ca2+]i elevations and net [Ca2+]i elevation (area), suggesting that to prevent the consequences of Ca2+ overload during ischemia it is critical to control ASIC1a activation. Consistent with these results, it has been suggested that the acidosis that occurs in vivo during ischemia exacerbates ischemic injury at later time points after the initial ischemic insult. The finding that ASIC1a activation interacts with ischemia to produce synergistic potentiation of Ca2+ dysregulation is a novel finding and suggests that this channel should be targeted even at delayed time points after stroke onset to prevent Ca2+ overload and neuronal apoptosis.

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159 CHAPTER 5 DISCUSSION Conclusions Stroke remains the 3rd leading cause of death and the leading cause of long term disability in the United States. Ischemic st roke, as a result of an embolus and restriction of blood flow to the brain, is the mo st common type of stroke accounting for over 80% of thes e cases. Thus far, only one drug (tissue plasminogen activator, tPA) has been appr oved by the FDA for the treatment of ischemia. This study has determined how -1 receptors modulate ASIC1a channels during physiologic al and pathophysiological conditions, and also suggest that this regulation could be one of the mechanisms of neuroprotection by receptor activation. Thus, receptors could be a pot ential therapeutic target for the treatment of stroke at delayed time points. ASIC are regulated by various factor s such as pH, membrane distention and arachidonic acid, and therefore, functi on as signal integrators in the CNS (Allen and Attwell, 2002; Lopez, 2002), but t hese factors elicit or potentiate ASICmediated responses. NMDA receptors modulate ASIC1a function via the activation of a CaMKII signaling cascade, but activation of this pathway results in an increase in currents through ASIC1a (G ao et al., 2005). Similarly, ASIC are

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160 also regulated by C kinase-1 (PICK-1), which binds to the C-terminus of several ASIC isoforms (Duggan et al., 2002). PI CK1 has been shown to promote the stimulation of homomeric ASIC2a and heteromultimeri c ASIC3/ASIC2b channels by protein kinase C (Baron et al., 2002; De val et al., 2004), while several protein kinase C isoforms have also been shown to inhibit ASIC1 (Berdiev et al., 2002). Protein kinase A phosphorylation of ASIC in terferes with the binding of PICK-1 to these channels, and disrupts PICK1-ASIC1 colocalizati on (Leonard et al., 2003). Three distinct kinases (CaMKII, protei n kinase C and protein kinase A) have all been shown to modulate the function of ASI C, by direct phosphorylation of the channel (Baron et al., 2002; Leonard et al., 2003; Gao et al., 2005). Kinase anchoring proteins such as PICK-1 and AKAP150 have also been shown to bind to ASIC subtypes, and appear to increase currents through these channels by facilitating protein kinase C and protein kinase A phosphorylation of the channels, respectively (Baron et al., 2002; Deval et al., 2004; Chai et al., 2007). Conversely, activation of receptors depresses ASIC1 a-mediated responses and remains the only reported instance of receptor mediated downregulation of ASIC activity. The responses observed throughout t hese studies are specifically mediated by ASIC1a channels and this conc lusion is supported by the inhibition produced by amiloride and the selective ASI C1a channel blocker, PcTx1 venom as well as the PcTx1 pepti de (Diochot et al., 2007). Fu rthermore, cultured cortical neurons from embryonic mice deficient in the ASIC1a subunit fail to show increases in [Ca2+]i or membrane currents at t he same proton concentrations

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161 used here (Xiong et al., 2004). ASIC2a and ASIC2b subunits are also expressed in the CNS, but homomeric ASIC2a channels are activated below pH 5.5, and ASIC2b does not generate currents in res ponse to low pH (Lingueglia et al., 1997). In addition, neither homomer ic ASIC2a nor heteromultimeric ASIC1a/ASIC2a channels conduct Ca2+, and thus could not account for the changes in [Ca2+]i observed (Yermolaieva et al., 2004). However, PcTx1 itself may not be an ideal pharmacological ag ent for stroke patients because of its size, stability and inability to cross t he blood brain barrier (Xiong et al., 2008). Therefore, the development of better compounds is un dergoing for the treatment of stroke. The -1 receptor subtype modulates neuronal responses to ASIC1a activation. Studies have shown that the affinity of carbetapentane for -1 receptors is >50-fold greater than for -2 receptors (Rothman et al., 1991; Vilner and Bowen, 2000). The calculated IC50 for carbetapentane inhibition of ischemiaevoked increases in [Ca2+]i via -1 receptor activation is 18.7 M (Katnik et al., 2006), which is comparable to the 13.8 M IC50 for CBP inhibition of ASIC1ainduced [Ca2+]i increases. Carbetapentane also inhibi ts epileptiform activity in rat hippocampal slices via -1 receptors with an IC50 value of 38 M (Thurgur and Church, 1998). Similarly, other -1 agonists, dextromethorphan (IC50 = 22 M) and PRE-084 (IC50 = 13.7 M), both of which have >100-fold greater affinities for -1 than -2 receptors, blocked ASIC1a-medi ated responses at concentrations consistent with those repor ted in the literature. De xtromethorphan inhibits spreading depression in rat neocor tical brain slices with an IC50 ~ 30 M

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162 (Anderson and Andrew, 2002), whereas PRE084 protects human retinal cells against oxidative stress with an IC50 ~ 10 M (Bucolo et al., 2006). The IC50 values determined here for carbetapent ane, dextromethorphan and PRE-084 are in the low M range and suggests that it is un likely these agonists are affecting ASIC1a activity via -2 receptors, since high M to mM concentrations of these compounds are required to stimulate -2 receptors. Moreover, -2-selective agonists (ibogaine and PB28) failed to inhibit ASIC1a-mediated responses at concentrations consistent with -2 specific effects and in a metaphit-insensitive manner. Pharmacological studies with the antagonists, me taphit and BD1063, confirm that -1 receptor activation modulates ASIC1a channels. Metaphit has been shown to bind irreversibly to -1 receptors with an IC50 value of 50 M (Wu, 2003). Preincubation in metaphit blocks -1 receptor modulation of voltage-gated K+ channels in intracardiac neurons and depression of ischemia-induced elevations in [Ca2+]i in cortical neurons (Zhang and Cuevas, 2005; Katnik et al., 2006). Preincubation of cortical neurons in 50 M metaphit antagonized CBP inhibition of ASIC1a by ~ 40%. BD1063 has a higher affinity for -1 than -2 receptors and attenuates the dystonia produced by DTG in rats in a dosedependent manner, suggesting this ligand acts as an antagonist at sites (Matsumoto et al., 1995). CBP is unable to block acid-induced increases in [Ca2+]i when co-applied with BD1 063, suggesting the effe cts are mediated by -1 receptors. In addition, metaphit fails to inhibit the effects of the -2 agonist, PB28, on ASIC1a-mediated responses. Ta ken together, these data show that

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163 increases in [Ca2+]i in response to ASIC1a activation are modulated only by -1 receptors. Inhibition of the second messenger calcineurin has been shown to potentiate currents through ASIC 1a and ASIC2a channels, and thus dephosphorylation of the channels by this phosphatase may be involved in downregulation of ASIC (Chai et al., 2007). The calcineuri n inhibitors, cyclosporin A and FK-506, dem onstrate that -1 receptor-mediated block of ASIC1a is dependent on activation of this phosphatase (Figure 5.1). Intere stingly, reports have shown that calcineurin may be acti vated by a pertussis toxin-sensitive G protein (Gromada et al., 2001). Consistent with this observation, the effects of receptor activation on ASIC1a-mediated [Ca2+]i increases are significantly lessened in cortical neurons following pr eincubation in pertussis toxin, suggesting a pertussis toxin-sensitive G protein is involved in the signaling cascade coupling -1 receptors to ASIC1a-mediated responses. This study demonstrates that -1 receptors modulate ASIC1a channel function via activation of a PTX-sensitive G protein, and these results are the first evidence of a receptor coupling to ASIC 1a through this mechanism (Figure 5.1). receptors regulate ion channel function vi a protein-protein in teractions (Aydar et al., 2002). Moreover, inhibi tion of G proteins either by cell dialysis with GDPS or preincubation in pertussi s toxin failed to affect -1 and -2 receptor modulation of voltage-gated K+ and Ca2+ channels in intracardiac neurons (Zhang and Cuevas, 2002; Zhang and Cuevas 2005). To date, nearly all reports of receptor modulation of ion channels suggest that the effects involve a

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164 membrane-delimited signaling pathway which could invo lve a protein-protein interaction. However, receptor activation has been shown to stimulate GTPase activity in mouse prefrontal membr anes (Tokuyama et al., 1997). A pertussis toxin-sensitive G protein has been im plicated in the modulation of NMDA receptors by receptors in rat CA3 dorsal hippocampus neurons (Monnet et al., 1994), whereas a cholera toxin-sensitive G protein has been suggested to couple -1 receptors to the channels mediati ng the A-current in frog pituitary melanotopes (Soriani et al., 1999) However, others have argued that receptors do not couple to G protei ns (Hopf et al., 1996).

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165 Figure 5.1 Signaling cascade linking ASIC1a channels and sigm a-1 receptors. Activation of -1 receptors inhibits ASIC1a-induced Ca2+ dysregulation via a PTX-sensitive G protein and a calcineurin/ AKAP complex, resulting in a decrease in [Ca2+]i elevations. Sigma-1 receptors dire ctly couple to ASIC1a channels via a PTX-sensitive G protein and a calcineurin/AKAP complex.

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166

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167 AKAP150 is involved in the regulation of receptor activity and localization, and in the regulation of synaptic stru cture during developmental synapse formation, in synaptic plasticity in learning and memory, and neuronal dysfunction and cell death dur ing pathophysiological conditi ons (Dell'Acqua et al., 2006). -1 receptors colocalize with both ASIC1a channels and AKAP150 in the plasma membrane of the cell body and along the neuronal processes of the cells. Consistent with this finding, a calci neurin/AKAP150 complex has been shown to modulate both ASIC1a and ASIC2a function (Chai et al., 2007). Furthermore, the similarity of the distributions of receptors colocalized with ASIC1a channels and with AKAP150 is consistent with previous studies showing colocalization of ASIC channels and AKAP (Chai et al., 2007). Disr uption of the actin cytoskeleton by chemical agents or NMDA receptor activa tion, results in the redistribution of AKAP150 away from the plasma membr ane (Gomez et al ., 2002), and thus, preventing -1 receptor modulation of ASIC1a-induced [Ca2+]i dysregulation (Figure 5.1). Depletion of Ca2+ from intracellular stores indica tes that most, if not all, of the acid-induced increases in [Ca2+]i is due to plasma memb rane influx. Multiple ion channels downstream of ASIC1a activa tion were shown to contribute to acidosis-induced elevations in [Ca2+]i, including NMDA and AMPA/kainate receptors and VGCC. receptors have been identifi ed in both presynaptic and postsynaptic sites (Gonzalez-Alvear and Wer ling, 1995; Alonso et al., 2000), and thus may modulate channels in both regions. In t he presence of specific inhibitors of ionotropic glutam ate receptors, activation of -1 receptors with CBP

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168 further decreased proton-evoked increases in [Ca2+]i, but the effects of CBP and the glutamate channel inhibitors were less than additive. Thus, -1 receptors also inhibit Ca2+ entry via NMDA and AMPA/kainate re ceptors directly by inhibiting these channels and indirectly by depressing ASIC1a activation. Application of the L-type VGCC inhibitor, nifedi pine, and the broad-spectrum Ca2+ channel inhibitor, cadmium, blocked ASIC1a-induced increases in [Ca2+]i by >70% and >90%, respectively. This observation indicates that most of the increases in [Ca2+]i produced upon ASIC1a activa tion is dependent on Ca2+ influx through VGCC. Co-application of CBP with nifedipine, but not with Cd2+, resulted in further reduction in the proton-evoked increases in [Ca2+]i. The activation of NMDA and AMPA/k ainate receptors following ASIC1a stimulation was observed even when neur onal conduction was inhibited with tetrodotoxin. This observation suggests a presynaptic localization of ASIC1a, whereby activation of the channel by prot ons results in synaptic transmission and subsequent activation of postsynaptic glut amatergic receptors. Consistent with this hypothesis, ASIC1a has been found to regulate neurotransmitter release probability in mouse hippocampal neurons (Cho and Askwith, 2008). AKAP150 has been shown to modulate the internalization of AMPA receptors, NMDA receptors duri ng long term potentiation and depression (Rosenmund et al., 1994; Westphal et al., 1999; Colledge et al., 2000; Gomez et al., 2002; Smith et al., 2006) and voltage-gated Ca2+ channels function (Oliveria et al., 2007). Since most of the elevations in [Ca2+]i triggered by acidosis are the result of Ca2+ channels opening downstream of ASIC1a activation, and

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169 stimulation of -1 receptors effectively suppresses these secondary Ca2+ fluxes, these results also raise the possibility that AKAP150 is a constituent in the modulation of these downstream ion channels (e.g. voltage-gated Ca2+ channels) and receptors (e.g. NMDA and AMPA) by -1 receptors (Figure 5.1). Simultaneous Ca2+ fluorometry and whole-ce ll patch clamp recordings confirmed that Ca2+ influx through ASIC1a channels itself contributed only a small fraction to the total observed [Ca2+]i increases. Cells voltage-clamped at -70 mV, which prevents NMDA recept or and VGCC activation, demonstrated minimal acid-evoked elevations in [Ca2+]i. Thus, the increases in [Ca2+]i evoked by ASIC1a activation are the result of synaptic transmission and subsequent opening of multiple Ca2+ channels, and that stimulation of -1 receptors downregulates all of these events. Howe ver, the fact that activation of -1 receptors depresses ASIC1a-medi ated currents in cells voltageclamped at -70 mV indicates that -1 receptors are functionally coupled to ASIC1a, and that the depression in acid-evoked increases in [Ca2+]i is not exclusively the result of -1 receptors blocking channels downstream of ASIC1a. The effects of receptor activation on ASIC1a-mediated membrane currents are significantly lessened in co rtical neurons following preincubation in pertussis toxin. These results suggest a pertussis toxin-sensitive G protein is involved in the signal ing cascade coupling receptors to ASIC1a channels (Figure 5.1). Moreover, -1 receptors couple to ASIC1a channels via a calcineurin/AKAP150 complex resulting in a decrease in acid-induced membrane currents (Figure 5.1). Whole-cell patch clamp experiments with VIVIT suggest

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170 that when this peptide com petes with calcineurin for binding to the PxIxIT-like motif in AKAP150, and thus prevents calcineurin binding to AKAP150, receptor activation loses its ability to regulate ASIC1 a function. Thus, the disruption of this interaction prevents -1 receptors from functionally coupling to ASIC1a channels. Similarly, VIVIT has been implicated in the disruption of calcineurin/AKAP150 modulation of L-type Ca2+ channels in hippocampal neurons (Oliveria et al., 2007). Pertinent to ischemia, ASIC1a is bei ng studied as a putative target for neuroprotection due to the observation that ASIC1a is involved in neuronal death following ischemic brain injury (Xiong et al., 2004; Yermolaieva et al., 2004). These studies also implicated calcium influx through these channels as a key mechanism leading to neurodegeneration (Waldmann et al., 1997b; Chu et al., 2002; Xiong et al., 2004; Yermolaieva et al., 2004). Transgenic mice deficient in ASIC1a have reduced infarct size in response to middle cerebral artery occlusion, relative to wild-type mice (X iong et al., 2004; Xiong et al., 2006). The increases in [Ca2+]i that are evoked by acidosis are also absent in these transgenic animals, giving insight into how these channels may contribute to neuronal injury (Xiong et al., 2004; Xiong et al., 2006). Furthermore, it has been shown that either blocking ASIC1a by amiloride or gene knockout of ASIC1a provides additional neur oprotection during oxygen glucose deprivation even in the presence of the NMDA receptor antagonist, memantine (Xiong et al., 2004; Xiong et al., 2006). It has also been est ablished that PcTx1 inhibits ASIC1a via chronic desensitization of the channel (Chen et al., 2005) and also diminishes the

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171 effects of NMDA-induced cell death (Xi ong et al., 2004; Gao et al., 2005). Ischemia has also been shown to enhance ASIC1a currents through phosphorylation at Ser478 or Ser479 by calcium/calmodulin protein kinase II (CaMKII), and specific blockade of CaMKII prevented potentiation of the ischemia-induced ASIC1a currents, cy toplasmic calcium dysregulation, and neuronal death (Gao et al., 2005). Neurons within the penumbra region re main viable for hours after the ischemic insult but are s ubject to apoptosis if perfusion is not re-established. Previous studies have shown that ischem ia and the acidosis that accompanies ischemia, produces marked increases in [Ca2+]i, which is one of the main mechanisms leading to cell death (Tombaugh and Sapolsky, 1993; Murai et al., 1997; Mattson, 2000; Xiong et al., 2004; Yermolaieva et al., 2004). ASIC1amediated currents in neurons have al so been shown to be potentiated by oxygen-glucose deprivation (Xiong et al., 2004; Gao et al., 2005). Though the effects of ASIC1a currents during ischemia have been established, this study is the first report showing how ASIC1a activa tion affects the responses to ischemia and how acidosis and ischemia interact with each other to produce a synergistic dysregulation in [Ca2+]i. Our in vitro model of ischem ia using the combination of azide in glucose-free PSS and acidosis (p H 6.0), resembles in vivo models of stroke since similar pH values have been observed during ischemia in whole animal studies (Nedergaard et al., 1991) Together, ischemia and acidosis produced elevations in [Ca2+]i that were greater than the sum of the changes evoked by acidosis and ischemia alone. There was also a noticeable difference

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172 in the kinetics of the observed [Ca2+]i transients resulting from acidosis, ischemia, and ischemia + acidosis. These differ ences cannot be explained by ischemia potentiating ASIC currents because ASIC1a channels desensitize within seconds. Thus, we conclude the effects se en minutes after the initial insult (steady state, rebound and net Ca2+ elevations) following ischemia + acidosis are the result of ASIC1a activation potentiating ischemia-mediated [Ca2+]i increases. Therefore, ischemia and ASIC1a activation interact to produce potentiated elevations in [Ca2+]i dysregulation. The fact that ASIC1a inhibitors am iloride and PcTx1 reduce the ischemiainduced increases in [Ca2+]i when the external solution was buffered to pH 7.4 suggests that the acidosis produced by meta bolic inhibition is not responsible for ASIC1a activation. Instead, it is reasoned that protons released from glutamate containing synaptic vesicles during neurot ransmission (DeVries, 2001; Traynelis and Chesler, 2001) are the source of ASIC1a channel activation. Therefore, there are two sources of protons associated with isch emia. First, an internal source as glucose and oxygen deprivat ion initiates a metabolic switch to anaerobic glycolysis to produce cellular ene rgy leading to the accumulation of lactic acid, the end product of anaerobic glycolysis. Second, protons released along with glutamate during synaptic transmi ssion. In our model, azide produces increased action potential firing and thus acidification of the synaptic cleft and the accumulation of lactic acid is mimicked by buffering external solutions to pH 6.0. Increased neurotransmission would activate more ASIC1a channels, inducing a feed-forward mechanism which promot es further synaptic transmission and

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173 greater Ca2+ accumulation. These increases in [Ca2+]i, if of sufficient duration, would produce Ca2+ overload and eventually lead to cell death. Consistent with these conclusions, the interaction between ischemia and acidosis leads to the synergistic potentiation of Ca2+ dysregulaton, which is what is expected to be seen in vivo following an ischemic stro ke (Tombaugh and Sapolsky, 1993; Murai et al., 1997; Mattson, 2000; Xiong et al., 2004; Yermolaieva et al., 2004). Several mechanisms are responsible for the different phases of Ca2+ responses to the combination of isc hemia and acidosis observed in these neurons. The strongest evidence that the initial peaks associated with ischemiainduced [Ca2+]i elevations reported here are due to activation of homomeric ASIC1a channels is the inhibition produced by both amiloride and the selective ASIC1a channel blocker PcTx1 (Diochot et al., 2007). The steady state [Ca2+]i is a function of the activated Ca2+ influx and efflux pathw ays. Interestingly, the steady state level of [Ca2+]i obtained at the end of ischemia applications increased as the pH was reduced from 7.4 to 7.0, but then remained constant as the pH was reduced to 6.0. Previous studi es suggest most of the elevations in [Ca2+]i triggered by acidosis are the result of Ca2+ channels (NMDA and AMPA receptors and voltage-gated Ca2+ channels) opening in response to ASIC1a mediated membrane depolarizati on. These results suggest that acidosis results in increased ASIC1a channel opening but t he effects of pH are countered by increased H+ block of other ion channels. The transient rebound increase in [Ca2+]i observed during washout of the acidosis + ischemia solution is most lik ely due to the removal of a block of Ca2+

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174 influx pathways by protons as the proton concentration is reduced to pH 7.4. Since this feature of the [Ca2+]i response is not observed following applications of azide in pH 7.4 PSS, it is unlikely that when observed at lower pH’s it is due to the washout of azide. Consistent with obs ervations in this study, the transient rebound is expected to be more robust at th e lower pH values due to the relief of a greater proton bl ock of these Ca2+ channels. Conditions which pr oduce large net [Ca2+]i elevations, or Ca2+ overload, are likely to lead to apoptosis. Acidosis alone, while producing a large [Ca2+]i increase, has a very small net Ca2+ elevation because the transient increase is short lived. Likewise, while ischemia produces a maintained elevated [Ca2+]i level in the presence of ischemia, it is onl y a moderately high level which returns quickly to baseline upon washout, and thus re sults in a small net elevation. The synergistic interaction of acidosis and ischemia, on the other hand, produces potentiated increases at all times duri ng the agonist-evoked application as well as an extenuated rebound increase and a sl ower decay back to baseline. All these contribute to produce net elevations in [Ca2+]i which are ~400% times greater than that of acidosis or ischemia alone. This pronounced Ca2+ overload would be more likely to trigger apoptosis, probably too due to endoplasmic recticulum stress. Blockade of voltage-gated Na+ channels by TTX and subsequent synaptic transmission inhibition, prevents isc hemia-induced elevations in [Ca2+]i at physiological pH values (Katnik et al., 2006). Interesti ngly, during the combination of ischemia and acidosis at pH 6.0, the inhibition of [Ca2+]i increases

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175 by TTX were overcome, most likely by the additional activation of ASIC1a channels. This observation suggests a presynaptic localiz ation of ASIC1a, whereby activation of the channel by prot ons results in synaptic transmission and subsequent activation of postsynaptic recept ors. Consistent wit h this theory, the activation of postsynaptic Ca2+ channels and receptors following ASIC1a stimulation during acidosis alone (p H 6.0) are observ ed even when neuronal conduction is inhibited by TTX. It has been proposed that ASIC1a activation may facilitate neurotransmission by compensat ing for the decrease in excitatory neurotransmission caused by direct inhibition of postsynaptic Na+ and Ca2+ channels by protons which are released dur ing exocytosis (Krishtal et al., 1987; Zha et al., 2006). Thus, action potential inhibi tion alone is not sufficient to prevent Ca2+ overload, as ASIC1a channel activation is able to overcome this block of glutamate release by depol arizing the presynaptic membrane to potentials capable of activating voltage-gated Ca2+ channels and thus, evoking synaptic transmission. Thus, even in the absence of glutamate release, ASIC1a would depolarize the cell and subsequent ly, activate voltage-gated Ca2+ channels to promote neurotransmission. In addition, thes e results also raise the possibility of ASIC1a activation resulting in enough Ca2+ influx to promote synaptic transmission and glutamate release. Similarl y, heteromeric complexes of alpha 5 and/or alpha 7 subunits of nicotinic re ceptors have been shown to conduct Ca2+ and thus, resulting in acetylcholine-induc ed presynaptic facilit ation (Girod et al., 1999).

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176 The -1 selective ligand, CBP, has previously been shown to inhibit ischemia-evoked increases in [Ca2+]i (Katnik et al., 2006) as well as ASIC1ainduced [Ca2+]i increases and membrane currents. Unlike the results seen with inhibition of ASIC1a with PcTx1 venom, CBP activation of -1 receptors is pH insensitive and significantly inhibit ed the synergistic increases in [Ca2+]i produced by ischemia and acidosis as measured by initial peak amplit ude, steady state, rebound and net elevations of [Ca2+]i. Overall Significance The finding that -1 receptors can inhibit AS IC1a channels has significant physiological implications (Figure 5. 2). It has been proposed that ASIC1a activation may facilitate neurotransmission by compensating for the decrease in excitatory neurotransmission caused by di rect inhibition of post-synaptic Na+ and Ca2+ channels by protons which are released during exocytosis (Krishtal et al., 1987; Zha et al., 2006) (Figure 5.2). Fu rthermore, the expression levels of ASIC1a have direct effects on the densit y of dendritic spines in hippocampal neurons (Zha et al., 2006). Thus, -1 receptors may influence cell-to-cell signaling in the CNS by a ffecting ASIC1a activity. One of the consequences of ASIC1a overexpression in mice is enhanc ed fear conditioning (Wemmie et al., 2004), whereas stimulation of -1 receptors is known to ameliorate conditioned fear stress (Kamei et al ., 1997). These observations, coupled with our current report, suggest that -1 receptor activation may pr oduce anxiolytic effects via the inhibition of ASIC1a channels.

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177 Figure 5.2 Presynaptic ASIC1a channe ls under physiological conditions. Neurotransmitter release leads to acidif ication of the synapt ic cleft, which will lead to decrease neuroexcitability due to the inhibitory effects of protons in the postsynaptic channels. ASIC1a provi de a positive feedback by increasing intracellular calcium following activation leading to increased neuroexcitability, and thus, preserving synaptic transmission.

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178

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179 The finding that -1 receptors can inhibit ASIC1a channels also has significant pathophysiolog ical implications -1 receptors functionally couple to ASIC1a channels via a PTX-sensitive G protein and an AKAP150/calcineurin complex in cortical neurons (Figure 5.1) This coupling results in decreases in both ASIC1a-mediated memb rane currents and concomitant elevations in cytosolic Ca2+ following -1 receptor activation. Furt hermore, these results also raise the possibility of -1 receptors coupling to downstream Ca2+ channels (voltage-gated Ca2+ channels) and receptors (N MDA and AMPA) via a similar signaling cascade. All of t hese channels and receptor s have been implicated in pathophysiological conditions. Moreov er, calcineurin may be a potential component of the neuroprot ective properties of receptors. -1 receptors are the first receptor shown to downregulate ASIC1a channel function and remain as the only receptor identified thus far to negatively modulate ASIC1a channels. Further evidence of the significance of -1 receptor modulation of ASIC1a channels during pathophysiological condi tions was the finding that ASIC1a activation at any point during an ischemic insult results in synergistic [Ca2+]i increases which are receptor sensitive (Figure 5.3). First, ASIC1a channels are activated by ischemia alone (pH 7.4), wh ich rapidly desensitize, but are able to be re-activated within less then 1 minute by application of an acidic external solution. This suggests that during pathophysi ological conditions it is possible to get repeated activation of ASIC1a channels if the extracellular media is buffered back to normal pH for even a short refr actory period. Consistent with this conclusion is the fact that lactate remains elevated and has been shown to

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180 Figure 5.3 Role of presynaptic ASIC 1a channels during pathological conditions. The combination of ischemia and acidosis interact to produce greater glutamate release leading to a synergistic potentiation of postsynaptic [Ca2+]i elevations, resulting in Ca2+ overload, excitotoxicity and ev entually cell death. Activation of -1 receptors prevents the synergist ic interaction between ischemia and acidosis, and thus, Ca2+ overload.

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181

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182 rebound days after the initial ischemic ev ent (Weinstein et al., 2004; Munoz Maniega et al., 2008). Even more dramatic though, was the observation that under ischemic conditions these repetit ive activations of ASIC1a channels synergistically interact with ischemia to produce potentiated elevations in peak, steady state, rebound and net Ca2+ elevations in [Ca2+]i. These results suggest that in order to prevent the consequences of Ca2+ overload during ischemia it is critical to control ASIC1a activation. E qually important is the finding that all phases of these cellular responses are modulated by receptor activation. Consistent with these results, it has been suggested that the acidosis that occurs in vivo during ischemia exacerbates ischemic injury at later ti me points after the initial ischemic insult (Figure 5.3). The finding that ASIC1a activation interacts with ischemia to produce sy nergistic potentiation in Ca2+ dysregulation is a novel finding and suggests that this channel and receptors should be targeted even at delayed time points after stroke onset to prevent Ca2+ overload and the resulting neuronal apoptosis. The inhibition of ASIC1a by -1 receptors is a potentially important component of the neuroprot ective properties of receptors, since activation of ASIC1a has been shown to contribute to stroke injury (Xiong et al., 2004). Moreover, -1 receptors inhibit [Ca2+]i increases following ASIC1a channel activation, ischemia-induced increases in [Ca2+]i as well as the synergistic potentiation of these increases observ ed during ischemia + acidosis insults (Figure 5.3). Importantly, inhibition of ASIC1a has been shown to be neuroprotective at delayed time points fo llowing ischemic stroke (Simon, 2006;

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183 Pignataro et al., 2007). Because acidic conditions exist during ischemic episodes, -1 receptors should be targeted fo r development of treatments for stroke and other pathophysiological conditi ons where ASIC1a channel activation are involved. -1 receptor mediated inhibi tion of ASIC1a channels and Ca2+ channels (NMDA and AMPA/kainate re ceptors and VGCC) activated by membrane depolarizations pr oduced by ASIC1a channel activation or metabolic inhibition and the ASIC1a channels themselves, may contribute to the enhanced neuronal survival observed fo llowing administration of receptor agonists 24 hr post-stroke in rats (Ajmo et al., 2006). All of these ion channels have been linked to ischemia-induced brain injury. Consist ent with this pleiotropic effect of receptor activation on neurons, is our observation that -1 receptor activation suppresses extracellular high K+-induced increases in [Ca2+]i, which would also activate these downstream effectors. For these reasons, -1 receptors can be targeted for therapeutic intervention in pathophysiological conditions involving ASIC1a activation (like ischemia) and expan d the therapeutic window of stroke treatment.

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204 APPENDICES

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205 Appendix A – Pharmaco logical Compounds Drug Name Definition and Effective/ Inhibitory Concentrations Mechanism of Action Reference Amiloride Nonspecific ASIC blocker (16 M) Binds to residues within the channel’s pore region (Kleyman et al., 1999; Alvarez de la Rosa et al., 2000; Xiong et al., 2004) Psalmotoxin1 venom or synthetic peptide (PcTx1) Homomeric ASIC1a channel selective blocker (500 ng/ml, 50 nM) Increases the affinity of the channel by H+ resulting in chromic desensitization of the channel (Escoubas et al., 2000; Chen et al., 2005; Diochot et al., 2007) 1,3-di-o-tolylguanidine (DTG) Pan-selective sigma receptor agonist (100 M, 65 M) Binds both sigma receptor subtypes (Klette et al., 1995; Soriani et al., 1998; Kume et al., 2002; Zhang and Cuevas, 2002; Zhang and Cuevas, 2005; Katnik et al., 2006) Carbetapentane citrate (CBP) Sigma-1 selective agonist (14 M, 38 M) Binds sigma-1 receptors with >50-fold greater affinity than for sigma-2 receptors (Rothman et al., 1991; Thurgur and Church, 1998; Vilner and Bowen, 2000; Katnik et al., 2006)

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206 Appendix A: (Continued) Opipramol Pan-selective sigma receptor agonist (10 M) Binds both sigma receptor subtypes (Muller and Siebert, 1998; Volz et al., 2000; Moller et al., 2001; Holoubek and Muller, 2003; Muller et al., 2004; Volz and Stoll, 2004) Dextromethorphan hydrobromide (DEX) Sigma-1 selective ligand (30 M) Binds sigma-1 receptors with >100-fold greater affinity than for sigma-2 receptors (Anderson and Andrew, 2002) 2-(4Morpholinethyl) 1phenylcyclohexane carboxylate hydrochloride (Pre084) Sigma-1 selective ligand (10 M) Binds sigma-1 receptors with >100-fold greater affinity than for sigma-2 receptors (Bucolo et al., 2006) Ibogaine Sigma-2 selective agonist (31 M) Binds sigma-2 receptors with >40-fold greater affinity than for sigma-1 receptors (Vilner and Bowen, 2000; Zhang and Cuevas, 2002) 1-Cyclohexyl-4-(3(5-methoxy1,2,3,4tetrahydronaphthal en-1-yl)-npropyl)piperazine dihydrochloride (PB28) Sigma-2 selective agonist (2 M) Potent sigma-2 receptors agonist (Berardi et al., 2004; Cassano et al., 2006) Metaphit (MET) Irreversible panselective sigma antagonist (50 M) Inhibits both sigma receptor subtypes (Zhang and Cuevas, 2002; Wu, 2003; Zhang and Cuevas, 2005; Katnik et al., 2006)

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207 Appendix A: (Continued) 1-[2-(3,4dichlorophenyl)eth yl]-4methylpiperazine dihydrochloride (BD1063) Sigma-1 selective receptor antagonist (10 nM, 1 M) Binds sigma-1 receptors with greater affinity than for sigma-2 receptors (Matsumoto et al., 1995; Hamabe et al., 2000; Nguyen et al., 2005) Thapsigargin (THAP) Depletes the ryanodine and IP3 sensitive stores (10 M, 20 M) Blocks the sarcoplasmic/ endoplasmic reticulum Ca2+ATPase (DeHaven and Cuevas, 2004; Katnik et al., 2006) Tetrodotoxin (TTX) Inhibits voltagegated Na+ channels (200 nM, 400 nM) Blocks action potentials by binding to the pores of the voltage-gated Na+ channels (DeHaven and Cuevas, 2004; Katnik et al., 2006) D-2-Amino-5phosphonovaleric acid (AP5) Selective NMDA receptors antagonist (100 M) Competitively inhibits NMDA receptors active site (Morris, 1989; Steele and Morris, 1999; MacGregor et al., 2003; Katnik et al., 2006) 1,4-Dihydro-2,6dimethyl-4-(2nitrophenyl)-3,5pyridinedicarboxyli c acid dimethyl ester (Nifedipine) Dihydropyridine calcium channel blocker (5 M, 10 M) L-type voltagegated Ca2+ channel blocker (Kim et al., 2008; Tu et al., 2009) Cadmium Broad spectrum Ca2+ channel blocker (100 M, 1 mM) Inhibits Ca 2 + channels (Ryan et al., 2007; Staruschenko et al., 2007) 6-Cyano-7nitroquinoxaline2,3-dione (CNQX) AMPA/kainate receptor antagonist (10 M) Competitively blocks AMPA and kainate receptors (MacGregor et al., 2003; Marino et al., 2007)

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208 Appendix A: (Continued) FK-506 Calcineurin inhibitor (1 M, 10 M) Forms immunophilin complex with FKBP to bind calcineurin and competitively inhibit its activity by blocking the catalytic groove (Dumont, 2000; MartinezMartinez and Redondo, 2004; Chai et al., 2007) CyclosporinA Calcineurin inhibitor (1 M 30 M) Forms immunophilin complex with cyclophilinA to bind calcineurin and competitively inhibit its activity by blocking the catalytic groove (MartinezMartinez and Redondo, 2004; Chai et al., 2007) Pertussis toxin (PTX) Inhibits G protein activation (100 ng/ml, 200 ng/ml) Catalyzes the ADP-ribosylation of the subunits of Gi/Go, preventing the G protein heterotrimers from interacting with receptors (Locht and Antoine, 1995; Jajoo et al., 2008) N -methyl-Daspartate (NMDA) NMDA receptor agonist (10 M) Selectively activates NMDA receptors and not other glutamate receptors (Gomez et al., 2002; Watkins and Jane, 2006) Latrunculin A Disrupts the actin cytoskeleton (5 M) Disrupts the actin cytoskeleton, resulting in AKAP150 dissociation from plasma membrane (Allison et al., 1998; Sattler et al., 2000; Zhou et al., 2001; Popp and Dertien, 2008)

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209 Appendix A: (Continued) VIVIT Inhibits calcineurin AKAP150 interaction (1 M, 10 M) Inhibits calcineurin/AKAP 150 interaction by disrupting calcineurin binding to a PxIxIT-docking motif in AKAP150 (Oliveria et al., 2007; Pereverzev et al., 2008)

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ABOUT THE AUTHOR Yelenis Herrera was born in Havana, Cuba. She is the daughter of Silvia and Roberto Herrera. She came to the Un ited States in 1994 at the age of 12 and started to pursue her sci entific career. In 2000, s he graduated as one of the top students in her class from Suncoast Community High School in West Palm Beach, FL. She continued her educati on and in 2004, she graduated Cum Laude earning Bachelors degrees in Molecula r Biology and Chemistry from Florida Atlantic University in Boca Raton, FL In 2005, she started her graduate studies in the laboratory of Dr. Javier Cuevas earning a Masters degree in 2007 and a PhD degree in the Spring semester of 2009 in Medical Science from the Department of Molecular P harmacology and Physiology.