University of South Florida Scholar Commons
Graduate Theses and Dissertations Graduate School
2-27-2009
Modulation of ASIC1a Function by Sigma-1 Receptors: Physiological and Pathophysiological Implications
Yelenis Herrera University of South Florida
Follow this and additional works at: https://scholarcommons.usf.edu/etd
Part of the American Studies Commons
Scholar Commons Citation Herrera, Yelenis, "Modulation of ASIC1a Function by Sigma-1 Receptors: Physiological and Pathophysiological Implications" (2009). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/2011
This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected].
Modulation of ASIC1a Function by Sigma-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 Pharmacology 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, whole-cell currents, AKAP150, calcineurin, G protein, neurotransmission, glutamate, VGCC, NMDA receptor
© Copyright 2009, Yelenis Herrera
DEDICATION
Those who have inspired me throughout my life and scientific career are
those that I love the most. This dissertation is dedicated to them - my husband
and family. I would like to thank my Mom and Dad for giving me the opportunity
to be free, succeed and pursue my education. To my brother, you are the best
and I am very proud of you; thank you for always being there and for your support. I would like to give a special thanks 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 possible. 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.
ACKNOWLEDGMENTS
First of all, I would to thank my mentor, Dr. Javier Cuevas, for giving me the opportunity to grow as a scientist, for his support, inspiration and encouragement throughout the years. I would also like to thank my committee members, Drs. Keith R. Pennypacker, Eric S. Bennett, 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 constructive comments on the dissertation.
I would like to thank all the members of Dr. Cuevas’ laboratory, Dr.
Christopher Katnik, Trimetria Bonds, Crystal 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 Nicholson, of the department of Molecular
Pharmacology and Physiology for all their assistance. Thank you all very much.
This work was funded in part by a McKnight Doctoral Fellowship from the
Florida Education Fund.
TABLE OF CONTENTS
LIST OF FIGURES ...... iii ABSTRACT ...... vii CHAPTER 1 INTRODUCTION ...... 1 Significance ...... 1 Background ...... 1 Pathobiology of Stroke ...... 4 Acid-Sensing Ion Channels (ASIC) ...... 8 ASIC1a Channels, Calcineurin and Scaffolding Proteins ...... 13 Sigma Receptors ...... 16 In Vitro Chemical Ischemia Models ...... 22 CHAPTER 2 SIGMA-1 RECEPTOR MODULATION OF ASIC1a CHANNELS AND ASIC1a-INDUCED Ca2+ INFLUX IN RAT CORTICAL NEURONS...... 25 Introduction ...... 25 Materials and Methods ...... 28 Primary Rat Cortical Neuron Preparation ...... 28 Calcium Imaging Measurements ...... 29 Electrophysiology Recordings ...... 30 Solutions and Reagents ...... 31 Data Analysis ...... 31 Results ...... 32 Discussion ...... 69 CHAPTER 3 SIGMA-1 RECEPTOR ACTIVATION INHIBITS ASIC1a CHANNELS VIA A PERTUSSIS TOXIN SENSITIVE G PROTEIN AND AN AKAP/CALCINEURIN COMPLEX ...... 75 Introduction ...... 75 Materials and Methods ...... 78 Primary Rat Cortical Neuron Preparation ...... 78 Calcium Imaging Measurements ...... 78 Electrophysiology Recordings ...... 79 Immunohistochemistry...... 80 Confocal Microscopy ...... 81 Solutions and Reagents ...... 82 Data Analysis ...... 82
i Results ...... 83 Discussion ...... 107 CHAPTER 4 SIGMA-1 RECEPTOR INHIBITION OF INTRACELLULAR Ca2+ DYSREGULATION DUE TO SYNERGISTIC INTERACTION BETWEEN ACIDOSIS AND ISCHEMIA ...... 113 Introduction ...... 113 Materials and Methods ...... 117 Primary Rat Cortical Neuron Preparation ...... 117 Calcium Imaging Measurements ...... 117 Electrophysiology Recordings ...... 118 Solutions and Reagents ...... 118 Data Analysis ...... 119 Results ...... 120 Discussion ...... 150 CHAPTER 5 DISCUSSION ...... 159 Conclusions ...... 159 Overall Significance ...... 176 LIST OF REFERENCES ...... 184 APPENDICES ...... 204 Appendix A – Pharmacological Compounds ...... 205 ABOUT THE AUTHOR ...... End Page
ii
LIST OF FIGURES
Figure 1.1 Pathobiology of ischemic stroke.………………………………………7
2+ Figure 2.1 ASIC1a blockers inhibit proton-evoked increases in [Ca ]i in cultured cortical neurons from embryonic (E18) rats………………46
Figure 2.2 Pan-selective sigma agonists inhibit proton-evoked transient 2+ increases in [Ca ]i…………………………………………………….48
Figure 2.3 Activation of σ-1 receptors inhibits ASIC1a-induced increases in 2+ [Ca ]i……………………………………...…………………………....50
Figure 2.4 Inhibition of σ-1 receptors blocks CBP-mediated suppression of 2+ proton-evoked increases in [Ca ]i……………………………….…..52
Figure 2.5 Sigma-2 receptor ligands inhibit ASIC1a-mediated elevations in 2+ [Ca ]i at concentrations inconsistent with σ-2 mediated effects and in a metaphit-insensitive manner……………………………….54
Figure 2.6 Intracellular Ca2+ stores are not involved in DTG modulation of 2+ ASIC1a-induced increases in [Ca ]i………………………………...56
2+ Figure 2.7 ASIC1a-mediated [Ca ]i increases are membrane potential dependent………………………………………………………………58
Figure 2.8 ASIC1a activation promotes membrane depolarization...…………60
iii Figure 2.9 Sigma-1 receptor activation inhibits Ca2+ channels downstream of ASIC1a……………………………………………………………….62
Figure 2.10 Multiple plasma membrane ion channels downstream of ASIC1a 2+ activation contribute to acidosis-evoked [Ca ]i increases…….…..64
Figure 2.11 ASIC1a blockers inhibit acidosis-mediated currents in voltage- clamped neurons……………………………………………………....66
Figure 2.12 Sigma receptor agonists inhibit ASIC1a-mediated currents in voltage-clamped neurons……………………………………………..68
Figure 3.1 Sigma-1 receptors colocalize with both ASIC1a channels and AKAP150 in cortical neurons…………………………………………91
Figure 3.2 Calcineurin inhibition prevents σ-1 receptor modulation of ASIC1a………………………………………………………………….94
2+ Figure 3.3 Sigma-1 receptors inhibit acid-induced elevations in [Ca ]i via a PTX-sensitive G protein……………………………………………….96
Figure 3.4 AKAP150 dissociation from the plasma membrane prevents 2+ σ-1 receptor modulation of acid-induced elevations in [Ca ]i…….98
Figure 3.5 Disruption of the actin cytoskeleton prevents σ-1 receptor 2+ inhibition of acid-induced increases in [Ca ]i……………………..100
Figure 3.6 Latrunculin A preincubation disrupts the actin cytoskeleton…….102
iv Figure 3.7 Sigma receptor-mediated inhibition of ASIC1a currents are blocked by preincubation in pertussis toxin……………………….104
Figure 3.8 Sigma-1 receptors couple to ASIC1a channels via an AKAP150/calcineurin complex……………………………………...106
2+ Figure 4.1 ASIC activation contributes to ischemia-evoked [Ca ]i increases in cultured rat cortical neurons………………………….130
Figure 4.2 ASIC activation and ischemia interact to produce a synergistic 2+ potentiation of elevations in [Ca ]i…………………………………132
2+ Figure 4.3 Protons potentiate ischemia-induced increases in [Ca ]i……….134
Figure 4.4 Inhibition of homomeric ASIC1a channels decreases ischemia 2+ + acidosis-induced elevations in [Ca ]i at pH values ranging from 7.4 to 6.0………………………………………...………………136
Figure 4.5 Heat inactivated PcTx1 venom does not inhibit ischemia- 2+ induced increases in [Ca ]i………………………………………....138
2+ Figure 4.6 PcTx1 peptide inhibits ischemia-induced elevations in [Ca ]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 ranging from 7.4 to 6.0………………145
Figure 4.9 Effects of synaptic transmission inhibition are overcome by presynaptic ASIC1a channels ……………………………………...147
v Figure 4.10 TTX inhibits Na+ currents at pH 7.4 and 6.0..……………………..149
Figure 5.1 Signaling cascade linking 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 during pathological conditions…..……………………………………...... 181
vi
Modulation of ASIC1a Function by Sigma-1 Receptors: Physiological and Pathophysiological Implications
Yelenis Herrera
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
2+ 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 (σ) receptors regulate multiple ion channels that become activated during ischemia. Second,
ASIC1a remain functionally active hours beyond the ischemic insult and σ receptors have been shown to be neuroprotective at delayed time points following stroke.
Ratiometric Ca2+ fluorometry and whole-cell patch clamp experiments
2+ showed that σ-1 receptor activation depresses elevations in [Ca ]i and
vii membrane currents mediated by ASIC1a channels in cortical neurons.
2+ Furthermore, most of the elevations in [Ca ]i triggered by acidosis are the result
of Ca2+ channels opening downstream of ASIC1a activation. Stimulation of σ-1 receptors effectively suppressed these secondary Ca2+ fluxes both by inhibiting
ASIC1a and the other channels directly.
The signaling cascade linking σ-1 receptors and ASIC1a was determined
to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein
2+ 150/calcineurin complex, which resulted in a decrease of acid-induced [Ca ]i elevations and ASIC1a-mediated currents. Furthermore, immunohistochemical studies confirmed that σ-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
2+ synergistic potentiation of [Ca ]i dysregulation. Although ASIC1a activation does
not induce long-lived priming of synaptic vesicles for release, channel activation
2+ 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
2+ enhance postsynaptic [Ca ]i elevations. σ -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.
viii
CHAPTER 1
INTRODUCTION
Significance
ASIC are proton activated ion channels expressed in the brain and are involved in several physiological functions 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 would result in neuroprotection. σ receptors are known to regulate multiple ion channels in neurons, all of which are activated during ischemia. Taken together, these observations demonstrate the importance of studying the regulation of ASIC function by σ receptors under physiological as well as during ischemic conditions and to determine the signaling cascade involved. Results from this study will identify potential therapeutic 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
1 the most common type of stroke, accounting for almost 88% of all strokes, and is caused by a clot or other blockage within a cerebral artery. Following a stroke, there are two major regions of injury within the ischemic cerebrovascular bed: the core ischemic region and the ischemic penumbra region (Dirnagl et al., 1999).
Within minutes of the ischemic insult, the core area forms. The core region is an area characterized by reduced blood flow, leading to a loss of adequate supply of oxygen and glucose resulting in rapid depletion of cellular energy stores. Ischemia results in neuronal death and supporting cellular elements such as glial cells by necrosis (characterized by cell swelling, cell rupture and activation of inflammatory response) within 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 the penumbra, a rim of compromised ischemic tissue lying between 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 demand for oxygen and glucose indefinitely.
As time goes by, hours and days after the insult, apoptosis in the penumbra
results in cell death and expansion of the core (Dirnagl et al., 1999; Lo et al.,
2005). Most of research taking place today focuses on the prevention of the
apoptotic death that occurs within the penumbra region. This may be achieved by
2 restoring blood flow to the penumbral zone and/or by disrupting several pathways that are activated resulting in neuronal death.
The thrombolytic agent, tissue plasminogen activator (tPA), is the only drug approved for clinical treatment of acute stroke. tPA cleaves the precursor molecule plasminogen producing the active 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 intracranial hemorrhage associated with its use, and the short therapeutic window in which it must be administered (≤ 3 hours)
(Dirnagl et al., 1999). Additionally, tPA can not be administered 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 acute stroke therapy has increased the demand for the development of alternative 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, there 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 receptors and voltage-gated Ca2+ channels) have failed in clinical trials.
3 Pathobiology of Stroke
To better understand neurodegeneration during 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 necrotic neuronal death in the
affected infarct area (core region) and delayed apoptotic neuronal death in the
penumbra. Brain ischemia is associated 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 acidotoxicity eventually results in cell death
(Xiong et al., 2004; Yermolaieva et al., 2004). Dysregulation 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 depolarization of neurons promotes Ca2+ entry, which in
turn evokes the release of neurotransmitters, such as glutamate (Figure 1.1).
Glutamate, acting via postsynaptic receptors, 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 leads to over-stimulation of N-methyl-D-
aspartate (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 accumulation of extracellular K+ allows
surrounding neurons to depolarize, whereas the buildup of intracellular Na+
4 results in cell swelling, or oedema (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, lipolysis, and the production of reactive oxygen
species (Mattson, 2000) (Figure 1.1). Ca2+ overload and energy depletion leads
to activation of secondary events including 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, cell death and expansion of the core region is also enhanced by inflammation caused by the immune response.
Activated microglia and reactive astrocytes are capable of entering the central nervous system since the blood brain barrier is degraded (Stoll et al., 1998).
These cells then exacerbate damage by activating proinflammatory mediators, releasing nitric oxide and glutamate (Heales et al., 1999; Hertz et al., 2001; Bal-
Price et al., 2002; Trendelenburg and Dirnagl, 2005). All these events are also detrimental to the neurons and result in subsequent expansion of the core.
5 Figure 1.1 - Pathobiology of ischemic stroke. Intracellular calcium plays a critical role during ischemia. Ischemia leads to acidosis and acidotoxicity in the ischemic
2+ region, which results in further increases in [Ca ]i. Calcium dysregulation ultimately leads to the activation of all these events that result in cell death and subsequent expansion of the core region.
6
7 Acid-Sensing Ion Channels (ASIC)
Recently, the acid-sensing ion channel has been shown to be a major
contributor to ischemic injury in the central nervous system. Physiological extracellular and intracellular pH is maintained at ~ 7.3 and 7.0 through various
proton transporting mechanisms. Under pathological conditions, like stroke, the
metabolic switch to anaerobic glycolysis produces lactic acid leading to a drop in
pH and acidotoxicity in the ischemic region. 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 brain acidosis (Xiong et al., 2004;
Pignataro et al., 2007). Thus, activation of ASIC has now been unequivocally
linked to brain injury resulting from ischemic stroke. It has been suggested that
low tissue pH may lead to protein and nucleic acid denaturation, trigger cell
swelling via activation of proton exchangers, lead to excitotoxicity by inhibiting
astrocyte glutamate reuptake, 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 extracellular low pH. Therefore, during ischemia,
8 other calcium influx pathways may exist independently of NMDA receptor
function.
ASIC are a class of ligand-gated ion channels 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 result of low extracellular pH caused
by ischemia (Xiong et al., 2004; Yermolaieva et al., 2004). Four genes (ASIC1 –
ASIC4) encoding for six ASIC subunits have been cloned, and four of those
subunits can form functional homomultimeric 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). The 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; Alvarez de la Rosa et al., 2003). ASIC1a is
a nonselective cation channel that is sodium and calcium permeable and
generates fast activating and inactivating 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 inactivating currents compared to the
9 ASIC1a subtype. ASIC3 has a pH0.5 = 6.5, and it is predominantly expressed in
neurons of the dorsal root ganglia (Waldmann 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 functional homomeric channels, but ASIC2b
can associate with other subunits and modulate their activity, and thus
biophysical properties of the channels. For example, studies of heteromeric
ASIC1a/2a channels have shown that ASIC1a establishes the current amplitude,
while ASIC2a influences desensitization, recovery from desensitization, and pH
sensitivity of the channel (Askwith et al., 2004).
ASIC contain two transmembrane domains flanked by a large cysteine-
rich extracellular loop and short intracellular N- and C- termini (Waldmann et al.,
1997b; Alvarez de la Rosa et al., 2000; Saugstad et al., 2004). In the peripheral nervous system, ASIC localize to neurons innervating skin, heart, gut, and muscle, and have also been detected in the eye, ear, taste buds, and bone (see review (Wemmie et al., 2006)). These channels are known to be involved in various processes such as mechanoreception (Price et al., 2000), taste transduction (Ugawa et al., 2003), maintenance of retinal integrity (Ettaiche et al.,
2004), and nociception (Allen and Attwell, 2002), particularly in the ischemic myocardium where ASIC are believed to transduce anginal pain (Benson et al.,
1999).
ASIC1a have a wide spread distribution in the brain including hippocampus, cerebral cortex, cerebellum, striatum, habenula, amygdale, and
10 olfactory (Wemmie et al., 2002; Alvarez de la Rosa et al., 2003). The ASIC1a
subtype have been localized to dendrites and dendritic spines, axons, and
throughout neurons with no preferential distribution to synapses (Wemmie et al.,
2003). In the central nervous system, ASIC1a is involved in synaptic plasticity,
fear conditioning, and learning and memory (Wemmie et al., 2002; Wemmie et
al., 2003). It has been shown that loss of ASIC1a disrupted hippocampal- dependent long term potentiation and spatial memory, impaired cerebellum-
dependent learning, and reduced fear response in the amygdala (freezing behavior) (Wemmie et al., 2003). Conversely, overexpression of ASIC1a in the amygdala and different regions of the brain leads to increased ASIC1a currents and enhanced context of fear conditioning (anxiety) (Wemmie et al., 2004).
ASIC1 and ASIC1 gene knockout studies have shown that this ion channel is calcium permeable and is involved in acidosis-mediated ischemic brain injury and neuronal death (Xiong et al., 2004; Yermolaieva et al., 2004; Pignataro et al.,
2007).
ASIC also belong to the amiloride-sensitive superfamily and studies have shown that inhibition of ASIC by this nonspecific blocker leads to diminished
2+ [Ca ]i elevations during acidosis. It has been shown that amiloride dose-
dependently blocked ASIC currents in mouse cortical neurons with an IC50 of
16.4 µM (Xiong et al., 2004). It has also been demonstrated that amiloride and its
derivatives benzamil and ethylisopropylamiloride (EIPA) reversibly blocked
proton-activated inward ASIC currents in sensory neurons (Kd = 10 µM)
(Waldmann et al., 1997b). Mutation studies have suggested that residues within
11 the pore and extracellular domain are critical for amiloride binding (Kleyman et
al., 1999). This observation suggests that the sites that interact with amiloride
within the channel’s extracellular domain may be in close proximity to residues
within the channel’s pore region. Supporting the notion that amiloride blocks the
outer pore of ASIC and Deg/ENac include 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 extracellular sodium concentrations, indicating
a competitive mechanism for the pore binding between amiloride and its
permeable cation (Alvarez de la Rosa et al., 2000).
Studies with the venom of the South American tarantula Psalmopoeus
cambridgei (psalmotoxin 1, PcTx1) have provided great insight about the role of
ASIC1a during ischemia. PcTx1 is the first 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 current 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, the 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 occlusion (MCAO) produced a significant
reduction in infarct size and volume (Xiong et al., 2004). It has been suggested
that the mechanism by which PcTx1 inhibits ASIC1a channels is via chronic
12 desensitization of the channel caused by increased affinity of the channel for
protons (Chen et al., 2005). Interestingly, this PcTx1-induced shift of the pH-
dependent inactivation of ASIC1a is Ca2+-dependent, where increasing
extracellular Ca2+ results in a decrease of the PcTx1 inhibition.
Acidosis occurs within minutes of stroke onset (mainly affecting the core
region but also the inner penumbral zone) (Back et al., 2000) and ASIC1a remain
functionally active beyond 4 hrs following the ischemic insult (Pignataro et al.,
2007). Additionally, pharmacological blockade of ASIC1a by amiloride or PcTx1
and administration of PcTx1 even 5 hrs after middle cerebral artery occlusion
(MCAO) diminishes stroke injury (Xiong et al., 2004; Pignataro et al., 2007).
These observations are consistent with studies showing the presence of lactate
72 hours following transient MCAO in rats (Weinstein et al., 2004). It remains to
be elucidated whether endogenous receptors 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 that 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 proteases (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
13 C-terminal domain of ASIC1a for calcium/calmodulin protein kinase II (CaMKII)
(Gao et al., 2005), protein kinase C (Baron et al., 2002) and protein kinase A
(PKA) (Leonard et al., 2003) to bind. However, phosphorylation of ASIC1a has
been shown to potentiate ASIC1a function in neurons (Xiong et al., 2004; Gao et al., 2005).
Calcineurin-dependent dephosphorylation of ASIC1a channels is the only
modulator of ASIC1a that results in downregulation of channel function (Chai et
al., 2007). Calcineurin, a second messenger, is a serine/threonine phosphatase
that is activated by calcium-calmodulin, 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 consisting of a catalytic domain, calmodulin- binding domain, and an autoinhibitory domain. The regulatory subunit, calcineurin B subunit, encodes the calcium-binding domain. The immunosuppressive drugs, cyclosporine A and FK-506, are well known calcineurin inhibitors. Cyclosporine A-cyclophilin A and FK-506-FKBP form drug- immunophilin complexes that bind to the calcineurin active site heterodimer to inhibit calcineurin activity competitively 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 protein family with more than 50 members which are abundantly expressed in the brain (Feliciello et al., 2001). Neuronal
14 AKAP150 (rat) and AKAP79 (human) share a high degree of sequence homology, differing primarily 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 phosphotases (calcineurin) that are inhibited when bound. AKAP150 targets these proteins to specific subcellular 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 (phosphotidylinositol-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 structure during synapse formation during development, synaptic plasticity in learning and memory, and neuronal dysfunction and cell death under pathophysiological conditions (Dell'Acqua et al.,
2006). In addition to ASIC1a regulation, AKAP150 modulates internalization of
AMPA and NMDA receptors during 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). All these proteins are possible constituents of the signaling cascade involving ASIC1a channels and σ receptors.
15 Sigma Receptors
Sigma receptors have been shown to regulate multiple ion channel function in neurons. This observation raises the possibility that σ receptors could modulate ASIC1a function. σ 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 σ receptors prefer to bind the (+) enantiomer. Moreover, in
vitro and in vivo studies suggested that the opioid antagonists, naloxone or
naltrexone, do not affect σ receptor-mediated events (Iwamoto, 1981; Brady et
al., 1982; Slifer and Balster, 1983; Vaupel, 1983; Katz et al., 1985). Thus, σ
receptors were then classified as their own unique receptor type.
Two σ receptor subtypes have been identified on the basis of
pharmacology. σ receptors have been shown to bind a range of substances including opiates, antipsychotics, antidepressants, phencyclidine (PCP)-related compounds, and neurosteroids (Walker et al., 1990; Bowen, 2000). σ-1 receptors have a higher affinity for the positive isomer of benzomorphans such as (+)- pentazocine and (+)-SKF-10,047, and the σ-2 receptors have high affinity for ibogaine and its congeners (Bowen, 2000; Vilner and Bowen, 2000). σ ligands like 1,3-di-o-tolyguanidine (DTG) and haloperidol have been shown to be pan- selective and bind to both σ receptor subtypes with high affinity. Evidence suggest that progesterone, neuropeptide Y, peptide YY, and zinc are possible endogenous σ ligands (Su et al., 1988; Roman et al., 1989; Connor and Chavkin,
1992).
16 To date, only the σ-1 receptor has been cloned (Hanner et al., 1996). The
structure of the σ-1 receptor subtype is thought to consist of 2 transmembrane-
spanning domains with internal C- and N-terminals (Aydar et al., 2002). While σ-
1 receptors do not posesses sequence homology to other mammalian proteins, it does have similar homology to fungal sterol isomerases (Hanner et al., 1996;
Kekuda et al., 1996; Seth et al., 1998). The cDNA of σ-1 receptors encodes a
protein of 223 amino acids with a molecular weight of 25 kDa. In humans, the σ-1
receptor gene is located on chromosome 9p13, a region commonly associated
with psychiatric disorders (Prasad et al., 1998). σ -1 receptors are found in the
plasma membrane as well as in intracellular membranes like the endoplasmic
reticulum (Hanner et al., 1996; Kekuda et al., 1996; Aydar et al., 2002).
In contrast to σ-1 receptors, the molecular identity of σ-2 receptors
remains to be determined. Reports suggest that a splice variant of σ-1 receptors
displays σ-2 receptor binding activities (Monassier and Bousquet, 2002).
However, studies with σ-1 receptor knockout mouse showed that σ-1 and σ-2
receptors were not isoforms of the same protein because σ-2 effects were still
seen in the absence of the σ-1 receptor subtype (Langa et al., 2003). Similar to
σ-1 receptors, pharmacological experiments suggest that σ-2 receptors are
located in the intracellular membrane of the endoplasmic reticulum and the
mitochondria (Bowen, 2000).
σ receptors are ubiquitously expressed in the immune system, the kidney
and liver, as well as throughout the central nervous system (Wolfe and De
Souza, 1993; Hellewell et al., 1994). Microglia activation is one of the main
17 contributors of the immune response 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 since these cells contribute to the demise
of compromised neurons, and thus, exacerbate 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, and release cytokine (Hall et al., 2008).
Furthermore, in vivo studies also suggest that sigma receptor activation
decreases reactive gliosis following stroke (Ajmo et al., 2006).
In the central nervous system, σ receptors are found in brainstem areas
that regulate motor function, limbic structures, some predominantly sensory
areas, and areas associated with endocrine function (della Puppa and London,
1989; Matsumoto et al., 1989; Matsumoto et al., 1990b; Walker et al., 1990;
Elsinga et al., 2004). σ receptors have been implicated in numerous physiological
and pathophysiological processes such as learning and memory (Senda et al.,
1996; Maurice and Privat, 1997), movement disorders (Matsumoto 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 (Casellas et al., 2004; Hayashi and Su,
2004).
18 σ -2 receptors have been shown to regulate sodium and calcium
channels. σ receptor activation blocks several types of calcium channels
including N-, T-, P/Q- and 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 hippocampal neurons have demonstrated that
σ-2 ligands inhibit voltage-gated calcium channels while σ-1 selective agonists
failed to show the same effect, indicating the inhibition is mediated via σ-2
receptors (Fletcher et al., 1995).
In contrast, σ-1 receptors regulate inositol 1,4,5-triphosphate (IP3) receptors and calcium signaling at the endoplasmic reticulum, mobilization of cytoskeletal adaptor proteins, modulation of nerve growth factor-induced neurite sprouting, modulation of neurotransmitter release and neuronal firing, and modulation of potassium channels as regulatory subunits (see review, (Su and
Hayashi, 2003)). σ -1 agonists are known to cause amplification of signal transduction incurred upon activation of the glutamatergic, dopaminergic, IP3-
related metabotropic, or nerve growth factor-related systems (Su and Hayashi,
2003). Immunohistochemistry studies have shown that σ-1 receptor, IP3 type-3
receptor, and ankyrin B are colocalized in NG-108 cells in perinuclear areas and regions of cell-to-cell communication, suggesting that σ-1 ligands may play a role in cells by controlling the function of cytoskeleton proteins and regulating calcium signaling may represent a site of action for neurosteroids and cocaine (Hayashi and Su, 2001).
19 σ -1 receptors have been implicated in the regulation of various ion
channels. σ -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 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 σ receptors has
been shown to be dependent on protein-protein interaction (Aydar et al., 2002).
The σ 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 calcium-
dependent K+ current (Kennedy and Henderson, 1990).
Activation of sigma receptors regulate multiple ion channels subtypes in
neurons (Aydar et al., 2002; Zhang and Cuevas, 2002; Zhang and Cuevas,
2005). This regulation of ion channels may result in neuroprotection during
ischemia. It has been suggested that the neuroprotective properties of σ ligands
2+ depend in part on their ability to depress elevations in [Ca ]i associated with
glutamate receptor-mediated excitotoxicity (Klette et al., 1995; Klette et al., 1997;
Katnik et al., 2006), neurotransmitter release regulation (Matsuno et al., 1993;
Couture and Debonnel, 1998), and modulation of multiple ion channel subtypes.
The studies of σ receptor modulation of glutamate evoked changes in
20 intracellular calcium have resulted in considerable controversy in the literature.
There are conflicting reports as to whether σ receptor ligands exert their effects
as a result of σ receptor activation (Hayashi et al., 1995) or through non-specific interaction with other targets, in particular, NMDA receptors (Kume et al., 2002).
σ 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 σ 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
2+ [Ca ]i (Katnik et al., 2006). σ -1 receptor activation has also been shown to
prevent neuronal death associated with glutamate 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 pro-
inflammatory factors (TNFα) and activate the expression of anti-inflammatory
agents like IL-10 (Bourrie et al., 1995; Bourrie et al., 1996; Bourrie et al., 2002).
Stimulation of σ 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 glutamate receptors (Monnet et al., 2003). All of these channel types are involved in the dysregulation of intracellular calcium homeostasis accompanying ischemia, and blocking the function of these
channels provides neuroprotection (Schurr, 2004). Thus, σ receptor ligands have
been targeted for the treatment of stroke due to the neuroprotective properties
21 observed in both in vivo and in vitro models 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 σ 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 source of energy (Vannucci et al., 1994).
This is energetically unfavorable and results in rapid depletion of ATP along with subsequent activation of deliterious biochemical cascades. All of these events are similar to those observed in cultured neurons (in vitro): (1) Ca2+ dysregulation
due to glutamate accumulation and activation 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 dysfunction leading to activation of
apoptotic pathways (Northington et al., 2001), and (5) activation of secondary
mechanisms including release of proinflammatory 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 ischemia in vivo. These models include: (1) oxygen
glucose deprivation (Monyer et al., 1989; Laake et al., 1999) and the addition of
22 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. These ischemic models are associated with
Ca2+ overload triggered by the excess influx of extracellular Ca2+ through Ca2+-
sensitive ion channels and receptors (Monyer et al., 1989; Goldberg and Choi,
1993; Katnik et al., 2006). All of these models affect the electron transport chain
in the mitochondria, which is the site of oxidative phosphorylation in eukaryotes.
Oxidative phosphorylation is the main pathway that uses energy released by the oxidation of nutrients to drive the production of adenosine triphosphate (ATP), which supplies energy for metabolism. Inhibition 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 cyanide 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 across 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 transport chain and both prevent the aerobic production of ATP as the source of cellular energy. However, the main disadvantage of cyanide is that it is a very toxic and lethal compound to humans since cyanide
23 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) uncouples the electron transport from oxidative phosphorylation, which disrupts 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 depletion, (2) difficult to completely remove oxygen and thus requires long incubation to produce significant injury (Rothman, 1984), and (3) requires using a closed-chamber, making patch-clamp experiments difficult. The major advantage of the sodium azide/glucose deprivation model over the oxygen/glucose deprivation model is that it elicits neurochemical responses that are significantly more rapid and robust, thus facilitating the recording of changes
2+ in [Ca ]i increases (Katnik et al., 2006). Disadvantages of this model are that azide has multiple affects including inhibition of cytochrome aa3, superoxide
dismutase and DNA synthesis (Varming et al., 1996). Azide breaks down to nitric
oxide and thus enhances excitatory neurotransmission (Varming et al., 1996).
Moreover, because of the short duration of the application of azide, this only
models acute ischemia. This azide/glucose deprivation model has previously
been used in rat cortical neurons to study σ receptor inhibition of ischemia-
2+ induced [Ca ]i elevations (Katnik et al., 2006). Moreover, calcium influx through
the plasma membrane and not release from the mitochondria or endoplasmic
2+ reticulum, accounts for most of the ischemia-mediated [Ca ]i increases in cortical
neurons using this azide model of ischemia (Katnik et al., 2006).
24
CHAPTER 2
SIGMA-1 RECEPTOR MODULATION OF ASIC1a CHANNELS AND ASIC1a- INDUCED Ca2+ INFLUX IN RAT CORTICAL NEURONS
Introduction
Acid-sensing ion channels are a class of ligand-gated channels that are
members of the degenerin/epithelial sodium channel superfamily and are
expressed in both peripheral and central nervous system 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 functional 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 (Askwith et al., 2004). These channels
are activated by pH ≤ 7 and have a pH of half-maximal 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; Yermolaieva et al., 2004). This
25 ASIC subtype has been implicated in a number 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 occlusion (MCAO) relative to wild-type mice
(Xiong et al., 2004). Moreover, pharmacological inhibition of ASIC1a with either
amiloride or psalmotoxin1, which is selective for homomultimeric ASIC1a
channels (Diochot et al., 2007), diminishes ischemic brain injury (Xiong et al.,
2004). Several studies have suggested that Ca2+ influx through these channels is
a key mechanism leading to neurodegeneration (Xiong et al., 2004; Yermolaieva et al., 2004).
Despite efforts to determine the function of ASIC1a and the role of these channels in ischemia, little is known about endogenous mechanisms which control ASIC1a activity. Thus far, only 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).
σ receptor activation has been shown to modulate multiple cell membrane
ion channels in neurons. σ -1 receptors regulate ionotropic glutamate receptors and voltage-gated K+ channels, whereas σ-2 receptors modulate voltage-gated
26 Ca2+ channels (Hayashi et al., 1995; Aydar et al., 2002; Zhang and Cuevas,
2002; Zhang and Cuevas, 2005). The inhibition of ionotropic glutamatergic
2+ receptors by σ receptors prevents elevations in [Ca ]i associated with glutamate-
induced excitotoxicity (Klette et al., 1995). All of these voltage-gated ion channels and NMDA receptors have been shown to contribute to the demise of neurons during an ischemic insult. Our laboratory has recently shown that σ-1 receptors
inhibit Ca2+ dysregulation evoked by ischemia and that activation of σ receptors
is neuroprotective at delayed time points in a rat model of ischemic stroke (Ajmo
et al., 2006; Katnik et al., 2006). Interstitial pH in the brain remains low several
hours after an ischemic event (Nedergaard 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 σ receptors may regulate ASIC1a function
and ASIC1a-induced intracellular calcium transients, and provide neuroprotection
when stimulated at delayed time points after an ischemic insult.
Experiments were conducted to determine the effects of σ receptors on
2+ ASIC-mediated membrane currents and transient [Ca ]i elevations. It was
2+ determined that σ receptor agonists inhibit acidosis-induced increases in [Ca ]i
and peak membrane currents in cells expressing homomeric ASIC1a channels.
Pharmacological studies demonstrated that the σ-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 σ-1 receptors also
27 diminished Ca2+ entry via these channels. Therefore, σ-1 receptors couple to
ASIC1a channels to inhibit channel function and also block ASIC1a-induced
2+ [Ca ]i dysregulation. Our findings suggest that σ-1 receptors represent potential
targets for improving outcome of stroke injury and expanding the therapeutic
window for ischemic stroke treatment.
Materials and Methods
Primary Rat Cortical Neuron Preparation
Primary cortical neurons from embryonic (E18) rats were cultured as
previously described by our laboratory (Katnik et al., 2006). All procedures were done in accordance with the regulations of the University of South Florida
Institutional Animal Care and Use Committee. 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 with NaOH. Cortices were excised and
minced, followed by digestion in isotonic buffer containing 0.25% trypsin/EDTA
for 10 minutes at 37°C and added to 3X volume of Dulbecco’s modified Eagle’s
Medium (DMEM) (Invitrogen Inc, Carlsbad, CA) supplemented with 10% (v/v)
fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were
counted using a hemocytometer, plated (0.5 X 106 cells/well) on 18-mm pre-
treated poly-L-lysine coverslips, and incubated at 37°C 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)
28 to minimize astrocyte proliferation 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 with B27 (Invitrogen) and 0.5 mM L-
glutamine, 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), 330 ± 10 mOsm. Both solutions contained 3 μM acetoxymethyl ester fura-2 and 0.3 % dimethyl sulfoxide. The coverslips were washed in PSS (fura-2 free) prior to the experiments being carried 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 Instruments Co., Novato,
CA) was used to apply alternating excitation light. Fluorescent emission was captured using a Sensicam digital CCD 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
2+ changes in [Ca ]i from the intensity of the emitted fluorescence following excitation with 340 nm and 380 nm light, respectively, using the Grynkiewicz
29 2+ equation: [Ca ]i = Kd *Q(R-Rmin)/(Rmax-R), where R represents the fluorescence intensity ratio (F340/F380) as determined during experiments, Q is
2+ the ratio of Fmin to Fmax at 380 nm, and Kd is the Ca dissociation constant for
fura-2 (224 μM). The system was calibrated using a Fura-2 Calcium Imaging
Calibration Kit (Molecular Probes; Eugene, OR) and values for Fmin/Fmax,
Rmin, and Rmax were determined.
Electrophysiology Recordings
Neurons plated on glass coverslips (as described above) were transferred
to a recording chamber mounted on a Zeiss Axiovert 200 and visualized at 400x.
Membrane currents were amplified using an Axopatch 200B (Axon Instruments),
filtered at 1 kHz, digitized at 5 kHz with a Digidata 1322A (Axon), and acquired
using Clampex 8 (Axon). Electrical access was achieved using the amphotericin
B perforated-patch method to preserve intracellular integrity of neurons (Rae et
al., 1991). An amphotericin B stock solution (60 mg/ml in DMSO) was made
daily, kept on ice, light protected, and diluted to 240 µg/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 glass (World Precision Instruments Inc.,
Sarasota, FL) using a Sutter Instruments P-87 pipette puller (Novato, CA) and had resistances of 1.0–1.5 MΩ. Access resistance (Rs) were monitored
throughout experiments for stable values ≤ 20 MΩ and were always
compensated at 40% (lag, 10 µs). All cells were voltage-clamped at -70 mV.
30 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 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 (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 grade. The following drugs were used: DTG,
opipramol, ibogaine, metaphit, nifedipine, 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 Scientific, Fair Lawn, NJ); and fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR).
Data Analysis
Analyses of measured intracellular Ca2+ and membrane current responses
were conducted using Clampfit 9 (Axon instruments). Fluorescence intensities were recorded from fura-2-loaded neuronal cell bodies. Time-lapse imaging data files collected with SlideBook 4.02 (Intelligent Imaging Innovations, Inc.) were converted to a text format and imported into Clampfit for subsequent analysis. Statistical
31 analysis was conducted using SigmaPlot 9 and SigmaStat 3 software (Systat
Software, Inc.). Statistical differences were determined using paired and unpaired
t-tests for within group and between group experiments, respectively, and were
considered significant if p < 0.05. For multiple group comparisons either a 1-way or
a 2-way ANOVA, with or without repeat measures, were used, as appropriate.
When significant differences were determined with an ANOVA, post-hoc analysis
was conducted using a Tukey Test to determine differences 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
2+ ASIC subtype(s) affecting [Ca ]i in our rat cortical neuron model. Figure 2.1A
2+ shows representative traces of [Ca ]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 µM), and following a 10 minute washout of drug (Wash). The general ASIC inhibitor, amiloride, reversibly blocked ASIC-mediated increases in
2+ [Ca ]i by 88 ± 1 % (Figure 2.1B). Psalmotoxin1 (PcTx1) from the venom of the
tarantula Psalmopoeus cambridgei has been shown to be a selective blocker of
homomultimeric ASIC1a channels (Diochot et al., 2007). Figure 2.1C shows
32 2+ representative traces of [Ca ]i as a function of time recorded from a neuron prior
to (Control), following a 10 - 20 min preincubation 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
2+ ASIC1a-mediated elevations in [Ca ]i (57 ± 2 %) (Figure 2.1D). The effects
produced by this concentration of PcTx1 are 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
2+ elevations in [Ca ]i that are mediated via the activation of homomultimeric
ASIC1a channels, as reported for cultured cortical neurons from embryonic mice
(Xiong et al., 2004).
Activation of σ receptors has been shown to inhibit numerous plasma membrane ion channels in neurons (Hayashi et al., 1995; Zhang and Cuevas,
2002; Zhang and Cuevas, 2005). Therefore, experiments were carried out to
study the effects of σ receptor activation on ASIC1a function using the pan-
selective σ receptor agonist, DTG. Figure 2.2A shows representative traces of
2+ [Ca ]i recorded from a single neuron during acidosis in the absence (Control) and presence of 100 μM DTG, a concentration previously 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
2+ reversibly inhibited the low pH-induced transient increases in [Ca ]i. In identical
experiments, 100 μM DTG produced a statistically significant decrease (46 ± 3
2+ %) in ASIC1a-mediated elevations in [Ca ]i (Figure 2.2B). Opipramol (10 µM),
33 another pan-selective σ-1/σ-2 agonist, also inhibited ASIC1a-mediated increases
2+ in [Ca ]i by 57 ± 1 % (Figure 2.2B). The concentration-response relationship for
DTG inhibition of ASIC1a was determined to confirm that the actions of DTG on
ASIC1a are consistent with σ receptor activation. Increasing DTG concentrations
2+ resulted in further depression of ASIC1a-mediated increases in [Ca ]i (Figure
2.2C). A plot of the concentration-response relationship obtained from
measurements made in multiple 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 µM
and a Hill coefficient of 0.9. These values are consistent with the effects of DTG
being mediated via activation of σ receptors (Zhang and Cuevas, 2002; Zhang
and Cuevas, 2005; Katnik et al., 2006). Taken together these results suggest that
2+ σ receptors depress acid-induced increases in [Ca ]i and modulate ASIC1a
function.
σ receptor subtype-selective agonists were used to identify the specific σ
2+ receptor subtype(s) mediating these effects. Representative traces of [Ca ]i as a
function of time recorded from two cells during acidosis in the absence (Control)
and presence of the σ-1 selective agonists carbetapentane (CBP) and
dextromethorphan (DEX) at the indicated concentrations are shown in Figures
2.3A and 2.3B, respectively. σ-1 selective agonists blocked the low pH-induced
2+ elevations in [Ca ]i in a concentration dependent and reversible manner.
2+ Concentration-response plots for mean changes in peak [Ca ]i recorded in
identical experiments using the σ-1 selective ligands CBP, DEX, and PRE-084,
are shown in Figure 2.3C. The data were best fit using the Langmuir-Hill equation
34 and values obtained for IC50 and Hill coefficients were 13.8 μM and 0.7 (CBP), 22
µM and 0.8 (DEX), and 13.7 μM and 0.6 (PRE-084), respectively. Presented for comparison is the best fit to the data obtained for CBP inhibition, via σ-1
2+ receptors, of chemical ischemia-induced increases in [Ca ]i in cortical neurons
(dotted line, IC50 =18.7 µM; Hill coefficient, 0.8) (Katnik et al., 2006). This curve
superimposes on the responses to CBP observed in the current study.
To provide further evidence that σ-1 receptors modulate ASIC1a-induced
Ca2+ elevations experiments were conducted using the irreversible σ antagonist,
metaphit, and a selective σ-1 antagonist, BD1063, in combination with the σ-1
agonist, CBP. Cells were exposed to acidosis in the absence and presence of
CBP (30 μM), with or without preincubation in metaphit (50 μM; 30 min -1hr,
2+ 23˚C). CBP decreased the acid-induced elevations in [Ca ]i in control cells, and
this inhibition was lessened by preincubation with metaphit (Figure 2.4A). Figure
2.4B shows results from several similar experiments determining percent
2+ inhibition of acid-induced increases in [Ca ]i observed in the presence of CBP in
control neurons (Control) and neurons preincubated with metaphit (MET).
2+ Whereas CBP decreased the elevations in [Ca ]i evoked by acidosis in control
cells by 52 ± 2 %, the σ-1 receptor agonist only reduced the response by
30 ± 3 % in cells preincubated in the irreversible σ receptor antagonist. This
>40% decrease in the effects of CBP following metaphit preincubation was
statistically significant (p < 0.001). The selective σ-1 antagonist, BD1063, showed
2+ more pronounced effects. Figure 2.4C shows representative traces of [Ca ]i as a function of time recorded from two neurons during acidosis in absence (Control)
35 and presence of CBP (30 µM), without (PSS) and with co-application in BD1063
(BD1063, 10 nM). In identical experiments, the σ agonist CBP completely loses
2+ its ability to block the low pH-induced elevations in [Ca ]i in the presence of the σ antagonist BD1063 when compared to 40% block in control cells (Figure 2.4D).
Inhibition of σ-1 receptors by BD1063 significantly blocked the effects of CBP on
2+ ASIC1a-mediated [Ca ]i elevations when compared to control cells (p <0.001).
These data confirm the effects of σ-1 agonists on ASIC1a mediated increases in
2+ [Ca ]i are the result of these compounds acting on σ-1 receptors.
Further experiments were carried out using the σ-2 selective ligands
ibogaine and PB28 to determine if the σ-2 receptor subtype also affects ASIC1a
2+ function. Figure 2.5A shows representative traces of [Ca ]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 σ-2 ligands inhibited acidosis-evoked increases in mean
2+ peak changes in [Ca ]i in a concentration dependent manner (Figure 2.5B). Best
fits to the data demonstrated that application of ibogaine and PB28 resulted in
2+ inhibition of ASIC1a-mediated increases in [Ca ]i with IC50 values of 69 µM and
11 μM, and Hill coefficients of 0.86 and 0.85, respectively. For comparison, the
best fit to the data obtained for ibogaine inhibition of ICa-induced increases in
2+ [Ca ]i is presented, which has been shown to be mediated by σ-2 receptors
(dashed line; IC50 = 31 µM; Hill coefficient, 1.1) (Zhang and Cuevas, 2002).
Unlike the similar concentration-response relationship observed for CBP
inhibition of ASIC1a and ischemia responses, there is a discrepancy between the
36 ibogaine block of responses mediated by ASIC1a and by voltage-gated Ca2+ channels (VGCC). To determine if the effects of these σ-2 ligands were mediated by activation of σ-2 receptors, experiments using metaphit were carried out.
2+ PB28 (20 μM) produced an inhibition of ASIC1a-mediated increases in [Ca ]i in
both control (PSS) and metaphit (+MET) treated cells (Figure 2.5C). PB28
2+ blocked ASIC1a-mediated increases in [Ca ]i by 67 ± 1% and 60 ± 2 % in the
absence and presence of metaphit preincubation, respectively (Figure 2.5D). The
~10% reduction in the effects of PB28 produced by metaphit was not statistically
significant (p = 0.64). Furthermore, BD1063 (10 nM) failed to block the effects of
2+ PB28 (20 µM) on ASIC1a-induced elevations in [Ca ]i (Figure 2.5D). These
results demonstrate that the effects of PB28 on ASIC1a-medited increases in
2+ [Ca ]i are not mediated by σ-2 receptors since the inhibition of these responses
to acidosis by PB-28 is metaphit-insensitive and occurs at concentrations
inconsistent with σ-2 receptor activation.
The activation of σ receptors has previously been shown to directly affect
Ca2+ release from intracellular stores (Cassano et al., 2006). Thus, experiments were conducted to resolve if σ receptor activation reduces acid-induced
2+ increases in [Ca ]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 ryanodine- and IP3-sensitive stores. Figure 2.6A shows representative
2+ traces of [Ca ]i as a function of time recorded from a neuron during acidosis in
37 the absence (Control) and presence of 100 µM DTG (DTG), while Figure 2.6B shows traces in the absence and presence of DTG following pre-incubation (1 hr,
23˚C) in 10 μM thapsigargin (THAP and THAP + DTG, respectively).
2+ Thapsigargin alone did not decrease the elevations in [Ca ]i produced by
2+ acidosis, and DTG depressed the increases in [Ca ]i under both conditions (± thapsigargin preincubation). Analysis of the data collected in identical experiments indicates that preincubation with thapsigargin does not significantly
2+ alter the effects of DTG on acid-mediated increases in [Ca ]i (Figure 2.6C).
2+ Thus, DTG does not decrease the low pH-induced elevations in [Ca ]i by affecting release of calcium from intracellular stores. The fact that depletion of
2+ intracellular stores fails to depress increases in [Ca ]i evoked by acidosis suggests that Ca2+ influx through the plasma membrane accounts for most, if not
2+ all, of the increases in [Ca ]i evoked by ASIC1a activation.
Simultaneous Ca2+ fluorometry and whole-cell patch clamp recordings were performed to study ASIC1a-mediated 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
2+ 2+ Ca transient measured in a patched cell ([Ca ]i V-Clamp). In contrast, a
2+ second cell ([Ca ]i Control) in the same field of view, which was not voltage
2+ clamped, had a significantly larger increase in [Ca ]i. Acidosis also resulted in a large inward current in the patched cell (Figure 2.7A, inset). The cumulative
38 results from several similar experiments demonstrate that ASIC1a activation of
2+ neurons voltage clamped at -70 mV resulted in elevations in [Ca ]i which were
an order of magnitude smaller 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 itself and that the majority of the
2+ 2+ acid-induced [Ca ]i increases are mediated by downstream Ca influx
pathways.
To test the possibility that ASIC1a channels promote cell depolarization,
cortical neurons were held under current-clamp mode. Figures 2.8A and 2.8C
show representative traces of membrane potential as a function of time recorded
from a single cell in absence (Control) and presence of amiloride (100 µM) and
from a different neuron in the absence (Control) 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 peptide, 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 rapid depolarization of neurons held under
current-clamp mode which, unlike the case in voltage-clamped neurons, was
2+ associated with pronounced elevations in [Ca ]i. Thus, ASIC1a channels are
likely promoting significant Ca2+ influx into the neurons by depolarizing the cells.
39 To mimic this change in membrane potential evoked upon ASIC1a activation,
cells were exposed to high K+ (40 mM) extracellular solution. Figure 2.9A shows
2+ + representative [Ca ]i traces recorded in response to high K o application in the
absence (Control) and presence of CBP (100 μM, +CBP). Depolarizing the
2+ neurons in this manner evoked robust [Ca ]i elevations which were blocked by
+ addition of CBP. In identical experiments CBP reduced the high K o-evoked
2+ increases in [Ca ]i by 83 ± 1% and this decrease was statistically significant (p <
0.001) (Figure 2.9B). Thus, σ receptor activation inhibits Ca2+ channels
downstream of ASIC1a in addition to the proton-gated channels themselves.
To determine the specific ion channels contributing to the ASIC1a-induced
2+ [Ca ]i influxes, several inhibitors of plasma 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
2+ elevate [Ca ]i. Thus, tetrodotoxin (TTX, 500 nM) was used to inhibit voltage-
gated Na+ channels to prevent the genesis of action potentials. Figure 2.10A
shows that application of TTX inhibited ~ 10% of the acid-induced increases in
2+ 2+ [Ca ]i, but did not significantly affect CBP (50 µM) modulation of the Ca responses, when compared to the PSS group. Because TTX had no effect on
the σ-1/ASIC1a interaction, all subsequent experiments included 500 nM TTX in
the bath solutions to prevent spontaneous action potentials from contributing to
2+ the measured increases in [Ca ]i. Inhibition of NMDA receptors by AP5 (100 µM)
2+ significantly reduced the acid-induced elevations in [Ca ]i, and co-application
2+ with CBP (50 µM) resulted in a further decrease in [Ca ]i which was statistically
40 significant (Figure 2.10A). These results show that ~ 30% of ASIC1a-induced
2+ [Ca ]i increases are dependent on NMDA receptor activation. Furthermore, since the effects of AP5 and CBP are less than additive, activation of σ-1
2+ receptors depresses ASIC1a-evoked increases in [Ca ]i in part by blocking this
NMDA receptor-dependent component. Blockade of L-type VGCC by nifedipine
2+ (10 µM) significantly inhibited (>75%) acid-induced [Ca ]i elevations, and CBP
2+ (50 µM) continued to provide further blockade of the residual increases in [Ca ]i
(Figure 2.10A). Cadmium (100 µM), a broad-spectrum blocker of plasma membrane calcium channels, inhibited ~ 90% of the acid-induced increases in
2+ 2+ [Ca ]i and CBP (50 µM) had no effect on the remaining Ca influx (Figure
2.10A). Thus, voltage-gated Ca2+ channels either directly (influx through the
channel) or indirectly (facilitating glutamate release) account for most of the
2+ [Ca ]i increases resulting from ASIC1a activation, and σ-1 receptor activation provides an inhibition similar to that observed with Cd2+.
Further experiments were conducted to determine if AMPA/kainate
2+ receptors are involved in the [Ca ]i elevations elicited upon ASIC1a activation.
2+ Figure 2.10B shows relative changes in [Ca ]i in the absence (Control) and
presence of the AMPA/kainate receptor blocker CNQX (10 µM), and the effects
of CBP at two different concentrations (50 µM and 300 µM). Maximal blockade of
AMPA/kainate receptors alone produced ~ 40% reduction of the elevations of
2+ [Ca ]i and 50 µM CBP did not provide additional, statistically significant block.
Increasing the CBP concentration to 300 µM, however, did produce an additional block of the Ca2+ response on top of the effects of CNQX. Figure 2.10B shows
41 that co-application of 300 µM CBP and CNQX blocked >80% of the acid-induced
2+ increases in [Ca ]i and this was statistically significant when compared to CNQX
2+ 2+ alone. Thus, [Ca ]i elevations elicited upon ASIC1a activation involve Ca influx through both ASIC1a and other Ca2+ permeable plasma membrane ion channels
expressed in cortical neurons. Importantly, it is also evident that activation of σ-1
receptors results in depression of Ca2+ influx through all of these sources, either
by inhibiting ASIC1a or the downstream 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 amiloride (100 µM, top traces, i), and from a different neuron in the absence (Control) and presence 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. Amiloride 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 reductions 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 acidosis in the absence (PSS) and presence of the indicated drugs. These data demonstrate that neither TTX, AP5, CNQX,
42 nifedipine, nor cadmium have any direct effects on ASIC1a channels.
σ receptor activation has been shown to modulate multiple cell membrane
ion channels in neurons that are activated following ASIC1a stimulation (Hayashi
et al., 1995; Zhang and Cuevas, 2002). This fact raises the possibility that σ-1 receptors only couple to the secondary events that result from ASIC1a activation and not to ASIC1a itself. To investigate if σ receptors affect ASIC1a function, whole-cell patch clamp recordings were performed in the presence of σ agonists.
Figure 2.12A shows representative membrane current traces as a function of time recorded from a cell in the absence (Control) and presence of the pan-
selective σ agonist DTG (100 µM). Results from several similar experiments
show that activation of σ 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 effects of DTG on ASIC1a currents were
mediated by σ-1 or σ-2 receptors, the effects of the σ-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 µM). After multiple experiments, analysis of the measured current densities showed that control cells had statistically significant larger current densities than CBP treated cells (Figure 2.12D). In the presence of
50 μM CBP, ASIC1a-mediated currents were decreased by 30 ± 5 % relative to control. In contrast, the σ-2 selective agonist, PB28 (20 µM), failed to inhibit
ASIC1a-mediated membrane currents, suggesting σ-2 receptors do not regulate
ASIC1a function (Figure 2.12D). Therefore, these results demonstrate that σ-1
43 receptors functionally couple to ASIC1a channels in cortical neurons as well as to targets downstream of ASIC1a activation.
44 2+ Figure 2.1 - ASIC1a blockers inhibit proton-evoked increases in [Ca ]i in cultured
2+ cortical neurons from embryonic (E18) rats. A, Representative traces of [Ca ]i as a function of time recorded from a single cell during acidosis in the absence
(Control, Wash) and presence of 100 μM Amiloride (Amiloride). B, Mean change
2+ in peak [Ca ]i (± SEM) measured in response to low pH solution (pH 6.0) under
2+ the indicated conditions (n = 69). C, Representative traces of [Ca ]i as a function of time recorded from a neuron during acidosis (Control, Wash) and following 20 minutes preincubation in 500 ng/ml Psalmotoxin1 venom (PcTx1). Psalmotoxin1 venom was only present in the pH 7.4 conditioning solution. D, Mean change in
2+ peak [Ca ]i (± SEM) measured during acidosis under the indicated conditions (n
= 158). Asterisks denote significant difference from Control and Wash groups in
(B) and (D) (p <0.001).
45
46 Figure 2.2 - Pan-selective sigma agonists inhibit proton-evoked transient
2+ 2+ increases in [Ca ]i. A, Representative traces of [Ca ]i as a function of time
recorded from a single neuron during acidosis in the absence (Control, Wash)
2+ and presence of 100 μM DTG (DTG). B, Mean change in peak [Ca ]i (± SEM)
measured during acidosis in the absence (Control, n = 119) and presence of 100
μM DTG (DTG; n = 99) or 10 μM 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
2+ [Ca ]i as a function of time recorded from a neuron during acidosis in the
absence (Control) and presence of 100 μM and 300 μM DTG. D, Concentration-
2+ response relationship for DTG inhibition of mean change in peak [Ca ]i (± SEM).
Values obtained for each cell were normalized to their respective controls (no
DTG) (n = 117-124). Line represents a best fit to the data using a single-site
Langmuir-Hill equation.
47 48 Figure 2.3 - Activation of σ-1 receptors inhibits ASIC1a-induced increases in
2+ 2+ [Ca ]i. Representative traces of [Ca ]i as a function of time recorded from two
cells during acidosis in the absence (Control) and presence of 10 μM and 100
μM carbetapentane (A, CBP), or in the absence (Control) and presence of 10 μM
and 100 μM dextromethorphan (B, DEX). C, Concentration-response
2+ relationships for mean change in peak [Ca ]i (± SEM) measured during acidosis
in the presence of the indicated σ-1 selective ligands DEX, PRE-084, and CBP.
Values were normalized to control (absence of σ 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 obtained for CBP inhibition of chemical
2+ ischemia-induced (4 mM azide in glucose-free PSS) increases in [Ca ]i, and is
shown for comparison (Katnik et al., 2006). For each condition n > 73.
49 50 Figure 2.4 - Inhibition of σ-1 receptors blocks CBP-mediated suppression of
2+ 2+ proton-evoked increases in [Ca ]i. A, Representative traces of [Ca ]i as a function of time recorded from 2 different neurons during acidosis in the absence
(Control) and presence of 30 µM CBP (CBP), without (PSS, left traces) and with
1hr preincubation in 50 µM metaphit (Metaphit, right traces). B, Percent inhibition
2+ of [Ca ]i increases (± SEM) by CBP measured during acidosis under control conditions (Control, n = 135) and following metaphit preincubation (MET, n =
2+ 113). C, Representative traces of acid-induced increases in [Ca ]i as a function of time recorded from 2 different neurons in the absence (Control) and presence of 30 µM CBP (CBP), without (PSS, left traces) and with co-application of 10 nM
2+ BD1063 (BD1063, right traces). D, Percent inhibition of [Ca ]i increases (±
SEM) by CBP under control conditions (Control, 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).
51 52 Figure 2.5 - Sigma-2 receptor ligands inhibit ASIC1a-mediated elevations in
2+ [Ca ]i at concentrations inconsistent with σ-2 mediated effects and in a metaphit-
2+ insensitive manner. A, Representative traces of [Ca ]i as a function of time recorded from two neurons during acidosis in the absence (Control) and presence of 10 µM and 100 µM ibogaine (IBO, left traces), or in the absence
(Control) and presence of 1 µM and 10 µM PB28 (right traces). B, Mean change
2+ in peak [Ca ]i (± SEM) measured during acidosis in the presence of the σ-2
selective ligands PB28 and IBO. Solid lines represent best fits to the data using single-site Langmuir-Hill equations. Dashed line represents best fit to the data obtained for IBO inhibition of ICa, and is shown for comparison (Zhang and
2+ Cuevas, 2002). For each condition, n > 108 cells. C, [Ca ]i as a function of time recorded from 2 different neurons during acidosis in the absence (Control) and presence of 20 µM PB28 (PB28), without (PSS) and with (+MET) 1hr
2+ preincubation in 50 µM metaphit. D, Percent inhibition of [Ca ]i (± SEM) by PB28
measured during acidosis under control conditions (Control, n = 324), following
metaphit preincubation (+MET, n = 276) and co-applied with 10 nM BD1063
(BD1063, n = 139). There is no significant difference between Control and +MET
groups, (p = 0.64) or Control and BD1063 groups (p = 0.70).
53
54 Figure 2.6 - Intracellular Ca2+ stores are not involved in DTG modulation of
2+ 2+ ASIC1a-induced increases in [Ca ]i. A, Representative traces of [Ca ]i as a
function of time recorded from a neuron during acidosis in the absence (Control)
2+ and presence of 100 µM DTG (DTG). B, Representative traces of [Ca ]i as a
function of time recorded from a neuron during acidosis following preincubation in
10 μM thapsigargin (1 hr, 23º C) in the absence (THAP) and presence of 100 μM
2+ DTG (THAP + DTG). C, Mean change in peak [Ca ]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 μM DTG (DTG).
Asterisks denote significant difference from Control group (p < 0.001). There was
no significant difference between PSS and THAP groups (p = 0.48).
55 56 2+ Figure 2.7 - ASIC1a-mediated [Ca ]i increases are membrane potential
2+ dependent. A, Representative traces of [Ca ]i recorded in response to ASIC1a
activation from a neuron electrically accessed using the perforated patch whole-
2+ cell configuration and held at -70 mV ([Ca ]i V-Clamp), and from a second
2+ neuron in the same field of view which was not electrically accessed ([Ca ]i
Control). Inset, whole-cell current trace recorded simultaneously from the voltage-clamped neuron. Lines above traces indicate application of pH 6.0
2+ solution. Scale bars: 500 pA, 5 sec. B, Mean change in peak [Ca ]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).
57 58 Figure 2.8 - ASIC1a activation promotes membrane depolarization. A,
Representative traces of ASIC1a-mediated membrane potential changes as a function of time recorded from a neuron held under current-clamp mode in the absence (Control) and presence of 100 μM amiloride (Amiloride). B, Changes in peak membrane potential (± SEM) recorded from neurons in normal PSS
(Control) or in PSS containing 100 μM amiloride (Amiloride, n = 3). Asterisk denotes significant difference from Control group (p < 0.05). C, Representative traces of ASIC1a-mediated membrane potential 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 peptide (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).
59 60 Figure 2.9 - Sigma-1 receptor activation inhibits Ca2+ channels downstream of
2+ ASIC1a. A, Representative traces of [Ca ]i recorded in response to application of extracellular high K+ (40 mM) solution in the absence (Control) and presence
2+ of 100 μM CBP. B, Bar graph of mean change in peak [Ca ]i (± SEM) measured after multiple experiments (n = 59). Asterisks denote significant difference between groups (p < 0.001).
61 62 Figure 2.10 - Multiple plasma membrane ion channels downstream of ASIC1a
2+ activation contribute to acidosis-evoked [Ca ]i increases. A, Relative changes in
2+ [Ca ]i (± SEM) during acidosis in the absence (PSS) and presence of 500 nM
tetrodotoxin (TTX) alone or TTX (500 nM) co-applied with 100 μM AP5, 10 μM
nifedipine or 100 μM cadmium. All combinations were done without (Control) and
with 50 µM 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
2+ from TTX group (p < 0.001). B, Relative changes in [Ca ]i (± SEM) during
acidosis in the absence (Control) and presence of 10 μM CNQX (CNQX) without
CBP (PSS) or with CBP at the indicated concentrations. For each condition n >
95. Asterisks denote significant differences from PSS group within Control or
CNQX groups (p < 0.001), pound symbols indicate significance differences between 50 μM and 300 μM CBP groups within Control or CNQX groups (p <
0.001), and daggers denote significant differences between Control and CNQX within PSS (p < 0.001) and 300 µM CBP (p < 0.05) groups.
63 64 Figure 2.11 - ASIC1a blockers inhibit acidosis-mediated currents in voltage-
clamped neurons. A, Representative traces of ASIC1a-mediated currents as a
function of time recorded from two neurons held at -70 mV in the absence
(Control) and presence of 100 μM amiloride (Amiloride) (i.) or 500 ng/ml
Psalmotoxin1 venom (PcTx1) (ii.). B, Relative mean peak proton-gated currents
(± SEM) recorded from neurons in normal PSS (Control) or in PSS containing either 100 μM 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 proton-
gated currents (I/Imax, ± SEM) recorded in the absence (PSS) and presence of
500 nM tetrodotoxin (TTX, n = 4) alone and TTX with 100 μM AP-5 (n = 3), 10
μM CNQX (n = 4), 10 μM nifedipine (Nif, n = 6) or 100 μM cadmium (Cd, n = 3) inthe bath solution. Cells were voltage-clamped at -70 mV. No significant differences between the groups were noted (p = 0.826).
65 66 Figure 2.12 - Sigma receptor agonists inhibit ASIC1a-mediated currents in
voltage-clamped neurons. A, Representative 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 μM DTG (DTG). B, Mean peak proton-gated current densities (± SEM) measured from neurons held at -70 mV without (Control) or with 100 μM DTG (DTG) in the bath solution (n = 6). C,
Representative traces of ASIC1a-mediated currents as a function of time recorded from a voltage-clamped neuron (-70 mV) in the absence (Control) and presence of 50 μM CBP (CBP). D, Mean peak proton-gated current densities (±
SEM) recorded from neurons held at -70 mV without (Control) or with 50 μM CBP
(CBP, n = 6) or 20 μM PB28 (PB28, n = 4). Asterisks in (B) and (C) denote significant differences from respective Control groups (p < 0.001).
67 68 Discussion
Activation of σ receptors depresses membrane currents and elevations in
2+ [Ca ]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 σ-1 receptor subtype. Furthermore, most of the
2+ 2+ elevations in [Ca ]i triggered by acidosis are the result of Ca channels opening downstream of ASIC1a activation. Stimulation of σ-1 receptors effectively suppressed these secondary Ca2+ fluxes both by inhibiting ASIC1a and the other channels directly.
ASIC are regulated by various factors such as pH, membrane distention and arachidonic acid, and therefore, function as signal integrators in the CNS
(Allen and Attwell, 2002; Lopez, 2002). All of these factors elicit or potentiate
ASIC-mediated responses. Information on endogenous mechanisms that inhibit
ASIC function is lacking. It has been shown that 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 (Gao et al.,
2005). Thus, our finding that activation of σ receptors depresses ASIC1a- mediated 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 deficient in the ASIC1a subunit fail to show
2+ increases in [Ca ]i or membrane currents at the proton concentrations used here
(Xiong et al., 2004). ASIC2a and ASIC2b subunits are also expressed in the
69
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 ASIC2a nor heteromultimeric ASIC1a/ASIC2a
2+ 2+ channels conduct Ca , and thus could not account for the changes in [Ca ]i observed here (Yermolaieva et al., 2004).
Results from Ca2+ imaging experiments suggest that it is specifically the
σ-1 receptor subtype that modulates neuronal responses to ASIC1a activation.
Studies have shown that the affinity of carbetapentane for σ-1 receptors is
>50-fold greater than for σ-2 receptors (Rothman et al., 1991; Vilner and Bowen,
2000). The calculated IC50 for carbetapentane inhibition of ischemia-evoked
2+ increases in [Ca ]i via σ-1 receptor activation is 18.7 µM (Katnik et al., 2006), which is comparable to the 13.8 μM IC50 for CBP inhibition of ASIC1a-induced
2+ [Ca ]i increases. Carbetapentane also inhibits epileptiform activity in rat
hippocampal slices via σ-1 receptors with an IC50 value of 38 µM (Thurgur and
Church, 1998). Similarly, we show that the σ-1 agonists dextromethorphan
(IC50 = 22 μM) and PRE-084 (IC50 = 13.7 μM), both of which have >100-fold greater affinities for σ-1 than σ-2 receptors, block ASIC1a-mediated responses at concentrations consistent with those reported in the literature.
Dextromethorphan inhibits spreading depression in rat neocortical brain slices with an IC50 ~ 30 μM (Anderson and Andrew, 2002), whereas PRE-084 protects
human retinal cells against oxidative stress with an IC50 ~ 10 μM (Bucolo et al.,
2006). The fact that IC50 values determined here for carbetapentane,
dextromethorphan and PRE-084 are in the low μM range suggests that it is
70 unlikely these agonists are affecting ASIC1a activity via σ-2 receptors, since high
μM to mM concentrations of these compounds are required to stimulate σ-2
receptors. Moreover, σ-2-selective agonists failed to inhibit ASIC1a-mediated
responses at concentrations consistent with σ-2 specific effects.
The strongest evidence that σ-1 receptor activation modulates ASIC1a
comes from experiments using the σ antagonists, metaphit and BD1063.
Metaphit has been shown to bind irreversibly to σ-1 receptors with an IC50 value
of 50 μM (Wu, 2003). Preincubation in metaphit blocks σ-1 receptor mediated
modulation of voltage-gated K+ channels in intracardiac neurons and depression
2+ of ischemia-induced elevations in [Ca ]i in cortical neurons (Zhang and Cuevas,
2005; Katnik et al., 2006). Preincubation of cortical neurons in 50 μM metaphit antagonized CBP inhibition of ASIC1a by ~ 40%. BD1063 has been shown to have a higher affinity for σ-1 than σ-2 receptors and attenuates the dystonia
produced by DTG in rats in a dose-dependent manner, suggesting this ligand
acts as an antagonist at σ sites (Matsumoto et al., 1995). Here we show that
2+ CBP is unable to block acid-induced increases in [Ca ]i when co-applied with
BD1063. In addition, we found that metaphit fails to inhibit the effects of the σ-2
agonist, PB28, on ASIC1a-mediated responses. Taken together, these data
2+ show that increases in [Ca ]i in response to ASIC1a activation are modulated
only by σ-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 indicates that most, if not all, of
71
2+ the acid-induced increases in [Ca ]i is due to plasma membrane influx. However,
our results show that multiple ion channels downstream of ASIC1a activation
2+ contribute to acidosis-induced elevations in [Ca ]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 tetrodotoxin. This observation suggests a
presynaptic localization of ASIC1a, whereby activation of the channel by protons
results in synaptic transmission and subsequent activation of postsynaptic
glutamatergic receptors. Consistent with this hypothesis, ASIC1a has been found
to regulate neurotransmitter release probability in mouse hippocampal neurons
(Cho and Askwith, 2008).
σ 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 the presence of specific inhibitors of
ionotropic glutamate receptors, activation of σ-1 receptors with CBP further
2+ decreased proton-evoked increases in [Ca ]i, but the effects of CBP and the
glutamate channel inhibitors were less than additive. Thus, σ-1 receptors also
inhibit Ca2+ entry via NMDA and AMPA/kainate receptors directly by inhibiting these channels and indirectly by depressing ASIC1a activation. Application of the
L-type VGCC inhibitor, nifedipine, and the broad-spectrum Ca2+ channel inhibitor,
2+ cadmium, blocked ASIC1a-induced increases in [Ca ]i by >70% and >90%,
2+ respectively. This observation indicates that most of the increases in [Ca ]i
produced upon ASIC1a activation is dependent on Ca2+ influx through VGCC.
72
Co-application of CBP with nifedipine, but not with Cd2+, resulted in further
2+ 2+ reduction in the proton-evoked increases in [Ca ]i. The conclusion that Ca
influx through ASIC1a channels itself contributed only a small fraction to the total
2+ 2+ observed [Ca ]i increases was confirmed with simultaneous Ca fluorometry
and whole-cell patch clamp recordings. Cells voltage-clamped at –70 mV, which
prevents NMDA receptor and VGCC activation, demonstrated minimal acid-
2+ evoked elevations in [Ca ]i. Taken together, our results show that the increases
2+ in [Ca ]i evoked by ASIC1a activation are the result of synaptic transmission and
subsequent opening of multiple Ca2+ channels, and that stimulation of σ-1
receptors downregulates all of these events. However, the fact that activation of
σ-1 receptors depressed ASIC1a-mediated currents in cells voltage-clamped at
-70 mV indicate that σ-1 receptors are functionally coupled to ASIC1a, and that
2+ the depression in acid-evoked increases in [Ca ]i is not exclusively the result of
σ-1 receptors blocking channels downstream of ASIC1a.
The finding that σ-1 receptors can inhibit ASIC1a channels has significant
physiological and pathophysiological implications. It has been proposed that
ASIC1a activation may facilitate neurotransmission by compensating for the
decrease in excitatory neurotransmission caused by direct inhibition of post-
synaptic 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 density of dendritic spines in hippocampal
neurons (Zha et al., 2006). Thus, σ-1 receptors may influence cell-to-cell signaling in the CNS by affecting ASIC1a activity. One of the consequences of
73
ASIC1a overexpression in mice is enhanced fear conditioning (Wemmie et al.,
2004), whereas stimulation of σ-1 receptors is known to ameliorate conditioned fear stress (Kamei et al., 1997). These observations, coupled with our current report, suggest that σ-1 receptor activation may produce anxiolytic effects via the
inhibition of ASIC1a channels.
The inhibition of ASIC1a by σ-1 receptors is a potential component of the neuroprotective properties of σ receptors, since activation of ASIC1a has been shown to contribute to stroke injury (Xiong et al., 2004). Importantly, inhibition of
ASIC1a has been shown to be neuroprotective at delayed time points following ischemic stroke (Simon, 2006). Thus, σ-1 receptor-mediated inhibition of ASIC1a may contribute to the enhanced neuronal survival following σ 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, σ-1 receptor activation may provide further neuroprotection by
reducing the activity of these channels which occurs subsequent to ASIC1a
stimulation. Consistent with this pleiotropic effect of σ-1 receptors is our
observation that σ-1 receptor activation suppressed extracellular high K+-induced
2+ increases in [Ca ]i, which would also activate these downstream effectors. In
conclusion, σ-1 receptors inhibit ASIC1a channel function and blunt acidosis-
2+ evoked ionic fluxes and increases in [Ca ]i. Thus, σ-1 receptors can be targeted
for therapeutic intervention in pathophysiological conditions involving ASIC1a
activation.
74
CHAPTER 3
SIGMA-1 RECEPTOR ACTIVATION INHIBITS 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 the degenerin/epithelial sodium channel (Deg/ENac)
superfamily (Waldmann et al., 1997b; Benos and Stanton, 1999). ASIC are
expressed in both peripheral and central nervous system neurons. Several
extracellular and intracellular modulators of ASIC have 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 (Andrey et al., 2005; Chu et al., 2006)
have been shown to interact with the extracellular 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 (CaMKII) (Gao et al., 2005), protein kinace C
(PKC) (Baron et al., 2002) and protein kinase A (PKA) (Leonard et al., 2003)
binding site. Phosphorylation of ASIC1a has been shown to potentiate ASIC1a
function in neurons (Xiong et al., 2004; Gao et al., 2005). In contrast, the second
75 messenger, calcineurin, dephosphorylates ASIC1a channels and results in
downregulation of channel function (Chai et al., 2007).
Dephosphorylation of ASIC1a is mediated through the interaction with
cytoskeletal anchoring protein, A-kinase anchoring protein (AKAP). Interestingly,
AKAP150 and calcineurin have been implicated 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 primarily 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 (calcineurin) that are inhibited when
bound. AKAP150 targets these proteins, through a unique targeting motif, to
specific subcellular sites and plasma membrane 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 during 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 channels have shown to be regulated by σ receptor
activation in neurons via various signaling cascade mechanisms. σ receptor activation has been shown to depress both membrane currents and elevations in
76
2+ [Ca ]i mediated by ASIC1a channels in cortical neurons. Furthermore, most of
2+ the elevations in [Ca ]i triggered by acidosis were determined to be the result of
Ca2+ channels opening downstream of ASIC1a activation, and activation of σ-1
receptors effectively suppressed these secondary Ca2+ fluxes by both inhibiting
ASIC1a and/or the Ca2+ channels directly. But the signaling cascade mechanism
by which σ-1 receptors regulate ASIC1a channels remains to be elucidated.
σ-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 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 σ receptors has been shown to be
dependent on protein-protein interactions (Aydar et al., 2002).
In this study, experiments were conducted to determine the signaling
cascade linking σ-1 receptors to ASIC1a channels and downstream Ca2+ channels. σ-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. Calcineurin inhibitors, cyclosporin A and FK-
506, and the G protein inhibitor pertussis toxin (PTX) diminished the downregulation of ASIC1a by σ-1 receptors, suggesting that σ-1 receptors exert their effect via calcineurin-dependent dephosphorylation of ASIC1a and a PTX-
77 sensitive G protein. Furthermore, disruption of the actin cytoskeleton or dissociation AKAP150 from the plasma membrane were shown to diminish σ-1
receptor mediated inhibition of ASIC1a channels. Moreover, whole-cell patch clamp experiments confirmed that preincubation in PTX or disruption of the
AKAP/calcineurin interaction with VIVIT, prevented σ-1 receptor modulation of
ASIC1a-mediated membrane currents, suggesting σ-1 receptors couple to
ASIC1a channels via a PTX-sensitive G protein and an AKAP150/calcineurin complex. This is the second report of receptor-mediated functional downregulation of ASIC1a channels. Thus far, σ-1 receptors are the only receptors identified to inhibit ASIC1a channel function.
Materials and Methods
Primary Rat Cortical Neuron Preparation
Primary cortical neurons from embryonic (E18) rats were cultured as previously described by our laboratory (Katnik et al., 2006). All procedures were done in accordance with the regulations of the University of South Florida
Institutional Animal Care and Use Committee. 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 ratiometric 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
78 imaged as we have previously described (DeHaven and Cuevas, 2004; Katnik et al., 2006). Briefly, cells plated on coverslips were incubated for 1 hour at room temperature in Neurobasal (Invitrogen) medium supplemented with B27
(Invitrogen) and 0.5 mM L-glutamine, 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 μM of fura-2,
acetoxymethylester (fura-2 AM) and 0.4 % dimethyl sulfoxide. The coverslips
were washed in PSS (fura-2-AM free) prior to the experiments being carried out.
Electrophysiology Recordings
ASIC1a-mediated membrane currents were recorded using the protocol
previously described by our laboratory. Briefly, neurons plated on glass
coverslips were transferred to a recording chamber and membrane currents were
amplified, filtered at 1 kHz, digitized at 5 kHz, and acquired using Clampex 8
(Axon). Electrical access was achieved using the amphotericin B perforated-
patch method to preserve intracellular integrity of neurons (Rae et al., 1991). An
amphotericin B stock solution (60 mg/ml in DMSO) was made fresh everyday,
kept on ice, light protected, and diluted to 240 µg/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 electrodes were pulled from thin-walled
borosilicate glass (World Precision Instruments Inc., Sarasota, FL) using a Sutter
Instruments P-87 pipette puller (Novato, CA) and had resistances of 1.0–1.5 MΩ.
Access resistances (Rs) were monitored throughout experiments for stable
79 values ≤ 20 MΩ and were always compensated at 40% (lag, 10 µs). All cells were voltage-clamped at -70 mV.
Immunohistochemistry
Cortical neurons plated on coverslips were first rinsed with phosphate buffer saline (PBS) to remove excess media. Cells were fixed by incubating in
95% ethanol and 5% acetic acid at -20°C for 20 minutes, followed by four 2-
minute incubations at room temperature in 95%, 75%, 50% and 0% ethanol.
Neurons were then permeabilized with PBS solution 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 cells were then blocked with 2% BSA in
PBS for 45 minutes at room temperature. Neurons were incubated in the primary
antibodies (ASIC1 guinea-pig polyclonal IgG at 1:500 dilution, AKAP150 goat
polyclonal IgG at 1:50 dilution and σ -1 rabbit polyclonal IgG at 1:500 dilution) in
0.5% BSA in PBS solution overnight at 4°C 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 antibodies were purchased from: AKAP150 (Santa Cruz
Biotechnology, Santa Cruz, CA) and ASIC1a (Lifespan Biosciences, Seattle,
WA). σ-1 receptor antibody was kindly provided by Drs. T. P. Su and Teruo
Hayashi (Baltimore, Maryland). Secondary antibodies were purchased from
Invitrogen (Eugene, OR): Alexa Fluor 488 anti-rabbit, Alexa Fluor 555 anti-goat,
and Alexa Fluor 633 anti-guinea-pig. Coverslips with labeled cells were then
80 mounted onto a microscope slide using VectaShield Hardset Media with DAPI purchased from Vector Laboratories (Burlingame, CA).
Confocal Microscopy
Confocal images of triple labeled neurons were collected using a Leica
DMI6000 inverted microscope, TCS SP5 confocal 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 used to minimize crosstalk between fluorchromes. Image sections at 0.4 μm were acquired with photomultiplier detectors, processed and analyzed using Leica LAS AF software version 1.8.2
(Leica Microsystems, Germany). 2-D images of fluorescence emission for each fluorophore (AF488, green; AF555, red; AF633, 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-section 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 outside the two straight lines are pixels with intensities greater than background in only one of the images. The Overlap
Coefficient (R), a correlation coefficient relating two images or regions within an image, is defined as
∑S1i • S2i R = i 2 2 ∑∑()S1i • (S2i ) ii (1)
81 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 colocalization, to 0, complete non-
colocalization and was calculated for entire images and within 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 chemicals used in this investigation were of
analytic grade. The following drugs were used: DTG and pertussis toxin (Sigma-
Aldrich, St. Louis, MO); carbetapentane and NMDA (Tocris Bioscience, Ellisville,
MO); latrunculin-A, cyclosporineA and VIVIT (Calbiochem, San Diego, CA); FK-
506 (LC Laboratories, Woburn, MA); tetrodotoxin (Alomone Labs, Jerusalem,
Israel); and fura-2 AM (Molecular Probes, Eugene, OR).
Data Analysis
Analysis of measured intracellular Ca2+ and membrane current responses
was conducted using Clampfit 9 (Axon instruments). Imaging data files collected
with SlideBook 4.02 (Intelligent 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 differences were determined using paired and
82 unpaired t-tests for within group and between 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 ANOVA, post-hoc analysis was conducted
using a Tukey’s Test to determine interaction between individual groups.
Results
To answer whether the signaling cascade linking σ receptors to ASIC
channels might involve protein-protein interactions, immunohistochemical
fluorescent staining experiments were performed on cortical neurons to
determine if σ-1 receptors, ASIC1a channels and AKAP150 colocalize. Figure
3.1A shows fluorescent images of neurons triple labeled for σ-1 receptors(i),
AKAP150 (ii) and ASIC1a channels (iii) from the same field of view. The white
pixels represent areas in the image where σ-1 receptor and AKAP150 (Figure
3.1B, i) labeling appear together as selected in the scatter plot (Figure 3.1B, ii).
The scatter plot maps pixel intensities from the σ-1 receptor labeling versus
intensities from the AKAP150 labeling. The overlap coefficient for σ-1 receptor
and AKAP150 colocalization calculated using Eq. 1 for the entire image was
calculated to be 0.74. Figure 3.1C, i, shows the merged image of σ-1 receptor
and ASIC1a labeling with colocalized pixels represented in white and the
colocalization scatter plot (Figure 3.1C, ii). The overlap coefficient for σ-1
receptors and ASIC1a channels for the entire image was calculated to be 0.78.
After multiple experiments (3 stains with a total of 59 cell bodies and 45 neuronal
83 processes), the mean overlap coefficient for σ-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 to be 0.696 ±
.013 and 0.751 ± .009, respectively (Figure 3.1D). These images suggest that σ-
1 receptors colocalize with both AKAP150 and ASIC1a in the cell body and in the
dendritic processes. The similarity of the 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 (Chai et al., 2007). Thus, experiments
were carried out to determine if calcineurin is a constituent of the signaling
cascade linking σ-1 receptors to ASIC1a channels. Figure 3.2A show
2+ characteristic traces of [Ca ]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 μM, ii) and cyclosporin A (1 μM, cyclo A, iii), all in the
absence and presence of CBP (10 μM). Both calcineurin inhibitors reversed the
effects of σ-1 receptor activation on ASIC1a function. A summary of data collected from identical experiments is shown in Figure 3.2B. Bath application of
FK-506 or cyclosporin A alone had no effects on control responses. However, following inhibition of calcineurin with either FK-506 or cyclosporin A, CBP blocked ASIC1a-mediated responses significantly less than under control conditions (Figure 3.2B). The percent-inhibition of ASIC1a-mediated responses by CBP was significantly decreased from 45 ± 1% in control cells to 31 ± 1% and
84
4 ± 2% in the presence of FK-506 and cyclosporin A, respectively (Figure 3.2C).
These results suggest σ-1 receptors are functionally linked to ASIC1a via a signaling cascade involving calcineurin. These observations are consistent with reports in the literature which suggest that dephosphorylation of ASIC1a and
ASIC2a channels by this phosphatase may be involved in the functional downregulation of ASIC (Chai et al., 2007).
Calcineurin has been shown to be activated by a pertussis toxin-sensitive
G protein, specifically Gi2 (Gromada et al., 2001). Previous reports suggest that
σ-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 proteins were also involved in σ-1 receptor
regulation of ASIC1a channels. Figures 3.3A and 3.3B show representative
2+ traces of [Ca ]i as a function of time recorded during acidosis in the absence
(Control) and presence of CBP (CBP, 50uM) without (A) and with PTX
preincubation (200 ng/ml for 24 hrs at 37°C, B). Results from multiple experiments suggest that PTX alone has some effect on ASIC1a-mediated
2+ [Ca ]i increases but it does not change the kinetics of the responses (Figure
3.3B). Preincubation in PTX significantly reduced σ-1 receptor mediated
2+ inhibition of ASIC1a-induced [Ca ]i increases (Figure 3.3C). While CBP inhibited
2+ 41 ± 1% of acid-induced increases in [Ca ]i in control cells, the σ-1 ligand only
blocked 26 ± 1% of the increases following PTX treatment. This inhibition was
statistically significant (Figure 3.3D). These results suggest that σ-1 receptor
85
2+ activation inhibits ASIC1a-induced increases in [Ca ]i via a PTX-sensitive G protein.
Having identified calcineurin and a PTX-sensitive G protein in the signaling pathway linking ASIC1a and σ-1 receptors, it was of interest to determine other constituents of the signaling 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
2+ (Gomez et al., 2002). Figures 3.4A and 3.4B show representative traces of [Ca ]i as a function of time recorded during acidosis in the absence (Control) and presence of CBP (CBP, 50 μM) without (PSS, A) and with NMDA (10 μM, B) pre- incubation. Pre-incubation in NMDA significantly reduced ASIC1a-mediated
2+ elevations in [Ca ]i (Figure 3.4C). In addition, pre-incubation in NMDA also
significantly reduced σ-1 receptor mediated inhibition of the remaining ASIC1a-
2+ induced [Ca ]i increase (Figure 3.4C). While CBP inhibited 44 ± 1% of acid-
2+ induced increases in [Ca ]i in control cells, it only inhibited 40 ± 1% in NMDA
treated cells and this inhibition was statistically significant (Figure 3.4D). The
small but significant block by NMDA of σ-1 receptor modulation 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
86 results are consistent with the theory that the scaffolding protein AKAP150 is
2+ involved in σ-1 receptors modulation of ASIC1a-induced increases in [Ca ]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; Feliciello et al., 2001; Wong and Scott,
2004). Latrunculin-A, a bioactive 2-thiazolidinone 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,
2+ 2008). Figures 3.5A and 3.5B show representative traces of [Ca ]i as a function
of time recorded during acidosis in the absence (Control) and presence of CBP
(CBP, 50 μM) without (DMSO, A) and with latrunculin-A (5 μM, B) preincubation
for 4 hours at 37°C. While latrunculin-A alone did not significantly reduce
2+ ASIC1a-mediated [Ca ]i elevations, disruption of the neuronal cytoskeleton more
substantially affected σ-1 receptor mediated inhibition of ASIC1a Ca2+
dysregulation (Figure 3.5C). In control cells CBP inhibited 47 ± 1% of acid-
2+ induced increases in [Ca ]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 cytoskeleton by latrunculin-A was confirmed using
phalloidin stain, which detects actin filaments cytochemically (Figure 3.6).
Results showed that control cells had preserved cytoskeleton staining around the
cell body and along the dendritic processes, while latrunculin-A treated cells
87 showed staining patterns consistent with retraction of the cytoskeleton from the
processes and synapses (Figure 3.6).
Previous studies have demonstrated that the major component of acid-
2+ 2+ induced increases in neuronal [Ca ]i is from influx through Ca channels
activated by ASIC1a-induced membrane depolarization. To isolate what direct
effects disruption of the σ-1/G-protein/calcineurin/AKAP150 protein structure has
on ASIC1a function, whole-cell patch clamp 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 σ agonist DTG was used instead of the σ-1 selective agonist CBP. Figures 3.7A and 3.7B show superimposed ASIC1a currents evoked by application of low pH solution (pH = 6)
in the absence (Control) and presence (DTG) of 100 μM 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 ASIC1a- mediated currents elicited by bath application of 100 μM DTG in neurons under control conditions and following preincubation in pertussis toxin (PTX, n = 7).
Preincubation in PTX significantly decreased the effects of DTG on acid-evoked currents, 10% versus 30% inhibition, respectively (Figure 3.7B). Therefore, in cortical neurons, the effects of σ-1 receptors on ASIC1a 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
88
(Oliveria et al., 2007). Moreover, VIVIT does not affect phosphatase activity
(Hogan et al., 2003; Im and Rao, 2004). To elucidate the effects of VIVIT on the signaling cascade linking ASIC1a channels to σ-1 receptors, cortical neurons
were voltage-clamped at -70 mV to isolate ASIC1a currents. Figure 3.8A shows
representative ASIC1a-mediated currents evoked by low pH solution in the
absence (Control) and presence of CBP (CBP, 100 μM). In contrast, Figure 3.8B
shows traces of ASIC1a-mediated current during acidosis from a different neuron
in the absence (Control), presence of preincubation in the cell permeable peptide
VIVIT (VIVIT, 10 nM, 5 minutes) and coapplication of VIVIT and CBP (VIVIT/CBP
100 μM). Analysis of the currents densities shows that while CBP significantly inhibited ASIC1a-mediated currents compared to control cells (Figure 3.8C),
VIVIT alone or in combination with CBP had no affect on ASIC1a-mediated currents (Figure 3.8D). Taken together, these data, consistent with Ca2+ imaging
results, suggest that σ-1 receptors couple to ASIC1a channels via a
calcineurin/AKAP complex in addition to a PTX-sensitive G protein.
89
Figure 3.1 - Sigma-1 receptors colocalize with both ASIC1a channels and
AKAP150 in cortical neurons. A, Representative fluorescent images of neurons
triple labeled for σ-1 receptors(i), AKAP150 (ii) and ASIC1a channels (iii) in the
same field of view. B, From A, merged image of σ-1 receptor and AKAP150. The
white pixels represent areas in the image where σ-1 receptor and AKAP150 labeling appear together (B, i) as selected in the scatter plot (B, ii). The scatter plot maps pixel intensities from the σ-1 receptor labeling versus intensities from the AKAP150 labeling. The overlap coefficient for σ-1 receptor and AKAP150 colocalization for the entire image was calculated to be 0.74. C, From A, merged image of σ-1 receptor and ASIC1a labeling with colocalized pixels represented in white (C, i) and the colocalization scatter plot (C, ii). The overlap coefficient for σ-
1 receptor and AKAP150 colocalization for the entire image was calculated to be
0.78. All scale bars (A, B, C) are 25 µm. D, Mean overlap coefficient for σ-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.
90
91
92
Figure 3.2 - Calcineurin inhibition prevents σ-1 receptor modulation of ASIC1a.
2+ A, Characteristic traces of [Ca ]i as a function of time recorded from three
neurons in the absence (Control) and presence of 10 µM carbetapentane (CBP)
during acidosis in normal PSS (i), or PSS containing 1 μM FK-506 (ii, FK-506) or
2+ 1 μM cyclosporin A (iii, Cyclo A). B, Mean peak proton-evoked changes in [Ca ]i recorded from neurons in the absence (Control) and presence of 10 µM carbetapentane (CBP) in normal PSS (n = 184), or PSS containing either 1 μM
FK-506 (n = 216) or 1 μM cyclosporin A (n = 155). Asterisks denote significant differences from respective control groups (p < 0.05), and pound symbols indicate significant differences from the PSS group within CBP (p < 0.05). C, Bar
2+ graph of mean CBP-induced inhibitions of peak proton-evoked changes in [Ca ]i
from the same experiments as B. Control group represents cells exposed to
acidosis in PSS alone. Asterisks denote significant differences from control group
(p < 0.05).
93
94
2+ Figure 3.3 - Sigma-1 receptors inhibit acid-induced elevations in [Ca ]i via a
2+ PTX-sensitive G protein. A, Representative traces of [Ca ]i as a function of time
recorded from a vehicle treated (PSS) neuron during acidosis in the absence
2+ (Control) and presence of 50 µM CBP (CBP). B, Representative traces of [Ca ]i
as a function of time recorded from a different neuron during acidosis following
preincubation in 200 ng/ml PTX (PTX, 24 hrs at 37º C) in the absence (Control)
2+ and presence of 50 μM CBP (CBP). C, Mean changes in peak [Ca ]i (± SEM)
measured in response to acidosis with (PTX, n = 246) or without (PSS, n = 188)
PTX preincubation in the absence (Control) and presence of 50 μM CBP (CBP).
Asterisks denote significant differences from respective Control groups (p <
0.05), pound symbol indicates significant difference between PSS and PTX
groups within CBP (p < 0.05), and dagger denotes significant difference between
2+ PSS and PTX groups within Control (p < 0.05). D, Percent inhibitions of [Ca ]i
increases (± SEM) by CBP under control conditions (Control) and in the
presence of PTX (PTX). Asterisk denotes significant difference between the
groups (p < 0.05).
95
96
Figure 3.4 - AKAP150 dissociation from the plasma membrane prevents σ-1
2+ receptor modulation of acid-induced elevations in [Ca ]i. A, Representative
2+ traces of [Ca ]i as a function of time recorded from a neuron during acidosis
(PSS) in the absence (Control) and presence of 50 µM CBP (CBP). B,
2+ Representative traces of [Ca ]i as a function of time recorded from a different
neuron during acidosis following preincubation in 10 μM NMDA (NMDA, 5 min -1 hr) in the absence (Control) and presence of 50 μM CBP (CBP). C, Mean
2+ changes in peak [Ca ]i (± SEM) measured in response to acidosis with (NMDA,
n = 398) or without (PSS, n = 198) NMDA preincubation in the absence (Control) and presence of 50 μM 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
2+ < 0.05). D, Percent inhibition of [Ca ]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).
97
98
Figure 3.5 - Disruption of the actin cytoskeleton prevents σ-1 receptor inhibition
2+ 2+ of acid-induced increases in [Ca ]i. A, Representative traces of [Ca ]i as a
function of time recorded from a vehicle (DMSO) treated neuron during acidosis
in the absence (Control) and presence of 50 µM CBP (CBP). B, Representative
2+ traces of [Ca ]i as a function of time recorded from a different neuron during acidosis following preincubation in 5 μM latrunculin A (Latrunculin A, 4 hrs at 37º
C) in the absence (Control) and presence of 50 μM CBP (CBP). C, Mean
2+ changes in peak [Ca ]i (± SEM) measured in response to acidosis with
(Latrunculin A, n = 222) or without (DMSO, n = 276) latrunculin A preincubation
in the absence (Control) and presence of 50 μM CBP (CBP). Asterisks denote
significant differences from respective Control groups (p < 0.05), pound symbol
indicates significant difference between DMSO and latrunculin A groups within
CBP (p < 0.05), and dagger denotes significant difference between DMSO and
2+ latrunculin A groups within Control (p < 0.05). D, Percent inhibition of [Ca ]i increases (± SEM) by CBP under control conditions (Control) and in the presence of latrunculin A (Latrunculin A). Asterisk denotes significant difference between the groups (p < 0.05).
99
100
Figure 3.6 - Latrunculin A preincubation disrupts the actin cytoskeleton. Cells were treated with either vehicle (A, DMSO) or latrunculin A (B, 5 μM) for 4 hours at 37°C. Cells were then stained with phalloidin to detect actin filaments cytochemically (A and B, right images). For comparison, bright field pictures are also shown (A and B, left images). All scale bars are 25 μm.
101
102
Figure 3.7 - Sigma receptor-mediated inhibition of ASIC1a currents are blocked by preincubation in pertussis toxin. A and B, Superimposed ASIC1a currents evoked by application of low pH solution (pH = 6) in the absence (Control) and presence of 100 μM 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 percent inhibitions (± SEM) of ASIC1a- mediated currents elicited by 100 μM DTG under control conditions (Control) or following preincubation in pertussis toxin (PTX), n = 7. Asterisk denotes significant difference from Control group (p < 0.05).
103
104
Figure 3.8 - Sigma-1 receptors couple to ASIC1a channels via an
AKAP150/calcineurin complex. 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 μM CBP (CBP). B, Representative traces of ASIC1a-mediated currents as a function of time recorded from a different cell in the absence (Control) and presence of 10 nM VIVIT (VIVIT) and during coapplication of VIVIT with 100 μM
CBP (VIVIT/CBP). C, Mean peak proton-gated current densities (± SEM) measured from neurons without (Control) or with 100 μM 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).
105
106
Discussion
Results from this study suggest the signaling cascade linking σ-1 receptors to ASIC1a channels in rat cortical neurons involves direct protein- protein interactions involving PTX-sensitive G proteins, calcineurin and
AKAP150. σ-1 receptors are shown to colocalize with both ASIC1a channels and the scaffolding protein AKAP150, 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 σ receptors affect ion channel function via mechanisms consistent with protein-protein interactions, σ-1 receptors modulate ASIC1a channels via a pertussis toxin-sensitive G protein-coupled signal transduction cascade. Furthermore, σ-1 receptors are also shown here to couple to ASIC1a channels via the second messenger calcineurin anchored to
AKAP150. This coupling results in decreases in both ASIC1a-mediated currents and concomitant elevations in cytosolic Ca2+ following σ-1 receptor activation.
σ 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 (CaMKII), has been shown to modulate
ASIC1a channels (Gao et al., 2005). Activation of NMDA receptors enhances
ASIC1a-mediated currents, which consequently exacerbates acidotoxicity during
ischemia (Gao et al., 2005). ASIC channels are also regulated in a similar
manner by C kinase-1 (PICK-1), which binds 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
107
by protein kinase C (PKCα) (Baron et al., 2002; Deval et al., 2004), while several
protein kinase C isoforms (PKCβI or PKCβII) have also been shown to inhibit
ASIC1 (Berdiev et al., 2002). Protein kinase A phosphorylation of ASIC interferes
with the binding of PICK-1 to these channels, and disrupts PICK1-ASIC1
colocalization (Leonard et al., 2003). Furthermore, various signaling molecules
have been linked to the regulation of ASIC. Three distinct kinases (CaMKII,
protein kinase C and protein kinase A) have all been shown to modulate the
function of ASIC, 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, inhibition of calcineurin has been shown to potentiate currents through ASIC1a and ASIC2a channels,
and thus dephosphorylation of the channels by this phosphatase may be involved in downregulation of ASIC (Chai et al., 2007). Our results with the
calcineurin inhibitors, cyclosporin A and FK-506, demonstrate that σ-1 receptor-
mediated block of ASIC1a is dependent on activation of this phosphatase.
Interestingly, reports have shown that calcineurin may be activated by a
pertussis toxin-sensitive G protein (Gromada et al., 2001). Consistent with this
observation, our study shows that a pertussis toxin-sensitive G protein is involved
in the signaling cascade coupling σ-1 receptors to ASIC1a. It has been proposed
that σ receptors regulate ion channel function via protein-protein interactions
108
(Aydar et al., 2002). Moreover, inhibition of G proteins either by cell dialysis with
GDP-β-S or preincubation in pertussis toxin failed to affect σ-1 and σ-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 σ receptor modulation of ion channels suggest that the effects involve a
membrane-delimited signaling pathway which likely involves a protein-protein
interaction. However, σ receptor activation has been shown to stimulate GTPase activity in mouse prefrontal membranes (Tokuyama et al., 1997). A pertussis toxin-sensitive G protein has been implicated in the modulation of NMDA receptors by σ receptors in rat CA3 dorsal hippocampus neurons (Monnet et al.,
1994), whereas a cholera toxin-sensitive G protein has been suggested to couple
σ-1 receptors to the channels mediating the A-current in frog pituitary melanotopes (Soriani et al., 1999). However, others have argued that σ receptors do not couple to G proteins (Hopf et al., 1996). Our findings demonstrate that σ-1
receptors can modulate ion channel function via activation of a PTX-sensitive G
protein, and these results are the first evidence 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 regulation of synaptic structure during
developmental synapse formation, in synaptic plasticity in learning and memory,
and neuronal dysfunction and cell death during pathophysiological conditions
(Dell'Acqua et al., 2006). Additionally, a calcineurin/AKAP150 complex has been
shown to modulate both ASIC1a and ASIC2a function (Chai et al., 2007). Our
109
immunohistochemical studies suggest that σ-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 similarity of the
distributions of σ receptors colocalized with ASIC1a channels and with AKAP150 is consistent with previous studies showing colocalization of ASIC channels and
AKAP (Chai et al., 2007). Disruption of the actin cytoskeleton 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 σ-1 receptor modulation of ASIC1a-
2+ induced [Ca ]i elevations. The small but significant block by NMDA of σ-1
receptor modulation of ASIC1a can probably be attributed to additional effects of
Ca2+ overload following preincubation in NMDA which led to increases in basal
calcium levels.
Previous results in our laboratory have shown that most of the elevations
2+ 2+ in [Ca ]i triggered by acidosis are the result of Ca channels (NMDA and AMPA
receptors, voltage-gated Ca2+ channels) opening downstream of ASIC1a
activation, and that activation of σ-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 (Rosenmund 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 σ-1 receptors functionally couple to ASIC1a
110
channels via AKAP150, which is consistent with all of these observations.
Moreover, these results also raise the possibility that AKAP150 is a component in
the modulation of other ion channels (voltage-gated Ca2+ channels) and
receptors (NMDA and AMPA) by σ-1 receptors.
The strongest evidence that σ-1 receptors couple to ASIC1a channels via
a calcineurin/AKAP150 complex comes from experiments using the VIVIT
peptide. Whole-cell patch clamp experiments with VIVIT suggest that when this
peptide competes with calcineurin for binding to the PxIxIT-like motif in AKAP150
and thus prevents calcineurin binding to AKAP150, σ receptor activation loses its
ability to regulate ASIC1a function. The disruption of this interaction prevents σ-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).
In conclusion, our data show that the signaling cascade between σ-1
receptors and ASIC1a channels involves a PTX-sensitive G protein and an
AKAP150/calcineurin complex in cortical neurons. This coupling results in
decreases in both ASIC1a-mediated membrane currents and concomitant
elevations in cytosolic Ca2+ following σ-1 receptor activation. Moreover,
calcineurin may be a potential component of the neuroprotective properties of σ
receptors. Furthermore, our results also raise the possibility of σ-1 receptors coupling to downstream Ca2+ channels (voltage-gated Ca2+ channels) and
receptors (NMDA and AMPA) via a similar signaling cascade. All of these
channels and receptors have been implicated in pathophysiological conditions,
111 such as stroke (Tanaka et al., 1997; Tanaka et al., 1999; Tanaka et al., 2002). σ-
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.
112
CHAPTER 4
SIGMA-1 RECEPTOR INHIBITION OF INTRACELLULAR Ca2+ DYSREGULATION DUE TO SYNERGISTIC INTERACTION BETWEEN ACIDOSIS AND ISCHEMIA
Introduction
Glucose and oxygen deprivation associated 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 region, activating Acid-Sensing Ion Channels
(ASIC) (Xiong et al., 2004; Gao et al., 2005; Pignataro et al., 2007). Synaptic vesicles contain not only the neurotransmitter glutamate but also protons that are released along with glutamate during neurotransmission. It remains to be elucidated if protons released during neurotransmission also activate ASIC.
ASIC subtypes are distinguishable by the pH of half-maximal activation,
Ca2+ permeability and tissue expression pattern. The predominant ASIC subtype in the central nervous system (CNS) contains the ASIC1a subunit, which can form homomultimeric or heteromultimeric 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
113 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 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 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 cerebral artery occlusion (MCAO) (Xiong et al.,
2004). Furthermore, pharmacological inhibition of ASIC1a with either the non- selective Na+ channel blocker amiloride or the homomultimeric ASIC1a selective inhibitor psalmotoxin1 (Diochot et al., 2007), diminishes ischemic brain injury
(Xiong et al., 2004). Thus, there is a direct correlation between infarct size, brain acidosis and ASIC activation, suggesting the 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 σ-1 receptors inhibit Ca2+ dysregulation evoked by ischemia (Katnik et al., 2006) as well as acidosis, and that activation of σ receptors is neuroprotective at delayed time points in a rat model of ischemic stroke (Ajmo et al., 2006). Moreover, σ-1 receptors regulate ionotropic glutamate receptors, voltage-gated K+ channels and voltage-gated Ca2+ channels (Hayashi
114
et al., 1995; Aydar et al., 2002; Zhang and Cuevas, 2002; Zhang and Cuevas,
2005). The inhibition of ionotropic glutamatergic receptors by σ receptors
2+ prevents elevations in [Ca ]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). Consistent with this observation, our
2+ laboratory has also shown that most of the elevations in [Ca ]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 σ-1 receptors effectively suppressed these secondary Ca2+ fluxes
by inhibiting both ASIC1a and these Ca2+ channels directly.
Previous reports have indicated that 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 whether 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 remain active for greater than 4 hours
following an ischemic insult (Pignataro et al., 2007). Pharmacological blockade of
ASIC1a by amiloride or PcTx1 administered even 5 hours after MCAO has been
shown to diminish stroke injury (Simon and Xiong, 2006; Pignataro et al., 2007).
115
These observations raise the possibility that σ receptors may provide
neuroprotection when stimulated several hours after an ischemic insult by directly
inhibiting ASIC1a channels as well as Ca2+ channels opening in response to
membrane depolarizations produced by ASIC1a activation.
Experiments were undertaken to determine whether ASIC1a activation
2+ and ischemia interact to produce potentiated elevations in [Ca ]i and to
determine if activation of σ-1 receptors is able to block this synergistic
potentiation. Ratiometric Ca2+ fluorometry experiments demonstrated that
acidification of the extracellular solution from pH 7.4 to 6.0 produced a
2+ potentiation of the ischemia-induced elevations in [Ca ]i. Inhibition of ASIC1a
channels with either amiloride or PcTx1, significantly decreased ischemia-
2+ induced elevations in [Ca ]i at pH values ranging from 7.4 to 6.0, suggesting that
homomeric ASIC1a channels are activated during ischemia alone and that these
2+ channels contribute to the pH dependence of these [Ca ]i increases.
Furthermore, inhibition of synaptic transmission with tetrodotoxin prevented
2+ ischemia-evoked increases in [Ca ]i but the effect of tetrodotoxin was overcome by acidification of the extracellular solution (pH 6.0). The selective σ-1 receptor agonist, carbetapentane, significantly decreased ischemia-mediated Ca2+ dysregulation at all pH values tested (7.4-6.0), suggesting that activation of σ-1
2+ receptors modulate ischemic- and ASIC1a-activated [Ca ]i increases individually
while also effecting the mechanism mediating the synergistic interaction between
these two initiators of Ca2+ influx pathways in cortical neurons.
116
Materials and Methods
Primary Rat Cortical Neuron Preparation
Primary cortical neurons from embryonic (E18) rats were cultured as
previously described by our laboratory (Katnik et al., 2006). All procedures were
done in accordance with the regulations of the University of South Florida
Institutional Animal Care and Use Committee. Cells were used after 10-21 days
in culture.
Calcium Imaging Measurements
The effects of acidosis and chemical ischemia on intracellular Ca2+
concentrations were examined in isolated 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 Neurobasal (Invitrogen) medium supplemented with B27 (Invitrogen) and 0.5 mM L-glutamine, 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 μM fura-2, acetoxymethylester (fura-2 AM) and 0.3 % dimethyl
sulfoxide. The coverslips were washed in PSS (fura-2-AM free) prior to
experiments being performed.
117
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 coverslips were transferred to a recording chamber and membrane currents were amplified, filtered at 1 kHz, digitized at 5 kHz, and acquired using Clampex 8 (Axon). 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. 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 protocols (Dr. Hongling Zhang, unpublished data, and (Mike et al., 2003)). Patch electrodes were pulled from thin-walled borosilicate glass (World Precision Instruments Inc., Sarasota, FL) using a Sutter
Instruments P-87 pipette puller (Novato, CA) and had resistances of 2.0 – 5.0
MΩ. Access resistances (Rs) were monitored throughout experiments for stable values ≤ 20 MΩ and were always compensated at 40% (lag, 10 µs). 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
118 applying PSS buffered to pH values of 7.4, 7.0, 6.5 and 6.0 (+/- drug). Chemical ischemia was induced by applying glucose-free PSS containing 4mM azide (+/- drug) and titrated to pH 7.4, 7.0, 6.5, and 6.0. Individual cells were exposed to ≤
3 ischemic or acidic+ischemic insults to prevent rundown of responses (Katnik et al., 2006). All chemicals used in this investigation were of analytical grade. The following drugs were used: carbetapentane (Tocris Bioscience, Ellisville, MO); psalmotoxin1 venom (Spider Pharm, Yarnelle, AZ); psalmotoxin1 peptide
(Peptide International, Louisville, KY); tetrodotoxin (Alomone Labs, Jerusalem,
Israel); amiloride (Alexis Biochemicals, Lausen, Switzerland); sodium-azide
(Sigma-Aldrich, St. Louis, MO); and fura-2 AM (Molecular Probes, Eugene, OR).
Data Analysis
Analysis of measured intracellular Ca2+ responses and Na+-current activation was performed using Clampfit 9 (Axon instruments). Imaging data files collected with SlideBook 4.02 (Intelligent 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 differences were determined using paired and unpaired t-tests for within group and between group experiments, respectively, and were considered significant if p < 0.05. For multiple group comparisons either a 1-way or a 2-way ANOVA, with or without repeat measures, were used, as appropriate.
When significant differences were determined with an ANOVA, post-hoc analysis was conducted using a Tukey Test to determine differences between individual groups.
119
Results
The low extracellular pH associated with ischemia can activate ASIC
(Xiong et al., 2004; Gao et al., 2005; Pignataro et al., 2007), and ASIC-mediated
currents are potentiated during ischemia (Xiong et al., 2004; Gao et al., 2005).
During neurotransmission, protons are released along with glutamate but it
remains to be elucidated whether these protons can activate ASIC at
physiological pH values. Experiments were carried out to determine if ASIC are
activated during ischemia at normal physiological pH (7.4) and to determine the
2+ effects of pathophysiological pH values (7.0 - 6.0) during ischemia on [Ca ]i
2+ dysregulation. Figures 4.1A and 4.1B show representative traces of [Ca ]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 blocker amiloride (100 µM). Similar experiments demonstrate that amiloride significantly inhibits ischemia-induced
2+ peak elevations in [Ca ]i by 48% in solutions buffered to pH 7.4 (Figure 4.1C).
These results indicate that protons released along with glutamate during
neurotransmission under physiological conditions activate ASIC. The application
2+ of ischemia + acidosis resulted in more robust peak increases in [Ca ]i and
these elevations were also inhibited by amiloride (Figure 4.1C). These data
suggest that ASIC are activated during ischemia and potentiate ischemia- induced Ca2+ dysregulation.
The kinetics of the Ca2+ responses to the combination of ischemia +
acidosis are noticeably different from those to ischemia or acidosis alone. To
compare the kinetics of these responses, a single cell was exposed to acidosis
120
(pH 6.0), ischemia at pH 7.4, and ischemia at pH 6.0. Figure 4.2A shows
2+ representative traces of [Ca ]i as a function of time from 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 + Ischemia) separated by 10 minute recovery
2+ periods. Acidosis alone produced an initial transient increase in [Ca ]i with a
rapid desensitization of the Ca2+ influx resulting in a return to basal levels within
seconds of the acid application. In contrast, ischemia alone resulted in smaller
2+ initial transient increase in [Ca ]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 produced a significant potentiation in the
2+ initial transient peak [Ca ]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
2+ rebound increase in [Ca ]i which then slowly decayed to baseline levels. To
quantitate the net elevations in the cytosolic Ca2+ concentration during these
2+ stimulations, increases in [Ca ]i were integrated over the time period surrounding the acidosis + ischemia challenge yielding the area under the curve.
2+ Figure 4.2B shows that there is a synergistic increase in [Ca ]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 potentiates the response to ischemia. This is
121
the first report showing the role of acidosis during ischemia and the effects of
2+ ASIC activation on ischemia-induced [Ca ]i elevations.
To better understand this synergistic interaction between acidosis and
2+ ischemia, experiments were performed to measure the [Ca ]i responses to ischemic insults as a function of pH. Four parameters were used to compare
2+ these responses: (1) magnitude of the initial [Ca ]i peak, (2) the steady state
2+ level of [Ca ]i at the end of the ischemia application, (3) the peak magnitude of
2+ the rebound transient [Ca ]i increase following washout of the ischemia solution, and (4) the net Ca2+ elevations measured in the cytosol as determined by
2+ integrating the area under the [Ca ]i vs. time curve. Results from multiple
2+ experiments show a significant increase in initial [Ca ]i peak (Figure 4.3A),
2+ rebound [Ca ]i peak (Figure 4.3C) and area (Figure 4.3D) as the pH of the ischemia solution was reduced from 7.4 to 6.0. Interestingly, the steady state
2+ level of [Ca ]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). These data suggest that ischemia-induced
2+ increases in [Ca ]i are pH dependent. Furthermore, acidosis significantly affects
2+ ischemia-induced [Ca ]i elevations such that greater synergy is observed as the
pH decreased from 7.4 to 6.0.
Psalmotoxin1 (PcTx1) from the 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 ASIC1a channels were mediating
2+ this synergistic potentiation of changes in [Ca ]i during ischemia at low pH
122
values, cells were preincubated in the absence (PSS, Control) and presence of
PcTx1 venom (PcTx1) in solutions buffered to the indicated pH values. The
venom was present in the conditioning solution (PSS pH 7.4) as well as all the
ischemia solutions (pH 7.4 - 6.0). PcTx1 venom inhibited the initial peak
2+ elevations in [Ca ]i to a statistically significant degree at all pH values tested
2+ (Figure 4.4A). The venom also blocked the rebound increases in [Ca ]i following
washout of the ischemia solution at pH values lower than 7.0 (Figure 4.4C).
Interestingly, at the lower pH values, the PcTx1 venom is unable to block
2+ 2+ elevated steady state levels of [Ca ]i or the net [Ca ]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 channel. In contrast, amiloride which directly blocks the
2+ channel, does inhibit steady state [Ca ]i elevations. These results show that
inhibition of ASIC1a channels prevents acidosis-associated synergistic
2+ potentiation of [Ca ]i dysregulation during ischemia.
As a negative control, PcTx1 venom was heat inactivated to denature all
2+ proteins. Figures 4.5A and 4.5B show representative traces of [Ca ]i as a
function of time recorded from a neuron during 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
2+ 2+ initial peak, steady state and rebound [Ca ]i, and net [Ca ]i (area) (± SEM) were
measured in response to ischemia at pH 7.0 and ischemia at pH 6.0 (Figure 4.5C
123
and 4.5D). These results show that heat inactivated PcTx1 venom do not
2+ significantly affect ischemia-induced [Ca ]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), Vanillotoxin 3 (VaTx3)) experiments were performed with pure PcTx1 peptide during acidosis alone (pH 6.0), ischemia at pH 7.0 and
2+ ischemia at pH 6.0. Figures 4.6A and 4.6B show representative traces of [Ca ]i as a function of time recorded from a neuron during ischemia at pH 7.0 and from a second neuron during ischemia + acidosis in the absence (Control) and presence of synthetic PcTx1 peptide (s.PcTx1). Similar to results from the venom experiments, PcTx1 peptide significantly inhibited changes in initial peak, steady
2+ 2+ state and rebound [Ca ]i, and net [Ca ]i (area) (± SEM) measured in response
to ischemia pH 7.0 (Figure 4.6C). Furthermore, the peptide inhibited initial and
2+ rebound [Ca ]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 channels are mediating
the synergistic potentiation seen during the combination of ischemia and
acidosis.
The synergistic interaction between ischemia and acidosis raises the
possibility that ASIC1a channels promote long-lived priming of presynaptic
vesicles, and could thus, enhance release of neurotransmitter if stimulated prior
to an ischemic event. To test this hypothesis, cells were exposed to acid alone
prior to the ischemia application. Figure 4.7A shows representative traces of
124
2+ [Ca ]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 activation alone does not promote long-lived
2+ vesicle priming since the mean changes in peak [Ca ]i were comparable (Figure
4.7B). These data suggest that 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 confirm that the synergistic potentiation in
Ca2+ observed is the result of the combination of ischemia + acidosis and that for
the potentiation to occur, the events must take place simultaneously. Consistent
with this observation, ischemia potentiates ASIC1a-mediated currents following
OGD preincubation (Xiong et al., 2004).
To further substantiate that the concurrent incidence of ischemia and
acidosis is required to produce synergistic potentiation of Ca2+ dysregulation, the temporal effects of acidosis on the ischemia responses were studied. Figure
2+ 4.7C shows representative traces of [Ca ]i as a function of time recorded from a
cell exposed to ischemia alone (black trace) and from a second neuron exposed to ischemia followed by the combination of ischemia + acidosis (gray trace).
Addition of protons resulted in a statistically significant increases in mean
2+ 2+ changes in [Ca ]i in peak, steady state, rebound peak, and net [Ca ]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,
125
these results indicate that ASIC1a channels can be activated again after the
initial ischemic onset, exacerbating ischemia-induced Ca2+ dysregulation.
Next we wanted to determine the mechanism responsible for the rebound
2+ increase in [Ca ]i following washout of ischemia and acidosis. Figure 4.7E
2+ shows representative traces of [Ca ]i as a function of time recorded from a neuron exposed to the combination of ischemia + acidosis (black trace) and from
a second cell exposed to the combination of ischemia + acidosis then rapidly
switched to ischemia (pH 7.4) alone (gray trace). In identical experiments,
populations of cells exposed to these two treatments did not exhibit significant
2+ differences comparing mean changes in peak, steady state and net [Ca ]i (area)
(Figure 4.7F). In contrast, mean changes in rebound were significantly different
(Figure 4.7F). Thus, these results suggest that the relief of proton block of downstream Ca2+ channels allows for the influx of Ca2+ contributing to the
2+ rebound [Ca ]i elevations.
σ 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 σ-1 receptor agonist carbetapentane (CBP) during ischemia at
the indicated pH values. CBP significantly inhibited the initial transient ischemia-
2+ induced [Ca ]i elevations (Figure 4.8A), consistent with previous results showing
σ-1 receptor inhibition of ASIC1a-activated Ca2+ increases. Similarly, CBP
significantly reduced steady state (Figure 4.8B) and rebound (Figure 4.8C) levels
2+ of [Ca ]i induced by ischemia at all pH values tested, consistent with σ-1
126
receptor inhibition of Ca2+ channels activated by ASIC1a-mediated
2+ depolarization. Likewise, CBP reduced the net [Ca ]i elevations (area), suggesting σ-1 receptor activation not only inhibit Ca2+ influx pathways but may
also modulate release from intracellular stores and/or extrusion pathways
(exchangers and ATPases) (Figure 4.8D). These results show that σ-1 receptor activation prevents the synergistic interaction between acidosis and ischemia.
Moreover, the σ-1 selective ligand inhibits ischemia-induced Ca2+ dysregulation
at all pH values (7.4 - 6.0).
Tetrodotoxin (TTX) is an inhibitor of voltage-gated Na+ channels, and thus, subsequent synaptic transmission. The activation of postsynaptic Ca2+ channels
and receptors following ASIC1a stimulation has been observed even when
neuronal conduction is inhibited by TTX, suggesting a presynaptic localization of
ASIC1a channels. Therefore, experiments were conducted to determine the
effects of presynaptic ASIC1a channels during ischemia in the presence of TTX.
2+ Figures 4.9A and 4.9B show representative traces of [Ca ]i as a function of time evoked by ischemia pH 7.4 (A) and ischemia pH 6.0 (B) in the absence (Control) and presence of tetrodotoxin (TTX, 500 nM). Similar to earlier reports, addition of
2+ TTX prevented ischemia-induced elevations in [Ca ]i by 69 ± 2% when
compared to control responses (Figure 4.9C and 4.9D) (Katnik et al., 2006).
2+ While in response to ischemia + acidosis, the effects of TTX on [Ca ]i
dysregulation were diminished, reducing only 9 ± 2% of the initial peak increase
2+ in [Ca ]i (Figure 4.9C and 4.9D). These data suggest that presynaptic ASIC1a
channels promote synaptic transmission during ischemia, and thus, overcome
127
2+ block of synaptic transmission and enhance postsynaptic [Ca ]i increases. This
observation is consistent with the hypothesis that ASIC1a channels are located presynapticly and regulate neurotransmitter release probability (Cho and
Askwith, 2008).
To confirm that the lack of TTX block of ischemia-induced increases in
2+ [Ca ]i by acidosis was not due to TTX being pH sensitive, whole-cell voltage-
clamp experiments were performed 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 stepping cells to -10 mV for 250 msec.
Figure 4.10A show representative current traces 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). Analysis of the measured current densities shows that TTX significantly inhibits Na+ currents at pH 7.4 and pH 6.0 (Figure 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 that presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission
2+ by TTX and enhancing postsynaptic [Ca ]i increases.
128
2+ Figure 4.1 - ASIC activation contributes to ischemia-evoked [Ca ]i increases in
2+ cultured rat cortical neurons. A, Representative traces of [Ca ]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 μM amiloride (Amiloride). B, Representative
2+ traces of [Ca ]i as a function of time recorded from a single neuron during
ischemia and acidosis (pH 6.0) in the absence (Control) and presence of 100 μM
2+ amiloride (Amiloride). C, Mean changes in peak [Ca ]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 μM 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).
129
130
Figure 4.2 - ASIC activation and ischemia interact to produce a synergistic
2+ 2+ potentiation of elevations in [Ca ]i. A, Representative traces of [Ca ]i as a
function of time recorded from a single cell during the indicated conditions. B,
2+ Quantification of net [Ca ]i elevations from multiple experiments identical to that
2+ 2+ in (A) (n = 84). Net [Ca ]i elevation is calculated by integrating Δ[Ca ]i over time.
Asterisks denote significant differences from the Acidosis group (p < 0.001), and
pound symbols indicates significant difference from Ischemia group (p < 0.001).
131
132
2+ Figure 4.3 - Protons potentiate ischemia-induced increases in [Ca ]i. Mean
2+ change in initial peak (A), steady state (B) and rebound peak (C) [Ca ]i, and net
2+ increase in [Ca ]i (area, D) (± SEM) measured in response 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 difference 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).
133
134
Figure 4.4 - Inhibition of homomeric ASIC1a channels decreases ischemia +
2+ acidosis-induced elevations in [Ca ]i at pH values ranging from 7.4 to 6.0. Mean
2+ change in initial peak (A), steady state (B) and rebound peak (C) [Ca ]i, and net
2+ [Ca ]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 PcTx1 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).
135
136
Figure 4.5 - Heat inactivated PcTx1 venom does not inhibit ischemia-induced
2+ 2+ increases in [Ca ]i. Representative traces of [Ca ]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 heat inactivated PcTx1 venom (h.PcTx1). Mean changes in initial peak (peak), steady
2+ 2+ state (SS) and rebound (Rbd) [Ca ]i, and net [Ca ]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 difference between any of the groups.
137
138
2+ Figure 4.6 - PcTx1 peptide inhibits ischemia-induced elevations in [Ca ]i.
2+ Representative traces of [Ca ]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).
2+ Mean changes in initial peak (peak), steady state (SS) and rebound (Rbd) [Ca ]i,
2+ and net [Ca ]i elevation (area) (± SEM) measured 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).
139
140
Figure 4.7 - Temporal effects of acidosis on ischemia-induced Ca2+
2+ dysregulation. A, Representative traces of [Ca ]i as a function of time recorded
from a cell during ischemia pH 7.4 (black 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, Mean changes in initial peak (peak), steady state (SS) and
2+ 2+ rebound (Rbd) peak [Ca ]i, (nM), and net [Ca ]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 calculated using the
two minute application of control protocol (A). Asterisks denote significant
2+ 2+ differences between the groups in peak [Ca ]i (p < 0.05), rebound [Ca ]i peak (p
2+ < 0.001) and area (p < 0.001). C, Representative traces of [Ca ]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
2+ + acidosis peaks (Peak), steady state (SS) and rebound (Rbd) [Ca ]i (nM), and
2+ net elevation in [Ca ]i (area, nM * min) (± SEM) 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
2+ between the groups (p < 0.001). E, Representative traces of [Ca ]i as a function
of time recorded from a neuron 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 initial peak (Peak), steady state
141
2+ 2+ (SS) and rebound (Rbd) [Ca ]i (nM), and net [Ca ]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 application of control protocol (D). Asterisks denote significant differences between the groups (p < 0.001).
142
143
Figure 4.8 - Sigma-1 receptor activation inhibits ischemia-mediated Ca2+ dysregulation at pH values ranging from 7.4 to 6.0. Mean changes in initial peak
2+ 2+ (A), steady state (B) and rebound peak (C) [Ca ]i, and net [Ca ]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 µM 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).
144
145
Figure 4.9 - Effects of synaptic transmission inhibition are overcome by
2+ presynaptic ASIC1a channels. A, Representative traces of [Ca ]i as a function of
time recorded from a neuron during ischemia (pH 7.4) in the absence (Control)
2+ and presence of 500 nM tetrodotoxin (TTX). B, Representative traces of [Ca ]i as a function of time recorded from a different cell during the combination of ischemia and acidosis (pH 6.0) in the absence (Control) and presence of 500 nM
2+ tetrodotoxin (TTX). C, Mean changes in peak [Ca ]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
2+ of changes in peak [Ca ]i by TTX measured during ischemia at pH 7.4 and pH
6.0. Asterisks denote significant differences from respective Control groups (p <
0.001).
146
147
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 between 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 difference between pH 7.4 and pH 6.0 groups,
(p = 0.683).
148
149
Discussion
The results from this study demonstrate that in rat cortical neurons,
acidosis and ischemia synergistically interact to produce potentiated elevations in
2+ [Ca ]i that are inhibited by σ-1 receptor activation. Acidification of the ischemia-
inducing extracellular solution to pathophysiological pH values (pH ≤ 7.0)
2+ produced elevations in [Ca ]i that were greater than the sum of the changes
evoked by acidosis and ischemia alone. Moreover, the kinetics of these
responses were significantly different. Inhibition of ASIC1a channels with either
2+ amiloride or PcTx1, significantly decreased ischemia-induced increases in [Ca ]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
2+ pH dependency of these [Ca ]i increases. While our data indicate that activation
of ASIC1a channels does not induce long-lived priming of synaptic vesicles for
release, channel activation does have a temporal effect on ischemia-mediated
2+ 2+ [Ca ]i increases after ischemia onset. Moreover, relief of proton block of Ca
2+ influx pathways mediates the rebound of [Ca ]i following washout of the acidic
ischemia solution. The selective σ-1 receptor agonist, carbetapentane,
decreased ischemia-mediated Ca2+ dysregulation at all pH values tested (7.4-
2+ 6.0). TTX was shown to block ~70% of the initial ischemia-induced [Ca ]i increases when the external solution was buffered to 7.4, but only produced
~10% block when the solution was acidified to pH 6.0, suggesting that presynaptic ASIC1a channels promote synaptic transmission which leads to
2+ postsynaptic [Ca ]i increases.
150
Previous studies have shown that ischemia and the acidosis that
2+ accompanies ischemia, produces marked increases in [Ca ]i (Katnik et al.,
2006), 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). ASIC1a-mediated currents in neurons have also 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 activation affects
the responses to ischemia and how acidosis and ischemia interact with each
2+ other to produce a synergistic dysregulation in [Ca ]i. Our in vitro model of
ischemia using the combination of azide in glucose-free PSS and acidosis (pH
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).
2+ Together, ischemia and acidosis produced elevations in [Ca ]i that were greater
than the sum of the changes evoked by acidosis and ischemia alone. There was
2+ also a noticeable difference in the kinetics of the observed [Ca ]i transients
resulting from acidosis, ischemia, and ischemia + acidosis. These differences
cannot be explained by ischemia potentiating ASIC currents because ASIC1a
channels desensitize within seconds. Thus, we conclude the effects seen
minutes after the initial insult (steady state, rebound and net Ca2+ elevations)
following ischemia + acidosis are the result of ASIC1a activation potentiating
2+ ischemia-mediated [Ca ]i increases. Therefore, ischemia and ASIC1a activation
2+ interact to produce potentiated elevations in [Ca ]i dysregulation.
151
The fact that ASIC1a inhibitors amiloride and PcTx1 reduce the ischemia-
2+ induced increases in [Ca ]i when the external solution was buffered to pH 7.4
suggests that the acidosis produced by metabolic inhibition is not responsible for
ASIC1a activation. Instead, it is reasoned that protons released from glutamate
containing synaptic vesicles during neurotransmission (DeVries, 2001; Traynelis
and Chesler, 2001) are the source of ASIC1a channel activation. Therefore,
there are two sources of protons associated with ischemia. First, an internal
source as glucose and oxygen deprivation initiates a metabolic switch to
anaerobic glycolysis to produce cellular energy leading to the accumulation of
lactic acid, the end product of anaerobic glycolysis. Second, protons released
along with glutamate during synaptic transmission. In our model, ischemia
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 promotes further synaptic
2+ 2+ transmission and greater Ca accumulation. These increases in [Ca ]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 stroke (Tombaugh and
Sapolsky, 1993; Murai et al., 1997; Mattson, 2000; Xiong et al., 2004;
Yermolaieva et al., 2004).
152
2+ The [Ca ]i responses to ischemia + acidosis were characterized by four
parameters, the initial transient peak amplitude, the steady state level just prior to
washout of the ischemia + acid solution, the transient rebound amplitude
2+ 2+ following washout and the net [Ca ]i elevations, calculated by integrating Δ[Ca ]i for the duration of the response. The strongest evidence that the initial peak in
2+ ischemia-induced [Ca ]i elevations reported here is 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
2+ 2+ [Ca ]i is a function of the activated Ca influx and efflux pathways. Interestingly,
2+ the steady state level of [Ca ]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 studies suggest most of the elevations in
2+ 2+ [Ca ]i triggered by acidosis are the result of Ca channels (NMDA and AMPA
receptors and voltage-gated Ca2+ channels) opening in response to ASIC1a
mediated membrane depolarization. These results suggest that acidosis results
in increased ASIC1a channel opening but the effects of pH are countered by
increased H+ block of other ion channels. The antioxidant MnTMPyP (25 nM) did
2+ not significantly affect the transient rebound increase in [Ca ]i, suggesting that
reactive oxygen species production do not account for the rebound mechanism.
2+ In contrast, the transient rebound increase in [Ca ]i observed during washout of
acidosis + ischemia solution is due to the removal of a block of Ca2+ influx
pathways by protons as the proton concentration is reduced to pH 7.4. Since this
2+ feature of the [Ca ]i response is not observed following applications of ischemia
153
at pH 7.4, it is unlikely that when observed at lower pH values it is due to the
washout of the ischemia solution. The transient rebound is expected to be more
robust at the lower pH values due to a greater proton block of these Ca2+ channels being relieved.
2+ 2+ Conditions which produce large net [Ca ]i elevations, or Ca overload,
2+ are likely to lead to apoptosis. Acidosis alone, while producing a large [Ca ]i increase, has a very small net Ca2+ elevation because the transient increase is
2+ short lived. Likewise, while ischemia produces a maintained elevated [Ca ]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 rebound increase and a slower decay back
2+ to baseline. All these contribute to produce net elevations in [Ca ]i which are
~400% times greater than that for acidosis or ischemia alone. This pronounced
Ca2+ overload would be more likely 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 voltage-gated channels (Na+, K+,
Ca2+) (Diochot et al., 2007). PcTx1 induces a shift in the steady-state desensitization of the channel. At normal pH (7.4) this shift puts ASIC1a channels in the inactive state (Chen et al., 2005). When studying ASIC1a
activation, it is sufficient to include the toxin only in the conditioning media
154
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 the low pH solutions. For this study,
however, the venom was included in the conditioning media as well as the test
solutions because the cellular response to ischemia lasted for the duration of the
ischemia application. PcTx1 venom blocks the initial ischemia + acidosis-
2+ mediated [Ca ]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
2+ [Ca ]i peak as well as disrupting the synergistic interaction between acidosis and
ischemia. Moreover, experiments performed with a synthetic PcTx1 peptide
provided similar results to those obtained with the venom. Therefore, ASIC1a
channels are mediating the synergistic potentiation seen during the combination
of ischemia and acidosis.
Similar to earlier reports, blockade of voltage-gated Na+ channels with
TTX and subsequent synaptic transmission inhibition, prevents ischemia-induced
2+ elevations in [Ca ]i at physiological pH (Katnik et al., 2006). Interestingly, with the more acidic conditions present during the combination of ischemia and
2+ acidosis at pH 6.0, the inhibition of [Ca ]i increases by TTX were overcome,
most likely by the additional activation of ASIC1a channels. This observation
suggests a presynaptic localization of ASIC1a, whereby activation of the channel by protons results in synaptic transmission and subsequent activation of postsynaptic receptors. Consistent with this theory, the activation of postsynaptic
155
Ca2+ channels and receptors following ASIC1a stimulation during acidosis alone
(pH 6.0) are observed even when neuronal conduction is inhibited by TTX. It has been proposed that ASIC1a activation may facilitate neurotransmission 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). Thus, action potential inhibition alone is not sufficient 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 transmission. In addition, these results also raise the possibility of ASIC1a activation resulting in enough Ca2+ influx to
promote synaptic transmission and glutamate release. Similarly, heteromeric
complexes of alpha 5 and/or alpha 7 subunits of nicotinic receptors have been
shown to conduct Ca2+ and thus, resulting in nicotine-induced presynaptic
facilitation (Girod et al., 1999).
σ 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. σ receptor activation has been shown to modulate the
function of multiple ion channels, several of which have been linked to neuronal
death following an ischemic insult. Studies have shown that the affinity of
carbetapentane for σ-1 receptors is >50-fold greater than for σ-2 receptors
(Rothman et al., 1991; Vilner and Bowen, 2000). The concentrations of CBP used in this study are consistent with CBP acting as a σ-1 receptor agonist
156
(Thurgur and Church, 1998). Moreover, similar concentrations of CBP have
2+ previously been shown to inhibit ischemia-evoked increases in [Ca ]i (Katnik et
2+ al., 2006) as well as ASIC1a-induced [Ca ]i increases and membrane currents.
Unlike the results seen with inhibition of ASIC1a by PcTx1 venom, CBP
activation of σ-1 receptors is pH insensitive and significantly inhibited the
2+ synergistic increases in [Ca ]i produced by ischemia and acidosis as measured
2+ by initial peak amplitude, steady state, rebound and net elevations of [Ca ]i.
2+ In conclusion, σ-1 receptors inhibit [Ca ]i increases following ASIC1a
2+ channel activation, ischemia-induced increases in [Ca ]i as well as the
synergistic potentiation during ischemia + acidosis. Because acidic conditions
exist during ischemic episodes, σ-1 receptors should be targeted for
development of treatments for stroke and other pathophysiological conditions
where ASIC1a channel activation are involved. σ-1 receptor mediated inhibition
of Ca2+ channels activated by membrane depolarizations produced by ASIC1a
channel activation or metabolic inhibition and the ASIC1a channels themselves,
may contribute to the enhanced neuronal survival observed following
administration of σ receptor agonists 24 hr post-stroke in rats (Ajmo et al., 2006).
For these reasons, σ-1 receptors should be targeted for therapeutic intervention during ischemia, expanding the therapeutic window of stroke treatment.
The finding that ASIC1a activation at any point during an ischemic insult
2+ results in synergistic [Ca ]i increases has significant physiological and
pathophysiological implications. Our data indicate that though ASIC1a channels
rapidly desensitize, they are able to be re-activated minutes into the ischemic
157
insult. This suggests that during pathophysiological 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 repetitive activations of ASIC1a channels
synergistically interact with ischemia to produce potentiated elevations in peak,
2+ 2+ steady state, and rebound [Ca ]i elevations and net [Ca ]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.
158
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 stroke, as a result of an
embolus and restriction of blood flow to the brain, is the most common type of
stroke accounting for over 80% of these cases. Thus far, only one drug (tissue
plasminogen activator, tPA) has been approved by the FDA for the treatment of
ischemia. This study has determined how σ-1 receptors modulate ASIC1a
channels during physiological and pathophysiological conditions, and also
suggest that this regulation could be one of the mechanisms of neuroprotection
by σ receptor activation. Thus, σ receptors could be a potential therapeutic target
for the treatment of stroke at delayed time points.
ASIC are regulated by various factors such as pH, membrane distention
and arachidonic acid, and therefore, function as signal integrators in the CNS
(Allen and Attwell, 2002; Lopez, 2002), but these factors elicit or potentiate ASIC-
mediated 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 (Gao et al., 2005). Similarly, ASIC are
159
also regulated by C kinase-1 (PICK-1), which binds 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
by protein kinase C (Baron et al., 2002; Deval 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 interferes with the binding of PICK-1 to these channels, and disrupts PICK1-ASIC1 colocalization (Leonard et al., 2003).
Three distinct kinases (CaMKII, protein kinase C and protein kinase A) have all
been shown to modulate the function of ASIC, 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 σ receptors depresses ASIC1a-mediated responses
and remains the only reported instance of receptor mediated downregulation of
ASIC activity.
The responses observed throughout these studies are specifically
mediated by ASIC1a channels and this conclusion is supported by the inhibition
produced by amiloride and the selective ASIC1a channel blocker, PcTx1 venom
as well as the PcTx1 peptide (Diochot et al., 2007). Furthermore, cultured cortical
neurons from embryonic mice deficient in the ASIC1a subunit fail to show
2+ increases in [Ca ]i or membrane currents at the same proton concentrations
160
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 response to low pH (Lingueglia et al.,
1997). In addition, neither homomeric ASIC2a nor heteromultimeric
ASIC1a/ASIC2a channels conduct Ca2+, and thus could not account for the
2+ changes in [Ca ]i observed (Yermolaieva et al., 2004). However, PcTx1 itself
may not be an ideal pharmacological agent for stroke patients because of its
size, stability and inability to cross the blood brain barrier (Xiong et al., 2008).
Therefore, the development of better compounds is undergoing for the treatment
of stroke.
The σ-1 receptor subtype modulates neuronal responses to ASIC1a
activation. Studies have shown that the affinity of carbetapentane for σ-1
receptors is >50-fold greater than for σ-2 receptors (Rothman et al., 1991; Vilner
and Bowen, 2000). The calculated IC50 for carbetapentane inhibition of ischemia-
2+ evoked increases in [Ca ]i via σ-1 receptor activation is 18.7 µM (Katnik et al.,
2006), which is comparable to the 13.8 μM IC50 for CBP inhibition of ASIC1a-
2+ induced [Ca ]i increases. Carbetapentane also inhibits epileptiform activity in rat
hippocampal slices via σ-1 receptors with an IC50 value of 38 µM (Thurgur and
Church, 1998). Similarly, other σ-1 agonists, dextromethorphan (IC50 = 22 μM)
and PRE-084 (IC50 = 13.7 μM), both of which have >100-fold greater affinities for
σ-1 than σ-2 receptors, blocked ASIC1a-mediated responses at concentrations
consistent with those reported in the literature. Dextromethorphan inhibits
spreading depression in rat neocortical brain slices with an IC50 ~ 30 μM
161
(Anderson and Andrew, 2002), whereas PRE-084 protects human retinal cells
against oxidative stress with an IC50 ~ 10 μM (Bucolo et al., 2006). The IC50 values determined here for carbetapentane, dextromethorphan and PRE-084 are in the low μM range and suggests that it is unlikely these agonists are affecting
ASIC1a activity via σ-2 receptors, since high μM to mM concentrations of these compounds are required to stimulate σ-2 receptors. Moreover, σ-2-selective agonists (ibogaine and PB28) failed to inhibit ASIC1a-mediated responses at concentrations consistent with σ-2 specific effects and in a metaphit-insensitive manner.
Pharmacological studies with the σ antagonists, metaphit and BD1063, confirm that σ-1 receptor activation modulates ASIC1a channels. Metaphit has been shown to bind irreversibly to σ-1 receptors with an IC50 value of 50 μM (Wu,
2003). Preincubation in metaphit blocks σ-1 receptor modulation of voltage-gated
K+ channels in intracardiac neurons and depression of ischemia-induced
2+ elevations in [Ca ]i in cortical neurons (Zhang and Cuevas, 2005; Katnik et al.,
2006). Preincubation of cortical neurons in 50 μM metaphit antagonized CBP inhibition of ASIC1a by ~ 40%. BD1063 has a higher affinity for σ-1 than σ-2 receptors and attenuates the dystonia produced by DTG in rats in a dose- dependent manner, suggesting this ligand acts as an antagonist at σ sites
(Matsumoto et al., 1995). CBP is unable to block acid-induced increases in
2+ [Ca ]i when co-applied with BD1063, suggesting the effects are mediated by σ-1
receptors. In addition, metaphit fails to inhibit the effects of the σ-2 agonist,
PB28, on ASIC1a-mediated responses. Taken together, these data show that
162
2+ increases in [Ca ]i in response to ASIC1a activation are modulated only by σ-1
receptors.
Inhibition of the second messenger calcineurin has been shown to
potentiate currents through ASIC1a and ASIC2a channels, and thus
dephosphorylation of the channels by this phosphatase may be involved in
downregulation of ASIC (Chai et al., 2007). The calcineurin inhibitors, cyclosporin
A and FK-506, demonstrate that σ-1 receptor-mediated block of ASIC1a is
dependent on activation of this phosphatase (Figure 5.1). Interestingly, reports
have shown that calcineurin may be activated by a pertussis toxin-sensitive G
protein (Gromada et al., 2001). Consistent with this observation, the effects of σ
2+ receptor activation on ASIC1a-mediated [Ca ]i increases are significantly
lessened in cortical neurons following preincubation in pertussis toxin, suggesting
a pertussis toxin-sensitive G protein is involved in the signaling cascade coupling
σ-1 receptors to ASIC1a-mediated responses.
This study demonstrates that σ-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 ASIC1a through this mechanism (Figure 5.1).
σ receptors regulate ion channel function via protein-protein interactions (Aydar
et al., 2002). Moreover, inhibition of G proteins either by cell dialysis with GDP-β-
S or preincubation in pertussis toxin failed to affect σ-1 and σ-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 σ receptor modulation of ion channels suggest that the effects involve a
163
membrane-delimited signaling pathway which could involve a protein-protein
interaction. However, σ receptor activation has been shown to stimulate GTPase activity in mouse prefrontal membranes (Tokuyama et al., 1997). A pertussis toxin-sensitive G protein has been implicated in the modulation of NMDA receptors by σ receptors in rat CA3 dorsal hippocampus neurons (Monnet et al.,
1994), whereas a cholera toxin-sensitive G protein has been suggested to couple
σ-1 receptors to the channels mediating the A-current in frog pituitary melanotopes (Soriani et al., 1999). However, others have argued that σ receptors do not couple to G proteins (Hopf et al., 1996).
164
Figure 5.1 - Signaling cascade linking ASIC1a channels and sigma-1 receptors.
Activation of σ-1 receptors inhibits ASIC1a-induced Ca2+ dysregulation via a
PTX-sensitive G protein and a calcineurin/AKAP complex, resulting in a decrease
2+ in [Ca ]i elevations. Sigma-1 receptors directly couple to ASIC1a channels via a
PTX-sensitive G protein and a calcineurin/AKAP complex.
165
166
AKAP150 is involved in the regulation of receptor activity and localization,
and in the regulation of synaptic structure during developmental synapse
formation, in synaptic plasticity in learning and memory, and neuronal
dysfunction and cell death during pathophysiological conditions (Dell'Acqua et al.,
2006). σ-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 calcineurin/AKAP150 complex has been shown to modulate both ASIC1a and ASIC2a function (Chai et al., 2007). Furthermore, the similarity of the distributions of σ receptors colocalized with ASIC1a channels and with AKAP150 is consistent with previous studies showing colocalization of ASIC channels and AKAP (Chai et al., 2007). Disruption of the actin cytoskeleton by chemical agents or NMDA receptor activation, results in the redistribution of
AKAP150 away from the plasma membrane (Gomez et al., 2002), and thus,
2+ preventing σ-1 receptor modulation of ASIC1a-induced [Ca ]i dysregulation
(Figure 5.1).
Depletion of Ca2+ from intracellular stores indicates that most, if not all, of
2+ the acid-induced increases in [Ca ]i is due to plasma membrane influx. Multiple
ion channels downstream of ASIC1a activation were shown to contribute to
2+ acidosis-induced elevations in [Ca ]i, including NMDA and AMPA/kainate
receptors and VGCC. σ 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 the presence of specific
inhibitors of ionotropic glutamate receptors, activation of σ-1 receptors with CBP
167
2+ further decreased proton-evoked increases in [Ca ]i, but the effects of CBP and
the glutamate channel inhibitors were less than additive. Thus, σ-1 receptors also
inhibit Ca2+ entry via NMDA and AMPA/kainate receptors directly by inhibiting these channels and indirectly by depressing ASIC1a activation. Application of the
L-type VGCC inhibitor, nifedipine, and the broad-spectrum Ca2+ channel inhibitor,
2+ cadmium, blocked ASIC1a-induced increases in [Ca ]i by >70% and >90%,
2+ respectively. This observation indicates that most of the increases in [Ca ]i
produced upon ASIC1a activation is dependent on Ca2+ influx through VGCC.
Co-application of CBP with nifedipine, but not with Cd2+, resulted in further
2+ reduction in the proton-evoked increases in [Ca ]i.
The activation of NMDA and AMPA/kainate receptors following ASIC1a
stimulation was observed even when neuronal conduction was inhibited with
tetrodotoxin. This observation suggests a presynaptic localization of ASIC1a,
whereby activation of the channel by protons results in synaptic transmission and
subsequent activation of postsynaptic glutamatergic 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 during 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
2+ et al., 2007). Since most of the elevations in [Ca ]i triggered by acidosis are the
result of Ca2+ channels opening downstream of ASIC1a activation, and
168
stimulation of σ-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 σ-1 receptors (Figure 5.1).
Simultaneous Ca2+ fluorometry and whole-cell patch clamp recordings
confirmed that Ca2+ influx through ASIC1a channels itself contributed only a
2+ small fraction to the total observed [Ca ]i increases. Cells voltage-clamped at
-70 mV, which prevents NMDA receptor and VGCC activation, demonstrated
2+ 2+ minimal acid-evoked elevations in [Ca ]i. Thus, the increases in [Ca ]i evoked
by ASIC1a activation are the result of synaptic transmission and subsequent opening of multiple Ca2+ channels, and that stimulation of σ-1 receptors
downregulates all of these events. However, the fact that activation of σ-1 receptors depresses ASIC1a-mediated currents in cells voltage-clamped at
-70 mV indicates that σ-1 receptors are functionally coupled to ASIC1a, and that
2+ the depression in acid-evoked increases in [Ca ]i is not exclusively the result of
σ-1 receptors blocking channels downstream of ASIC1a.
The effects of σ receptor activation on ASIC1a-mediated membrane currents are significantly lessened in cortical neurons following preincubation in pertussis toxin. These results suggest a pertussis toxin-sensitive G protein is involved in the signaling cascade coupling σ receptors to ASIC1a channels
(Figure 5.1). Moreover, σ-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
169
that when this peptide competes with calcineurin for binding to the PxIxIT-like
motif in AKAP150, and thus prevents calcineurin binding to AKAP150, σ receptor
activation loses its ability to regulate ASIC1a function. Thus, the disruption of this
interaction prevents σ-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 being 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 (Xiong et al., 2004; Xiong et al., 2006). The
2+ increases in [Ca ]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 neuroprotection 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 established that PcTx1 inhibits ASIC1a via
chronic desensitization of the channel (Chen et al., 2005) and also diminishes the
170
effects of NMDA-induced cell death (Xiong 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, cytoplasmic calcium dysregulation, and
neuronal death (Gao et al., 2005).
Neurons within the penumbra region remain viable for hours after the
ischemic insult but are subject to apoptosis if perfusion is not re-established.
Previous studies have shown that ischemia and the acidosis that accompanies
2+ ischemia, produces marked increases in [Ca ]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). ASIC1a-
mediated currents in neurons have also 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 activation affects the responses to ischemia
and how acidosis and ischemia interact with each other to produce a synergistic
2+ dysregulation in [Ca ]i. Our in vitro model of ischemia using the combination of
azide in glucose-free PSS and acidosis (pH 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
2+ produced elevations in [Ca ]i that were greater than the sum of the changes
evoked by acidosis and ischemia alone. There was also a noticeable difference
171
2+ in the kinetics of the observed [Ca ]i transients resulting from acidosis, ischemia,
and ischemia + acidosis. These differences cannot be explained by ischemia
potentiating ASIC currents because ASIC1a channels desensitize within
seconds. Thus, we conclude the effects seen minutes after the initial insult
(steady state, rebound and net Ca2+ elevations) following ischemia + acidosis
2+ are the result of ASIC1a activation potentiating ischemia-mediated [Ca ]i increases. Therefore, ischemia and ASIC1a activation interact to produce
2+ potentiated elevations in [Ca ]i dysregulation.
The fact that ASIC1a inhibitors amiloride and PcTx1 reduce the ischemia-
2+ induced increases in [Ca ]i when the external solution was buffered to pH 7.4
suggests that the acidosis produced by metabolic inhibition is not responsible for
ASIC1a activation. Instead, it is reasoned that protons released from glutamate
containing synaptic vesicles during neurotransmission (DeVries, 2001; Traynelis
and Chesler, 2001) are the source of ASIC1a channel activation. Therefore,
there are two sources of protons associated with ischemia. First, an internal
source as glucose and oxygen deprivation initiates a metabolic switch to
anaerobic glycolysis to produce cellular energy leading to the accumulation of
lactic acid, the end product of anaerobic glycolysis. Second, protons released
along with glutamate during synaptic transmission. 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 promotes further synaptic transmission and
172
2+ 2+ greater Ca accumulation. These increases in [Ca ]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 stroke (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 ischemia and acidosis observed in these neurons. The strongest evidence that the initial peaks associated with ischemia-
2+ induced [Ca ]i elevations reported here are due to activation of homomeric
ASIC1a channels is the inhibition produced by both amiloride and the selective
2+ ASIC1a channel blocker PcTx1 (Diochot et al., 2007). The steady state [Ca ]i is a function of the activated Ca2+ influx and efflux pathways. Interestingly, the
2+ steady state level of [Ca ]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 studies suggest most of the elevations in
2+ 2+ [Ca ]i triggered by acidosis are the result of Ca channels (NMDA and AMPA
receptors and voltage-gated Ca2+ channels) opening in response to ASIC1a
mediated membrane depolarization. These results suggest that acidosis results
in increased ASIC1a channel opening but the effects of pH are countered by
increased H+ block of other ion channels.
2+ The transient rebound increase in [Ca ]i observed during washout of the
acidosis + ischemia solution is most likely due to the removal of a block of Ca2+
173
influx pathways by protons as the proton concentration is reduced to pH 7.4.
2+ Since this feature of the [Ca ]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 observations in this study, the transient
rebound is expected to be more robust at the lower pH values due to the relief of
a greater proton block of these Ca2+ channels.
2+ 2+ Conditions which produce large net [Ca ]i elevations, or Ca overload,
2+ are likely to lead to apoptosis. Acidosis alone, while producing a large [Ca ]i increase, has a very small net Ca2+ elevation because the transient increase is
2+ short lived. Likewise, while ischemia produces a maintained elevated [Ca ]i level in the presence of ischemia, 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 rebound increase and a slower decay back to baseline. All
2+ these contribute to produce net elevations in [Ca ]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
2+ transmission inhibition, prevents ischemia-induced elevations in [Ca ]i at
physiological pH values (Katnik et al., 2006). Interestingly, during the
2+ combination of ischemia and acidosis at pH 6.0, the inhibition of [Ca ]i increases
174
by TTX were overcome, most likely by the additional activation of ASIC1a
channels. This observation suggests a presynaptic localization of ASIC1a,
whereby activation of the channel by protons results in synaptic transmission and
subsequent activation of postsynaptic receptors. Consistent with this theory, the
activation of postsynaptic Ca2+ channels and receptors following ASIC1a
stimulation during acidosis alone (pH 6.0) are observed even when neuronal
conduction is inhibited by TTX. It has been proposed that ASIC1a activation may
facilitate neurotransmission 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). Thus, action potential inhibition alone is not sufficient 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
transmission. Thus, even in the absence of glutamate release, ASIC1a would
depolarize the cell and subsequently, activate voltage-gated Ca2+ channels to
promote neurotransmission. In addition, these results also raise the possibility of
ASIC1a activation resulting in enough Ca2+ influx to promote synaptic
transmission and glutamate release. Similarly, heteromeric complexes of alpha 5
and/or alpha 7 subunits of nicotinic receptors have been shown to conduct Ca2+ and thus, resulting in acetylcholine-induced presynaptic facilitation (Girod et al.,
1999).
175
The σ-1 selective ligand, CBP, has previously been shown to inhibit
2+ ischemia-evoked increases in [Ca ]i (Katnik et al., 2006) as well as ASIC1a-
2+ induced [Ca ]i increases and membrane currents. Unlike the results seen with
inhibition of ASIC1a with PcTx1 venom, CBP activation of σ-1 receptors is pH
2+ insensitive and significantly inhibited the synergistic increases in [Ca ]i produced
by ischemia and acidosis as measured by initial peak amplitude, steady state,
2+ rebound and net elevations of [Ca ]i.
Overall Significance
The finding that σ-1 receptors can inhibit ASIC1a 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 direct 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). Furthermore, the expression levels of
ASIC1a have direct effects on the density of dendritic spines in hippocampal neurons (Zha et al., 2006). Thus, σ-1 receptors may influence cell-to-cell
signaling in the CNS by affecting ASIC1a activity. One of the consequences of
ASIC1a overexpression in mice is enhanced fear conditioning (Wemmie et al.,
2004), whereas stimulation of σ-1 receptors is known to ameliorate conditioned
fear stress (Kamei et al., 1997). These observations, coupled with our current
report, suggest that σ-1 receptor activation may produce anxiolytic effects via the inhibition of ASIC1a channels.
176
Figure 5.2 - Presynaptic ASIC1a channels under physiological conditions.
Neurotransmitter release leads to acidification of the synaptic cleft, which will lead to decrease neuroexcitability due to the inhibitory effects of protons in the postsynaptic channels. ASIC1a provide a positive feedback by increasing intracellular calcium following activation leading to increased neuroexcitability, and thus, preserving synaptic transmission.
177
178
The finding that σ-1 receptors can inhibit ASIC1a channels also has
significant pathophysiological implications σ-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 membrane currents and concomitant elevations in cytosolic Ca2+ following σ-1 receptor activation. Furthermore, these results also
raise the possibility of σ-1 receptors coupling to downstream Ca2+ channels
(voltage-gated Ca2+ channels) and receptors (NMDA and AMPA) via a similar
signaling cascade. All of these channels and receptors have been implicated in
pathophysiological conditions. Moreover, calcineurin may be a potential
component of the neuroprotective properties of σ receptors. σ-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 σ-1 receptor modulation of ASIC1a
channels during pathophysiological conditions was the finding that ASIC1a
2+ activation at any point during an ischemic insult results in synergistic [Ca ]i increases which are σ receptor sensitive (Figure 5.3). First, ASIC1a channels are activated by ischemia alone (pH 7.4), which 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 pathophysiological 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 refractory period. Consistent with this conclusion is the fact that lactate remains elevated and has been shown to
179
Figure 5.3 - Role of presynaptic ASIC1a channels during pathological conditions.
The combination of ischemia and acidosis interact to produce greater glutamate
2+ release leading to a synergistic potentiation of postsynaptic [Ca ]i elevations, resulting in Ca2+ overload, excitotoxicity and eventually cell death. Activation of
σ-1 receptors prevents the synergistic interaction between ischemia and acidosis, and thus, Ca2+ overload.
180
181
rebound days after the initial ischemic event (Weinstein et al., 2004; Munoz
Maniega et al., 2008). Even more dramatic though, was the observation that
under ischemic conditions these repetitive activations of ASIC1a channels
synergistically interact with ischemia to produce potentiated elevations in peak,
2+ 2+ steady state, rebound and net Ca elevations in [Ca ]i. These results suggest
that in order to prevent the consequences of Ca2+ overload during ischemia it is
critical to control ASIC1a activation. Equally important is the finding that all phases of these cellular responses are modulated by σ receptor 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 (Figure 5.3). The finding that ASIC1a activation interacts
with ischemia to produce synergistic potentiation in Ca2+ dysregulation is a novel
finding and suggests that this channel and σ 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 σ-1 receptors is a potentially important
component of the neuroprotective properties of σ receptors, since activation of
ASIC1a has been shown to contribute to stroke injury (Xiong et al., 2004).
2+ Moreover, σ-1 receptors inhibit [Ca ]i increases following ASIC1a channel
2+ activation, ischemia-induced increases in [Ca ]i as well as the synergistic potentiation of these increases observed during ischemia + acidosis insults
(Figure 5.3). Importantly, inhibition of ASIC1a has been shown to be neuroprotective at delayed time points following ischemic stroke (Simon, 2006;
182
Pignataro et al., 2007). Because acidic conditions exist during ischemic
episodes, σ-1 receptors should be targeted for development of treatments for
stroke and other pathophysiological conditions where ASIC1a channel activation
are involved. σ-1 receptor mediated inhibition of ASIC1a channels and Ca2+ channels (NMDA and AMPA/kainate receptors and VGCC) activated by membrane depolarizations produced by ASIC1a channel activation or metabolic inhibition and the ASIC1a channels themselves, may contribute to the enhanced neuronal survival observed following administration of σ 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. Consistent with this pleiotropic effect of σ receptor activation on neurons, is our observation that σ-1 receptor activation
+ 2+ suppresses extracellular high K -induced increases in [Ca ]i, which would also
activate these downstream effectors. For these reasons, σ-1 receptors can be targeted for therapeutic intervention in pathophysiological conditions involving
ASIC1a activation (like ischemia) and expand the therapeutic window of stroke treatment.
183
LIST OF REFERENCES
Ajmo CT, Jr., Vernon DO, Collier L, Pennypacker KR and Cuevas J (2006) Sigma receptor activation reduces infarct size at 24 hours after permanent middle cerebral artery occlusion in rats. Curr Neurovasc Res 3:89-98.
Akopian AN, Chen CC, Ding Y, Cesare P and Wood JN (2000) A new member of the acid-sensing ion channel family. Neuroreport 11:2217-2222.
Allen NJ and Attwell D (2002) Modulation of ASIC channels in rat cerebellar Purkinje neurons by ischaemia-related signals. J Physiol 543:521-529.
Allison DW, Gelfand VI, Spector I and Craig AM (1998) Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. J Neurosci 18:2423-2436.
Alonso G, Phan V, Guillemain I, Saunier M, Legrand A, Anoal M and Maurice T (2000) Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system. Neuroscience 97:155-170.
Alvarez de la Rosa D, Canessa CM, Fyfe GK and Zhang P (2000) Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol 62:573-594.
Alvarez de la Rosa D, Krueger SR, Kolar A, Shao D, Fitzsimonds RM and Canessa CM (2003) Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system. J Physiol 546:77-87.
Anderson TR and Andrew RD (2002) Spreading depression: imaging and blockade in the rat neocortical brain slice. J Neurophysiol 88:2713-2725.
Andrey F, Tsintsadze T, Volkova T, Lozovaya N and Krishtal O (2005) Acid sensing ionic channels: modulation by redox reagents. Biochim Biophys Acta 1745:1-6.
Askwith CC, Wemmie JA, Price MP, Rokhlina T and Welsh MJ (2004) Acid- sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons. J Biol Chem 279:18296-18305.
184
Aydar E, Palmer CP, Klyachko VA and Jackson MB (2002) The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. Neuron 34:399- 410.
Back T, Hoehn M, Mies G, Busch E, Schmitz B, Kohno K and Hossmann KA (2000) Penumbral tissue alkalosis in focal cerebral ischemia: relationship to energy metabolism, blood flow, and steady potential. Ann Neurol 47:485-492.
Bal-Price A, Moneer Z and Brown GC (2002) Nitric oxide induces rapid, calcium- dependent release of vesicular glutamate and ATP from cultured rat astrocytes. Glia 40:312-323.
Baron A, Deval E, Salinas M, Lingueglia E, Voilley N and Lazdunski M (2002) Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1. J Biol Chem 277:50463-50468.
Baron A, Schaefer L, Lingueglia E, Champigny G and Lazdunski M (2001) Zn2+ and H+ are coactivators of acid-sensing ion channels. J Biol Chem 276:35361-35367.
Bassler EL, Ngo-Anh TJ, Geisler HS, Ruppersberg JP and Grunder S (2001) Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b. J Biol Chem 276:33782-33787.
Beene DL and Scott JD (2007) A-kinase anchoring proteins take shape. Curr Opin Cell Biol 19:192-198.
Benos DJ and Stanton BA (1999) Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol 520 Pt 3:631-644.
Benson CJ, Eckert SP and McCleskey EW (1999) Acid-evoked currents in cardiac sensory neurons: A possible mediator of myocardial ischemic sensation. Circ Res 84:921-928.
Berardi F, Ferorelli S, Abate C, Colabufo NA, Contino M, Perrone R and Tortorella V (2004) 4-(tetralin-1-yl)- and 4-(naphthalen-1-yl)alkyl derivatives of 1-cyclohexylpiperazine as sigma receptor ligands with agonist sigma2 activity. J Med Chem 47:2308-2317.
Berdiev BK, Xia J, Jovov B, Markert JM, Mapstone TB, Gillespie GY, Fuller CM, Bubien JK and Benos DJ (2002) Protein kinase C isoform antagonism controls BNaC2 (ASIC1) function. J Biol Chem 277:45734-45740.
185
Bourrie B, Benoit JM, Derocq JM, Esclangon M, Thomas C, Le Fur G and Casellas P (1996) A sigma ligand, SR 31747A, potently modulates Staphylococcal enterotoxin B-induced cytokine production in mice. Immunology 88:389-393.
Bourrie B, Bouaboula M, Benoit JM, Derocq JM, Esclangon M, Le Fur G and Casellas P (1995) Enhancement of endotoxin-induced interleukin-10 production by SR 31747A, a sigma ligand. Eur J Immunol 25:2882-2887.
Bourrie B, Bribes E, De Nys N, Esclangon M, Garcia L, Galiegue S, Lair P, Paul R, Thomas C, Vernieres JC and Casellas P (2002) SSR125329A, a high affinity sigma receptor ligand with potent anti-inflammatory properties. Eur J Pharmacol 456:123-131.
Bowen WD (2000) Sigma receptors: recent advances and new clinical potentials. Pharm Acta Helv 74:211-218.
Brady KT, Balster RL and May EL (1982) Stereoisomers of N-allylnormetazocine: phencyclidine-like behavioral effects in squirrel monkeys and rats. Science 215:178-180.
Bucolo C, Drago F, Lin LR and Reddy VN (2006) Sigma receptor ligands protect human retinal cells against oxidative stress. Neuroreport 17:287-291.
Casellas P, Galiegue S, Bourrie B, Ferrini JB, Jbilo O and Vidal H (2004) SR31747A: a peripheral sigma ligand with potent antitumor activities. Anticancer Drugs 15:113-118.
Cassano G, Gasparre G, Contino M, Niso M, Berardi F, Perrone R and Colabufo NA (2006) The sigma-2 receptor agonist PB28 inhibits calcium release from the endoplasmic reticulum of SK-N-SH neuroblastoma cells. Cell Calcium 40:23-28.
Chai S, Li M, Lan J, Xiong ZG, Saugstad JA and Simon RP (2007) A kinase- anchoring protein 150 and calcineurin are involved in regulation of acid sensing ion channels ASIC1a and ASIC2a. J Biol Chem.
Chen CC, England S, Akopian AN and Wood JN (1998) A sensory neuron- specific, proton-gated ion channel. Proc Natl Acad Sci U S A 95:10240- 10245.
Chen X, Kalbacher H and Grunder S (2005) The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity. J Gen Physiol 126:71-79.
186
Chen X, Kalbacher H and Grunder S (2006) Interaction of acid-sensing ion channel (ASIC) 1 with the tarantula toxin psalmotoxin 1 is state dependent. J Gen Physiol 127:267-276.
Chew LJ, Takanohashi A and Bell M (2006) Microglia and inflammation: impact on developmental brain injuries. Ment Retard Dev Disabil Res Rev 12:105-112.
Cho JH and Askwith CC (2008) Presynaptic release probability is increased in hippocampal neurons from ASIC1 knockout mice. J Neurophysiol 99:426- 441.
Chu XP, Close N, Saugstad JA and Xiong ZG (2006) ASIC1a-specific modulation of acid-sensing ion channels in mouse cortical neurons by redox reagents. J Neurosci 26:5329-5339.
Chu XP, Miesch J, Johnson M, Root L, Zhu XM, Chen D, Simon RP and Xiong ZG (2002) Proton-gated channels in PC12 cells. J Neurophysiol 87:2555- 2561.
Chu XP, Wemmie JA, Wang WZ, Zhu XM, Saugstad JA, Price MP, Simon RP and Xiong ZG (2004) Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J Neurosci 24:8678-8689.
Colledge M, Dean RA, Scott GK, Langeberg LK, Huganir RL and Scott JD (2000) Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron 27:107-119.
Connor MA and Chavkin C (1992) Ionic zinc may function as an endogenous ligand for the haloperidol-sensitive sigma 2 receptor in rat brain. Mol Pharmacol 42:471-479.
Couture S and Debonnel G (1998) Modulation of the neuronal response to N- methyl-D-aspartate by selective sigma2 ligands. Synapse 29:62-71.
Cuevas J and Berg DK (1998) Mammalian nicotinic receptors with alpha7 subunits that slowly desensitize and rapidly recover from alpha- bungarotoxin blockade. J Neurosci 18:10335-10344.
Dawson LA, Galandak J and Djali S (2002) Attenuation of ischemic efflux of endogenous amino acids by the novel 5-HT(1A)/5-HT(2) receptor ligand adatanserin. Neurochem Int 40:203-209.
Dawson VL, Dawson TM, London ED, Bredt DS and Snyder SH (1991) Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci U S A 88:6368-6371.
187
DeHaven WI and Cuevas J (2004) VPAC receptor modulation of neuroexcitability in intracardiac neurons: dependence on intracellular calcium mobilization and synergistic enhancement by PAC1 receptor activation. J Biol Chem 279:40609-40621.
Dell'Acqua ML, Faux MC, Thorburn J, Thorburn A and Scott JD (1998) Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate. Embo J 17:2246-2260.
Dell'Acqua ML, Smith KE, Gorski JA, Horne EA, Gibson ES and Gomez LL (2006) Regulation of neuronal PKA signaling through AKAP targeting dynamics. Eur J Cell Biol 85:627-633. della Puppa A and London ED (1989) Cerebral metabolic effects of sigma ligands in the rat. Brain Res 505:283-290.
Deval E, Salinas M, Baron A, Lingueglia E and Lazdunski M (2004) ASIC2b- dependent regulation of ASIC3, an essential acid-sensing ion channel subunit in sensory neurons via the partner protein PICK-1. J Biol Chem 279:19531-19539.
DeVries SH (2001) Exocytosed protons feedback to suppress the Ca2+ current in mammalian cone photoreceptors. Neuron 32:1107-1117.
Diochot S, Salinas M, Baron A, Escoubas P and Lazdunski M (2007) Peptides inhibitors of acid-sensing ion channels. Toxicon 49:271-284.
Dirnagl U, Iadecola C and Moskowitz MA (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391-397.
Diviani D and Scott JD (2001) AKAP signaling complexes at the cytoskeleton. J Cell Sci 114:1431-1437.
Dodge K and Scott JD (2000) AKAP79 and the evolution of the AKAP model. FEBS Lett 476:58-61.
Dodge KL and Scott JD (2003) Calcineurin anchoring and cell signaling. Biochem Biophys Res Commun 311:1111-1115.
Duggan A, Garcia-Anoveros J and Corey DP (2002) The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J Biol Chem 277:5203-5208.
Dumont FJ (2000) FK506, an immunosuppressant targeting calcineurin function. Curr Med Chem 7:731-748.
188
Elsinga PH, Tsukada H, Harada N, Kakiuchi T, Kawamura K, Vaalburg W, Kimura Y, Kobayashi T and Ishiwata K (2004) Evaluation of [18F]fluorinated sigma receptor ligands in the conscious monkey brain. Synapse 52:29-37.
Escoubas P, De Weille JR, Lecoq A, Diochot S, Waldmann R, Champigny G, Moinier D, Menez A and Lazdunski M (2000) Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels. J Biol Chem 275:25116- 25121.
Ettaiche M, Guy N, Hofman P, Lazdunski M and Waldmann R (2004) Acid- sensing ion channel 2 is important for retinal function and protects against light-induced retinal degeneration. J Neurosci 24:1005-1012.
Feliciello A, Gottesman ME and Avvedimento EV (2001) The biological functions of A-kinase anchor proteins. J Mol Biol 308:99-114.
Fletcher EJ, Church J, Abdel-Hamid K and MacDonald JF (1995) Blockade by sigma site ligands of N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurones. Br J Pharmacol 116:2791-2800.
Fukui O, Kinugasa Y, Fukuda A, Fukuda H, Tskitishvili E, Hayashi S, Song M, Kanagawa T, Hosono T, Shimoya K and Murata Y (2006) Post-ischemic hypothermia reduced IL-18 expression and suppressed microglial activation in the immature brain. Brain Res 1121:35-45.
Gao J, Duan B, Wang DG, Deng XH, Zhang GY, Xu L and Xu TL (2005) Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death. Neuron 48:635-646.
Gao J, Wu LJ, Xu L and Xu TL (2004) Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons. Brain Res 1017:197-207.
Garcia-Anoveros J, Derfler B, Neville-Golden J, Hyman BT and Corey DP (1997) BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels. Proc Natl Acad Sci U S A 94:1459-1464.
Girod R, Crabtree G, Ernstrom G, Ramirez-Latorre J, McGehee D, Turner J and Role L (1999) Heteromeric complexes of alpha 5 and/or alpha 7 subunits. Effects of calcium and potential role in nicotine-induced presynaptic facilitation. Ann N Y Acad Sci 868:578-590.
189
Goldberg MP and Choi DW (1993) Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury. J Neurosci 13:3510-3524.
Gomez LL, Alam S, Smith KE, Horne E and Dell'Acqua ML (2002) Regulation of A-kinase anchoring protein 79/150-cAMP-dependent protein kinase postsynaptic targeting by NMDA receptor activation of calcineurin and remodeling of dendritic actin. J Neurosci 22:7027-7044.
Gonzalez-Alvear GM and Werling LL (1995) sigma1 Receptors in rat striatum regulate NMDA-stimulated [3H]dopamine release via a presynaptic mechanism. Eur J Pharmacol 294:713-719.
Goyagi T, Goto S, Bhardwaj A, Dawson VL, Hurn PD and Kirsch JR (2001) Neuroprotective effect of sigma(1)-receptor ligand 4-phenyl-1-(4- phenylbutyl) piperidine (PPBP) is linked to reduced neuronal nitric oxide production. Stroke 32:1613-1620.
Gromada J, Hoy M, Buschard K, Salehi A and Rorsman P (2001) Somatostatin inhibits exocytosis in rat pancreatic alpha-cells by G(i2)-dependent activation of calcineurin and depriming of secretory granules. J Physiol 535:519-532.
Grunder S, Geissler HS, Bassler EL and Ruppersberg JP (2000) A new member of acid-sensing ion channels from pituitary gland. Neuroreport 11:1607- 1611.
Hall AA, Herrera Y, Ajmo CT, Jr., Cuevas J and Pennypacker KR (2008) Sigma receptors suppress multiple aspects of microglial activation. Glia.
Hamabe W, Fujita R, Yasusa T, Yoneda F, Yoshida A and Ueda H (2000) (-)1- (Benzofuran-2-yl)-2-propylaminopentane shows survival effect on cortical neurons under serum-free condition through sigma receptors. Cell Mol Neurobiol 20:695-702.
Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J, Kempner E and Glossmann H (1996) Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci U S A 93:8072-8077.
Hayashi T, Kagaya A, Takebayashi M, Shimizu M, Uchitomi Y, Motohashi N and Yamawaki S (1995) Modulation by sigma ligands of intracellular free Ca++ mobilization by N-methyl-D-aspartate in primary culture of rat frontal cortical neurons. J Pharmacol Exp Ther 275:207-214.
Hayashi T and Su TP (2001) Regulating ankyrin dynamics: Roles of sigma-1 receptors. Proc Natl Acad Sci U S A 98:491-496.
190
Hayashi T and Su TP (2004) Sigma-1 receptor ligands: potential in the treatment of neuropsychiatric disorders. CNS Drugs 18:269-284.
Heales SJ, Bolanos JP, Stewart VC, Brookes PS, Land JM and Clark JB (1999) Nitric oxide, mitochondria and neurological disease. Biochim Biophys Acta 1410:215-228.
Hellewell SB, Bruce A, Feinstein G, Orringer J, Williams W and Bowen WD (1994) Rat liver and kidney contain high densities of sigma 1 and sigma 2 receptors: characterization by ligand binding and photoaffinity labeling. Eur J Pharmacol 268:9-18.
Hertz L, Hansson E and Ronnback L (2001) Signaling and gene expression in the neuron-glia unit during brain function and dysfunction: Holger Hyden in memoriam. Neurochem Int 39:227-252.
Hesselager M, Timmermann DB and Ahring PK (2004) pH Dependency and desensitization kinetics of heterologously expressed combinations of acid- sensing ion channel subunits. J Biol Chem 279:11006-11015.
Hogan PG, Chen L, Nardone J and Rao A (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17:2205-2232.
Holoubek G and Muller WE (2003) Specific modulation of sigma binding sites by the anxiolytic drug opipramol. J Neural Transm 110:1169-1179.
Hopf FW, Reddy P, Hong J and Steinhardt RA (1996) A capacitative calcium current in cultured skeletal muscle cells is mediated by the calcium- specific leak channel and inhibited by dihydropyridine compounds. J Biol Chem 271:22358-22367.
Im SH and Rao A (2004) Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells 18:1-9.
Immke DC and McCleskey EW (2001) Lactate enhances the acid-sensing Na+ channel on ischemia-sensing neurons. Nat Neurosci 4:869-870.
Iwamoto ET (1981) Locomotor activity and antinociception after putative mu, kappa and sigma opioid receptor agonists in the rat: influence of dopaminergic agonists and antagonists. J Pharmacol Exp Ther 217:451- 460.
Jajoo S, Mukherjea D, Brewer GJ and Ramkumar V (2008) Pertussis toxin B- oligomer suppresses human immunodeficiency virus-1 Tat-induced neuronal apoptosis through feedback inhibition of phospholipase C-beta by protein kinase C. Neuroscience 151:525-532.
191
Jasti J, Furukawa H, Gonzales EB and Gouaux E (2007) Structure of acid- sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449:316- 323.
Kamei H, Noda Y, Kameyama T and Nabeshima T (1997) Role of (+)-SKF- 10,047-sensitive sub-population of sigma 1 receptors in amelioration of conditioned fear stress in rats: association with mesolimbic dopaminergic systems. Eur J Pharmacol 319:165-172.
Katnik C, Guerrero WR, Pennypacker KR, Herrera Y and Cuevas J (2006) Sigma-1 receptor activation prevents intracellular calcium dysregulation in cortical neurons during in vitro ischemia. J Pharmacol Exp Ther 319:1355- 1365.
Katz JL, Spealman RD and Clark RD (1985) Stereoselective behavioral effects of N-allylnormetazocine in pigeons and squirrel monkeys. J Pharmacol Exp Ther 232:452-461.
Kekuda R, Prasad PD, Fei YJ, Leibach FH and Ganapathy V (1996) Cloning and functional expression of the human type 1 sigma receptor (hSigmaR1). Biochem Biophys Res Commun 229:553-558.
Kennedy C and Henderson G (1990) Inhibition of potassium currents by the sigma receptor ligand (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine in sympathetic neurons of the mouse isolated hypogastric ganglion. Neuroscience 35:725-733.
Kim S, Nah SY and Rhim H (2008) Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons. Biochem Biophys Res Commun 365:399-405.
Klette KL, DeCoster MA, Moreton JE and Tortella FC (1995) Role of calcium in sigma-mediated neuroprotection in rat primary cortical neurons. Brain Res 704:31-41.
Klette KL, Lin Y, Clapp LE, DeCoster MA, Moreton JE and Tortella FC (1997) Neuroprotective sigma ligands attenuate NMDA and trans-ACPD-induced calcium signaling in rat primary neurons. Brain Res 756:231-240.
Kleyman TR, Sheng S, Kosari F and Kieber-Emmons T (1999) Mechanism of action of amiloride: a molecular prospective. Semin Nephrol 19:524-532.
Krishtal OA, Osipchuk YV, Shelest TN and Smirnoff SV (1987) Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Brain Res 436:352-356.
192
Kume T, Nishikawa H, Taguchi R, Hashino A, Katsuki H, Kaneko S, Minami M, Satoh M and Akaike A (2002) Antagonism of NMDA receptors by sigma receptor ligands attenuates chemical ischemia-induced neuronal death in vitro. Eur J Pharmacol 455:91-100.
Laake JH, Haug FM, Wieloch T and Ottersen OP (1999) A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res Brain Res Protoc 4:173-184.
Langa F, Codony X, Tovar V, Lavado A, Gimenez E, Cozar P, Cantero M, Dordal A, Hernandez E, Perez R, Monroy X, Zamanillo D, Guitart X and Montoliu L (2003) Generation and phenotypic analysis of sigma receptor type I (sigma 1) knockout mice. Eur J Neurosci 18:2188-2196.
Leonard AS, Yermolaieva O, Hruska-Hageman A, Askwith CC, Price MP, Wemmie JA and Welsh MJ (2003) cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1. Proc Natl Acad Sci U S A 100:2029-2034.
Lingueglia E, de Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R and Lazdunski M (1997) A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J Biol Chem 272:29778-29783.
Lo EH, Broderick JP and Moskowitz MA (2004) tPA and proteolysis in the neurovascular unit. Stroke 35:354-356.
Lo EH, Moskowitz MA and Jacobs TP (2005) Exciting, radical, suicidal: how brain cells die after stroke. Stroke 36:189-192.
Locht C and Antoine R (1995) A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit. Biochimie 77:333-340.
Lockhart BP, Soulard P, Benicourt C, Privat A and Junien JL (1995) Distinct neuroprotective profiles for sigma ligands against N-methyl-D-aspartate (NMDA), and hypoxia-mediated neurotoxicity in neuronal culture toxicity studies. Brain Res 675:110-120.
Lopez JC (2002) ASIC-nal integrator. Nature Reviews Neuroscience 3:763.
MacGregor DG, Avshalumov MV and Rice ME (2003) Brain edema induced by in vitro ischemia: causal factors and neuroprotection. J Neurochem 85:1402- 1411.
193
Manders EEM, Verbeek, F.J., and Aten, J.A. (1993) Measurement of co- localization of objects in dual-colour confocal images. Journal of Microscopy - Oxford 169:375-382.
Marino S, Marani L, Nazzaro C, Beani L and Siniscalchi A (2007) Mechanisms of sodium azide-induced changes in intracellular calcium concentration in rat primary cortical neurons. Neurotoxicology 28:622-629.
Martin WR, Eades CG, Thompson JA, Huppler RE and Gilbert PE (1976) The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther 197:517- 532.
Martinez-Martinez S and Redondo JM (2004) Inhibitors of the calcineurin/NFAT pathway. Curr Med Chem 11:997-1007.
Matsumoto RR, Bowen WD, Tom MA, Vo VN, Truong DD and De Costa BR (1995) Characterization of two novel sigma receptor ligands: antidystonic effects in rats suggest sigma receptor antagonism. Eur J Pharmacol 280:301-310.
Matsumoto RR, Bowen WD and Walker JM (1989) Age-related differences in the sensitivity of rats to a selective sigma ligand. Brain Res 504:145-148.
Matsumoto RR, Bowen WD and Walker JM (1990a) Down-regulation of sigma receptors by chronic haloperidol. Prog Clin Biol Res 328:125-128.
Matsumoto RR, Hemstreet MK, Lai NL, Thurkauf A, De Costa BR, Rice KC, Hellewell SB, Bowen WD and Walker JM (1990b) Drug specificity of pharmacological dystonia. Pharmacol Biochem Behav 36:151-155.
Matsuno K, Matsunaga K, Senda T and Mita S (1993) Increase in extracellular acetylcholine level by sigma ligands in rat frontal cortex. J Pharmacol Exp Ther 265:851-859.
Mattson MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1:120-129.
Maurice T and Privat A (1997) SA4503, a novel cognitive enhancer with sigma1 receptor agonist properties, facilitates NMDA receptor-dependent learning in mice. Eur J Pharmacol 328:9-18.
McCracken KA, Bowen WD, de Costa BR and Matsumoto RR (1999a) Two novel sigma receptor ligands, BD1047 and LR172, attenuate cocaine-induced toxicity and locomotor activity. Eur J Pharmacol 370:225-232.
194
McCracken KA, Bowen WD and Matsumoto RR (1999b) Novel sigma receptor ligands attenuate the locomotor stimulatory effects of cocaine. Eur J Pharmacol 365:35-38.
Mike A, Karoly R, Vizi ES and Kiss JP (2003) Inhibitory effect of the DA uptake blocker GBR 12909 on sodium channels of hippocampal neurons. Neuroreport 14:1945-1949.
Moita MA, Lamprecht R, Nader K and LeDoux JE (2002) A-kinase anchoring proteins in amygdala are involved in auditory fear memory. Nat Neurosci 5:837-838.
Moller HJ, Volz HP, Reimann IW and Stoll KD (2001) Opipramol for the treatment of generalized anxiety disorder: a placebo-controlled trial including an alprazolam-treated group. J Clin Psychopharmacol 21:59-65.
Monassier L and Bousquet P (2002) Sigma receptors: from discovery to highlights of their implications in the cardiovascular system. Fundam Clin Pharmacol 16:1-8.
Monnet FP, Debonnel G, Bergeron R, Gronier B and de Montigny C (1994) The effects of sigma ligands and of neuropeptide Y on N-methyl-D-aspartate- induced neuronal activation of CA3 dorsal hippocampus neurones are differentially affected by pertussin toxin. Br J Pharmacol 112:709-715.
Monnet FP, Morin-Surun MP, Leger J and Combettes L (2003) Protein kinase C- dependent potentiation of intracellular calcium influx by sigma1 receptor agonists in rat hippocampal neurons. J Pharmacol Exp Ther 307:705-712.
Monyer H, Goldberg MP and Choi DW (1989) Glucose deprivation neuronal injury in cortical culture. Brain Res 483:347-354.
Morris RG (1989) Synaptic plasticity and learning: selective impairment of learning rats and blockade of long-term potentiation in vivo by the N- methyl-D-aspartate receptor antagonist AP5. J Neurosci 9:3040-3057.
Muller WE and Siebert B (1998) [Opipramol in comparison to other drugs--new pharmacologic data]. Fortschr Neurol Psychiatr 66 Suppl 1:S9-12.
Muller WE, Siebert B, Holoubek G and Gentsch C (2004) Neuropharmacology of the anxiolytic drug opipramol, a sigma site ligand. Pharmacopsychiatry 37 Suppl 3:S189-197.
195
Munoz Maniega S, Cvoro V, Chappell FM, Armitage PA, Marshall I, Bastin ME and Wardlaw JM (2008) Changes in NAA and lactate following ischemic stroke: a serial MR spectroscopic imaging study. Neurology 71:1993- 1999.
Murai Y, Ishibashi H, Koyama S and Akaike N (1997) Ca2+-activated K+ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia. J Neurophysiol 78:2674-2681.
Nedergaard M, Kraig RP, Tanabe J and Pulsinelli WA (1991) Dynamics of interstitial and intracellular pH in evolving brain infarct. Am J Physiol 260:R581-588.
Nguyen EC, McCracken KA, Liu Y, Pouw B and Matsumoto RR (2005) Involvement of sigma (sigma) receptors in the acute actions of methamphetamine: receptor binding and behavioral studies. Neuropharmacology 49:638-645.
Northington FJ, Ferriero DM, Graham EM, Traystman RJ and Martin LJ (2001) Early Neurodegeneration after Hypoxia-Ischemia in Neonatal Rat Is Necrosis while Delayed Neuronal Death Is Apoptosis. Neurobiol Dis 8:207-219.
Oliveria SF, Dell'Acqua ML and Sather WA (2007) AKAP79/150 anchoring of calcineurin controls neuronal L-type Ca2+ channel activity and nuclear signaling. Neuron 55:261-275.
Pereverzev A, Komarova SV, Korcok J, Armstrong S, Tremblay GB, Dixon SJ and Sims SM (2008) Extracellular acidification enhances osteoclast survival through an NFAT-independent, protein kinase C-dependent pathway. Bone 42:150-161.
Pidoplichko VI and Dani JA (2006) Acid-sensitive ionic channels in midbrain dopamine neurons are sensitive to ammonium, which may contribute to hyperammonemia damage. Proc Natl Acad Sci U S A 103:11376-11380.
Pignataro G, Simon RP and Xiong ZG (2007) Prolonged activation of ASIC1a and the time window for neuroprotection in cerebral ischaemia. Brain 130:151-158.
Poirot O, Vukicevic M, Boesch A and Kellenberger S (2004) Selective regulation of acid-sensing ion channel 1 by serine proteases. J Biol Chem 279:38448-38457.
196
Popp RL and Dertien JS (2008) Actin depolymerization contributes to ethanol inhibition of NMDA receptors in primary cultured cerebellar granule cells. Alcohol 42:525-539.
Prasad PD, Li HW, Fei YJ, Ganapathy ME, Fujita T, Plumley LH, Yang-Feng TL, Leibach FH and Ganapathy V (1998) Exon-intron structure, analysis of promoter region, and chromosomal localization of the human type 1 sigma receptor gene. J Neurochem 70:443-451.
Price MP, Lewin GR, McIlwrath SL, Cheng C, Xie J, Heppenstall PA, Stucky CL, Mannsfeldt AG, Brennan TJ, Drummond HA, Qiao J, Benson CJ, Tarr DE, Hrstka RF, Yang B, Williamson RA and Welsh MJ (2000) The mammalian sodium channel BNC1 is required for normal touch sensation. Nature 407:1007-1011.
Rae J, Cooper K, Gates P and Watsky M (1991) Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37:15-26.
Riepe MW, Schmalzigaug K, Fink F, Oexle K and Ludolph AC (1996) NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation. Brain Res 710:21-27.
Roman FJ, Pascaud X, Duffy O, Vauche D, Martin B and Junien JL (1989) Neuropeptide Y and peptide YY interact with rat brain sigma and PCP binding sites. Eur J Pharmacol 174:301-302.
Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD and Westbrook GL (1994) Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 368:853-856.
Rothman RB, Reid A, Mahboubi A, Kim CH, De Costa BR, Jacobson AE and Rice KC (1991) Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands. Mol Pharmacol 39:222-232.
Rothman S (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J Neurosci 4:1884-1891.
Ryan SK, Shotts LR, Hong SK, Nehra D, Groat CR, Armstrong JR and McClellan AD (2007) Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey. Dev Neurobiol 67:173-188.
197
Sattler R, Xiong Z, Lu WY, MacDonald JF and Tymianski M (2000) Distinct roles of synaptic and extrasynaptic NMDA receptors in excitotoxicity. J Neurosci 20:22-33.
Saugstad JA, Roberts JA, Dong J, Zeitouni S and Evans RJ (2004) Analysis of the membrane topology of the acid-sensing ion channel 2a. J Biol Chem 279:55514-55519.
Schurr A (2004) Neuroprotection against ischemic/hypoxic brain damage: blockers of ionotropic glutamate receptor and voltage sensitive calcium channels. Curr Drug Targets 5:603-618.
Senda T, Matsuno K, Okamoto K, Kobayashi T, Nakata K and Mita S (1996) Ameliorating effect of SA4503, a novel sigma 1 receptor agonist, on memory impairments induced by cholinergic dysfunction in rats. Eur J Pharmacol 315:1-10.
Seth P, Fei YJ, Li HW, Huang W, Leibach FH and Ganapathy V (1998) Cloning and functional characterization of a sigma receptor from rat brain. J Neurochem 70:922-931.
Shalak L and Perlman JM (2004) Hypoxic-ischemic brain injury in the term infant- current concepts. Early Hum Dev 80:125-141.
Shibasaki F, Hallin U and Uchino H (2002) Calcineurin as a multifunctional regulator. J Biochem 131:1-15.
Silver J and Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146-156.
Simon R and Xiong Z (2006) Acidotoxicity in brain ischaemia. Biochem Soc Trans 34:1356-1361.
Simon RP (2006) Acidotoxicity trumps excitotoxicity in ischemic brain. Arch Neurol 63:1368-1371.
Sircar R, Rappaport M, Nichtenhauser R and Zukin SR (1987) The novel anticonvulsant MK-801: a potent and specific ligand of the brain phencyclidine/sigma-receptor. Brain Res 435:235-240.
Slifer BL and Balster RL (1983) Reinforcing properties of stereoisomers of the putative sigma agonists N-allylnormetazocine and cyclazocine in rhesus monkeys. J Pharmacol Exp Ther 225:522-528.
198
Smith KE, Gibson ES and Dell'Acqua ML (2006) cAMP-dependent protein kinase postsynaptic localization regulated by NMDA receptor activation through translocation of an A-kinase anchoring protein scaffold protein. J Neurosci 26:2391-2402.
Soriani O, Foll FL, Roman F, Monnet FP, Vaudry H and Cazin L (1999) A- Current down-modulated by sigma receptor in frog pituitary melanotrope cells through a G protein-dependent pathway. J Pharmacol Exp Ther 289:321-328.
Soriani O, Vaudry H, Mei YA, Roman F and Cazin L (1998) Sigma ligands stimulate the electrical activity of frog pituitary melanotrope cells through a G-protein-dependent inhibition of potassium conductances. J Pharmacol Exp Ther 286:163-171.
Staruschenko A, Dorofeeva NA, Bolshakov KV and Stockand JD (2007) Subunit- dependent cadmium and nickel inhibition of acid-sensing ion channels. Dev Neurobiol 67:97-107.
Steele RJ and Morris RG (1999) Delay-dependent impairment of a matching-to- place task with chronic and intrahippocampal infusion of the NMDA- antagonist D-AP5. Hippocampus 9:118-136.
Stoll G, Jander S and Schroeter M (1998) Inflammation and glial responses in ischemic brain lesions. Prog Neurobiol 56:149-171.
Streit WJ, Mrak RE and Griffin WS (2004) Microglia and neuroinflammation: a pathological perspective. J Neuroinflammation 1:14.
Su TP and Hayashi T (2003) Understanding the molecular mechanism of sigma- 1 receptors: towards a hypothesis that sigma-1 receptors are intracellular amplifiers for signal transduction. Curr Med Chem 10:2073-2080.
Su TP, London ED and Jaffe JH (1988) Steroid binding at sigma receptors suggests a link between endocrine, nervous, and immune systems. Science 240:219-221.
Sullivan PG, Dube C, Dorenbos K, Steward O and Baram TZ (2003) Mitochondrial uncoupling protein-2 protects the immature brain from excitotoxic neuronal death. Ann Neurol 53:711-717.
Takahashi H, Kirsch JR, Hashimoto K, London ED, Koehler RC and Traystman RJ (1996) PPBP [4-phenyl-1-(4-phenylbutyl) piperidine] decreases brain injury after transient focal ischemia in rats. Stroke 27:2120-2123.
199
Tanaka E, Niiyama S, Uematsu K, Yokomizo Y and Higashi H (2002) The presynaptic modulation of glutamate release and the membrane dysfunction induced by in vitro ischemia in rat hippocampal CA1 neurons. Life Sci 72:363-374.
Tanaka E, Yamamoto S, Inokuchi H, Isagai T and Higashi H (1999) Membrane dysfunction induced by in vitro ischemia in rat hippocampal CA1 pyramidal neurons. J Neurophysiol 81:1872-1880.
Tanaka E, Yamamoto S, Kudo Y, Mihara S and Higashi H (1997) Mechanisms underlying the rapid depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J Neurophysiol 78:891- 902.
Thurgur C and Church J (1998) The anticonvulsant actions of sigma receptor ligands in the Mg2+-free model of epileptiform activity in rat hippocampal slices. Br J Pharmacol 124:917-929.
Tokuyama S, Hirata K, Ide A and Ueda H (1997) Sigma ligands stimulate GTPase activity in mouse prefrontal membranes: evidence for the existence of metabotropic sigma receptor. Neurosci Lett 233:141-144.
Tombaugh GC and Sapolsky RM (1993) Evolving concepts about the role of acidosis in ischemic neuropathology. J Neurochem 61:793-803.
Traynelis SF and Chesler M (2001) Proton release as a modulator of presynaptic function. Neuron 32:960-962.
Trendelenburg G and Dirnagl U (2005) Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia 50:307-320.
Tu P, Kunert-Keil C, Lucke S, Brinkmeier H and Bouron A (2009) Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons. J Neurochem 108:126- 138.
Ugawa S, Yamamoto T, Ueda T, Ishida Y, Inagaki A, Nishigaki M and Shimada S (2003) Amiloride-insensitive currents of the acid-sensing ion channel-2a (ASIC2a)/ASIC2b heteromeric sour-taste receptor channel. J Neurosci 23:3616-3622.
Van Lint P and Libert C (2007) Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation. J Leukoc Biol 82:1375-1381.
200
Vannucci RC, Yager JY and Vannucci SJ (1994) Cerebral glucose and energy utilization during the evolution of hypoxic-ischemic brain damage in the immature rat. J Cereb Blood Flow Metab 14:279-288.
Vannucci SJ and Hagberg H (2004) Hypoxia-ischemia in the immature brain. J Exp Biol 207:3149-3154.
Varming T, Drejer J, Frandsen A and Schousboe A (1996) Characterization of a chemical anoxia model in cerebellar granule neurons using sodium azide: protection by nifedipine and MK-801. J Neurosci Res 44:40-46.
Vaupel DB (1983) Naltrexone fails to antagonize the sigma effects of PCP and SKF 10,047 in the dog. Eur J Pharmacol 92:269-274.
Vilner BJ and Bowen WD (2000) Modulation of cellular calcium by sigma-2 receptors: release from intracellular stores in human SK-N-SH neuroblastoma cells. J Pharmacol Exp Ther 292:900-911.
Volz HP, Moller HJ, Reimann I and Stoll KD (2000) Opipramol for the treatment of somatoform disorders results from a placebo-controlled trial. Eur Neuropsychopharmacol 10:211-217.
Volz HP and Stoll KD (2004) Clinical trials with sigma ligands. Pharmacopsychiatry 37 Suppl 3:S214-220.
Vornov JJ (1995) Toxic NMDA-receptor activation occurs during recovery in a tissue culture model of ischemia. J Neurochem 65:1681-1691.
Vornov JJ, Tasker RC and Coyle JT (1994) Delayed protection by MK-801 and tetrodotoxin in a rat organotypic hippocampal culture model of ischemia. Stroke 25:457-464; discussion 464-455.
Waldmann R, Bassilana F, de Weille J, Champigny G, Heurteaux C and Lazdunski M (1997a) Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J Biol Chem 272:20975-20978.
Waldmann R, Champigny G, Bassilana F, Heurteaux C and Lazdunski M (1997b) A proton-gated cation channel involved in acid-sensing. Nature 386:173- 177.
Walker JM, Bowen WD, Walker FO, Matsumoto RR, De Costa B and Rice KC (1990) Sigma receptors: biology and function. Pharmacol Rev 42:355-402.
Wang W, Duan B, Xu H, Xu L and Xu TL (2006) Calcium-permeable acid- sensing ion channel is a molecular target of the neurotoxic metal ion lead. J Biol Chem 281:2497-2505.
201
Watkins JC and Jane DE (2006) The glutamate story. Br J Pharmacol 147 Suppl 1:S100-108.
Weinstein PR, Hong S and Sharp FR (2004) Molecular identification of the ischemic penumbra. Stroke 35:2666-2670.
Wemmie JA, Askwith CC, Lamani E, Cassell MD, Freeman JH, Jr. and Welsh MJ (2003) Acid-sensing ion channel 1 is localized in brain regions with high synaptic density and contributes to fear conditioning. J Neurosci 23:5496- 5502.
Wemmie JA, Chen J, Askwith CC, Hruska-Hageman AM, Price MP, Nolan BC, Yoder PG, Lamani E, Hoshi T, Freeman JH, Jr. and Welsh MJ (2002) The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory. Neuron 34:463-477.
Wemmie JA, Coryell MW, Askwith CC, Lamani E, Leonard AS, Sigmund CD and Welsh MJ (2004) Overexpression of acid-sensing ion channel 1a in transgenic mice increases acquired fear-related behavior. Proc Natl Acad Sci U S A 101:3621-3626.
Wemmie JA, Price MP and Welsh MJ (2006) Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci 29:578-586.
Westphal RS, Tavalin SJ, Lin JW, Alto NM, Fraser ID, Langeberg LK, Sheng M and Scott JD (1999) Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex. Science 285:93-96.
Wolfe SA, Jr. and De Souza EB (1993) Sigma and phencyclidine receptors in the brain-endocrine-immune axis. NIDA Res Monogr 133:95-123.
Wong W and Scott JD (2004) AKAP signalling complexes: focal points in space and time. Nat Rev Mol Cell Biol 5:959-970.
Wu Z, Gebreselassie, D., Williams, W., Bowen, W.D. (2003) Reexamination of metaphit as a tool for the study of sigma receptors, in Society for Neuroscience, Washington, DC.
Xiong ZG, Chu XP and Simon RP (2006) Ca2+ -permeable acid-sensing ion channels and ischemic brain injury. J Membr Biol 209:59-68.
Xiong ZG, Pignataro G, Li M, Chang SY and Simon RP (2008) Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases. Curr Opin Pharmacol 8:25-32.
202
Xiong ZG, Zhu XM, Chu XP, Minami M, Hey J, Wei WL, MacDonald JF, Wemmie JA, Price MP, Welsh MJ and Simon RP (2004) Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels. Cell 118:687-698.
Yermolaieva O, Leonard AS, Schnizler MK, Abboud FM and Welsh MJ (2004) Extracellular acidosis increases neuronal cell calcium by activating acid- sensing ion channel 1a. Proc Natl Acad Sci U S A 101:6752-6757.
Zha XM, Wemmie JA, Green SH and Welsh MJ (2006) Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines. Proc Natl Acad Sci U S A 103:16556-16561.
Zhang H and Cuevas J (2002) Sigma receptors inhibit high-voltage-activated calcium channels in rat sympathetic and parasympathetic neurons. J Neurophysiol 87:2867-2879.
Zhang H and Cuevas J (2005) sigma Receptor activation blocks potassium channels and depresses neuroexcitability in rat intracardiac neurons. J Pharmacol Exp Ther 313:1387-1396.
Zhang J, Tan Z and Tran ND (2000) Chemical hypoxia-ischemia induces apoptosis in cerebromicrovascular endothelial cells. Brain Res 877:134- 140.
Zhou Q, Xiao M and Nicoll RA (2001) Contribution of cytoskeleton to the internalization of AMPA receptors. Proc Natl Acad Sci U S A 98:1261- 1266.
203
APPENDICES
204
Appendix A – Pharmacological Compounds
Drug Name Definition and Mechanism of Reference Effective/ Action Inhibitory Concentrations Amiloride Nonspecific Binds to residues (Kleyman et ASIC blocker within the al., 1999; (16 μM) channel’s pore Alvarez de la region Rosa et al., 2000; Xiong et al., 2004) Psalmotoxin1 Homomeric Increases the (Escoubas et venom or synthetic ASIC1a channel affinity of the al., 2000; peptide (PcTx1) selective channel by H+ Chen et al., blocker (500 resulting in 2005; Diochot ng/ml, 50 nM) chromic et al., 2007) desensitization of the channel 1,3-di-o-tolyl- Pan-selective Binds both sigma (Klette et al., guanidine (DTG) sigma receptor receptor subtypes 1995; Soriani agonist (100 et al., 1998; μM, 65 μM) Kume et al., 2002; Zhang and Cuevas, 2002; Zhang and Cuevas, 2005; Katnik et al., 2006) Carbetapentane Sigma-1 Binds sigma-1 (Rothman et citrate (CBP) selective receptors with al., 1991; agonist (14 μM, >50-fold greater Thurgur and 38 μM) affinity than for Church, 1998; sigma-2 receptors Vilner and Bowen, 2000; Katnik et al., 2006)
205
Appendix A: (Continued)
Opipramol Pan-selective Binds both sigma (Muller and sigma receptor receptor subtypes Siebert, 1998; agonist (10 μM) Volz et al., 2000; Moller et al., 2001; Holoubek and Muller, 2003; Muller et al., 2004; Volz and Stoll, 2004) Dextromethorphan Sigma-1 Binds sigma-1 (Anderson and hydrobromide selective ligand receptors with Andrew, 2002) (DEX) (30 μM) >100-fold greater affinity than for sigma-2 receptors
2-(4- Sigma-1 Binds sigma-1 (Bucolo et al., Morpholinethyl) 1- selective ligand receptors with 2006) phenylcyclohexane (10 μM) >100-fold greater carboxylate affinity than for hydrochloride (Pre- sigma-2 receptors 084) Ibogaine Sigma-2 Binds sigma-2 (Vilner and selective receptors with Bowen, 2000; agonist (31 μM) >40-fold greater Zhang and affinity than for Cuevas, 2002) sigma-1 receptors 1-Cyclohexyl-4-(3- Sigma-2 Potent sigma-2 (Berardi et al., (5-methoxy- selective receptors agonist 2004; 1,2,3,4- agonist (2 μM) Cassano et tetrahydronaphthal al., 2006) en-1-yl)-n- propyl)piperazine dihydrochloride (PB28) Metaphit (MET) Irreversible pan- Inhibits both (Zhang and selective sigma sigma receptor Cuevas, 2002; antagonist (50 subtypes Wu, 2003; μM) Zhang and Cuevas, 2005; Katnik et al., 2006)
206
Appendix A: (Continued)
1-[2-(3,4- Sigma-1 Binds sigma-1 (Matsumoto et dichlorophenyl)eth selective receptors with al., 1995; yl]-4- receptor greater affinity Hamabe et al., methylpiperazine antagonist (10 than for sigma-2 2000; Nguyen dihydrochloride nM, 1 μM) receptors et al., 2005) (BD1063) Thapsigargin Depletes the Blocks the (DeHaven and (THAP) ryanodine and sarcoplasmic/ Cuevas, 2004; IP3 sensitive endoplasmic Katnik et al., stores (10 μM, reticulum Ca2+- 2006) 20 μM) ATPase Tetrodotoxin (TTX) Inhibits voltage- Blocks action (DeHaven and gated Na+ potentials by Cuevas, 2004; channels (200 binding to the Katnik et al., nM, 400 nM) pores of the 2006) voltage-gated Na+ channels D-2-Amino-5- Selective NMDA Competitively (Morris, 1989; phosphonovaleric receptors inhibits NMDA Steele and acid (AP5) antagonist (100 receptors active Morris, 1999; μM) site MacGregor et al., 2003; Katnik et al., 2006) 1,4-Dihydro-2,6- Dihydropyridine L-type voltage- (Kim et al., dimethyl-4-(2- calcium channel gated Ca2+ 2008; Tu et nitrophenyl)-3,5- blocker (5 μM, channel blocker al., 2009) pyridinedicarboxyli 10 μM) c acid dimethyl ester (Nifedipine) Cadmium Broad spectrum Inhibits Ca2+ (Ryan et al., Ca2+ channel channels 2007; blocker (100 Staruschenko μM, 1 mM) et al., 2007) 6-Cyano-7- AMPA/kainate Competitively (MacGregor et nitroquinoxaline- receptor blocks AMPA and al., 2003; 2,3-dione (CNQX) antagonist (10 kainate receptors Marino et al., μM) 2007)
207
Appendix A: (Continued)
FK-506 Calcineurin Forms (Dumont, inhibitor (1 μM, immunophilin 2000; 10 μM) complex with Martinez- FKBP to bind Martinez and calcineurin and Redondo, competitively 2004; Chai et inhibit its activity al., 2007) by blocking the catalytic groove CyclosporinA Calcineurin Forms (Martinez- inhibitor (1 μM , immunophilin Martinez and 30 μM) complex with Redondo, cyclophilinA to 2004; Chai et bind calcineurin al., 2007) and competitively inhibit its activity by blocking the catalytic groove Pertussis toxin Inhibits G Catalyzes the (Locht and (PTX) protein ADP-ribosylation Antoine, 1995; activation (100 of the α subunits Jajoo et al., ng/ml, 200 of Gi/Go, 2008) ng/ml) preventing the G protein heterotrimers from interacting with receptors
N-methyl-D- NMDA receptor Selectively (Gomez et al., aspartate (NMDA) agonist (10 μM) activates NMDA 2002; Watkins receptors and not and Jane, other glutamate 2006) receptors Latrunculin A Disrupts the Disrupts the actin (Allison et al., actin cytoskeleton, 1998; Sattler cytoskeleton (5 resulting in et al., 2000; μM) AKAP150 Zhou et al., dissociation from 2001; Popp plasma and Dertien, membrane 2008)
208
Appendix A: (Continued)
VIVIT Inhibits Inhibits (Oliveria et al., calcineurin calcineurin/AKAP 2007; AKAP150 150 interaction by Pereverzev et interaction (1 disrupting al., 2008) μM, 10 μM) calcineurin binding to a PxIxIT-docking motif in AKAP150
209
ABOUT THE AUTHOR
Yelenis Herrera was born in Havana, Cuba. She is the daughter of Silvia and Roberto Herrera. She came to the United States in 1994 at the age of 12 and started to pursue her scientific career. In 2000, she graduated as one of the top students in her class from Suncoast Community High School in West Palm
Beach, FL. She continued her education and in 2004, she graduated Cum Laude earning Bachelors degrees in Molecular 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 Pharmacology and Physiology.