0022-3565/08/3272-491–502$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 327, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 143974/3400789 JPET 327:491–502, 2008 Printed in U.S.A.

␴-1 Receptor Modulation of Acid-Sensing Ion Channel a (ASIC1a) and ASIC1a-Induced Ca2ϩ Influx in Rat Cortical Neurons

Yelenis Herrera, Christopher Katnik, Jael D. Rodriguez, Aaron A. Hall, Alison Willing, Keith R. Pennypacker, and Javier Cuevas Department of Molecular Pharmacology and Physiology (Y.H., C.K., J.D.R., A.A.H., A.W., K.R.P., J.C.) and Center for Aging and Brain Repair (A.W.), University of South Florida, College of Medicine, Tampa, Florida Received July 23, 2008; accepted August 21, 2008 Downloaded from

ABSTRACT Acid-sensing ion channels (ASICs) are proton-gated cation experiments using ␴ receptor-subtype-specific agonists dem- channels found in peripheral and central nervous system neu- onstrated that ␴-1, but not ␴-2, receptors inhibit ASIC1a- rons. The ASIC1a subtype, which has high Ca2ϩ permeability, induced Ca2ϩ elevations. These results were confirmed using jpet.aspetjournals.org is activated by ischemia-induced acidosis and contributes to the irreversible ␴ receptor antagonist metaphit (50 ␮M) and the the neuronal loss that accompanies ischemic stroke. Our lab- selective ␴-1 antagonist BD1063 (10 nM), which obtunded the oratory has shown that activation of ␴ receptors depresses ion inhibitory effects of the ␴-1 agonist, carbetapentane. Activation 2ϩ 2ϩ channel activity and [Ca ]i dysregulation during ischemia, of ASIC1a was shown to stimulate downstream Ca influx which enhances neuronal survival. Whole-cell patch-clamp pathways, specifically N-methyl-D-aspartate and (Ϯ)-␣-amino- electrophysiology and fluorometric Ca2ϩ imaging were used to 3-hydroxy-5-methylisoxazole-4-propionic acid/kainate recep- determine whether ␴ receptors regulate the function of ASIC in tors and voltage-gated Ca2ϩ channels. These subsequent cultured rat cortical neurons. Bath application of the selective Ca2ϩ influxes were also inhibited upon activation of ␴-1 recep- at ASPET Journals on January 27, 2020 ASIC1a blocker, psalmotoxin1, decreased proton-evoked tors. These findings demonstrate that ␴-1 receptor stimulation 2ϩ [Ca ]i transients and peak membrane currents, suggesting the inhibits ASIC1a-mediated membrane currents and consequent presence of homomeric ASIC1a channels. The pan-selective intracellular Ca2ϩ accumulation. The ability to control ionic ␴-1/␴-2 receptor agonists, 1,3-di-o-tolyl-guanidine (100 ␮M) imbalances and Ca2ϩ dysregulation evoked by ASIC1a activa- and (10 ␮M), reversibly decreased acid-induced el- tion makes ␴ receptors an attractive target for ischemic stroke 2ϩ evations in [Ca ]i and membrane currents. Pharmacological therapy.

Acid-sensing ion channels are a class of ligand-gated chan- al., 2006) that encode protein subunits that form functional nels that are members of the degenerin/epithelial sodium proton-gated homomultimeric or heteromultimeric channels channel superfamily and are expressed in both peripheral (Wemmie et al., 2006). The pH of half-maximal activation and central nervous system neurons (Waldmann et al., 1997). and the tissue expression patterns differ between each chan- Thus far, four genes (ASIC1–ASIC4) and two splice variants nel subtype. of ASIC1 and ASIC2 (a and b) have been cloned (Wemmie et 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 This study was supported by an American Heart Association Florida/Puerto Rico Affiliate Grant-In-Aid Award and by a University of South Florida Sig- ASIC2a (Askwith et al., 2004). These channels are activated nature Program in Neuroscience Award (to J.C.). by pH Յ 7 and have a pH of half-maximal activation of ϳ6.0 Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. to 6.5 (Waldmann et al., 1997; Hesselager et al., 2004). doi:10.1124/jpet.108.143974. ASIC1a homomultimeric channels differ from other ASIC

ABBREVIATIONS: ASIC, acid-sensing ion channel; CNS, central nervous system; NMDA, N-methyl-D-aspartate; AMPA, (Ϯ)-␣-amino-3-hydroxy- 5-methylisoxazole-4-propionic acid; VGCC, voltage-gated Ca2ϩ channel; PSS, physiological saline solution; DTG, 1,3-di-o-tolyl-guanidine; nifedipine, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester; AP5, D-2-amino-5-phosphonovaleric acid; PB28, 1-cyclohexyl-4-(3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl)piperazine dihydrochloride; BD1063, 1-[2-(3,4-dichlorophenyl)- ethyl]-4-methylpiperazine dihydrochloride; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; PRE-084, 2-(4-morpholinethyl) 1-phenylcyclohexane- carboxylate hydrochloride; PcTx1, psalmotoxin1; CBP, carbetapentane citrate; DEX, hydrobromide; MET, metaphit; IBO, ; THAP, thapsigargin; TTX, tetrodotoxin. 491 492 Herrera et al. subtypes in that they are highly permeable to both Naϩ and via these channels. Therefore, ␴-1 receptors couple to ASIC1a Ca2ϩ ions (Waldmann et al., 1997; Yermolaieva et al., 2004). channels to inhibit channel function and also block ASIC1a- 2ϩ ␴ This ASIC subtype has been implicated in a number of phys- induced [Ca ]i dysregulation. Our findings suggest that -1 iological processes such as synaptic plasticity, fear condition- receptors represent potential targets for improving outcome ing, and learning and memory (Wemmie et al., 2002, 2003, of stroke injury and expanding the therapeutic window for 2004). ASIC1a has also been shown to be activated after ischemic stroke treatment. cerebral ischemia and has been unequivocally linked to neu- ronal cell death (Xiong et al., 2004; Gao et al., 2005; Pigna- taro et al., 2007). Transgenic mice deficient in ASIC1a have Materials and Methods reduced infarct size in response to middle cerebral artery Primary Rat Cortical Neuron Preparation. Primary cortical occlusion relative to wild-type mice (Xiong et al., 2004). More- neurons from embryonic (E18) rats were cultured as previously de- over, pharmacological inhibition of ASIC1a with either scribed by our laboratory (Katnik et al., 2006). All procedures were amiloride or psalmotoxin1, which is selective for homomul- done in accordance with the regulations of the University of South timeric ASIC1a channels (Diochot et al., 2007), diminishes Florida Institutional Animal Care and Use Committee. Cells were ischemic brain injury (Xiong et al., 2004). Several studies used between 10 and 21 days in culture. have suggested that Ca2ϩ influx through these channels is a Calcium Imaging Measurements. The effects of acidosis on intracellular Ca2ϩ concentrations were examined in isolated cortical key mechanism leading to neurodegeneration (Xiong et al., ϩ neurons using fluorescent imaging techniques. Cytosolic free Ca2 2004; Yermolaieva et al., 2004). was measured using the Ca2ϩ-sensitive dye, fura-2, as we have Downloaded from Despite efforts to determine the function of ASIC1a and described previously (Katnik et al., 2006). Cells, plated on poly-L- the role of these channels in ischemia, little is known about lysine-coated coverslips, were incubated for1hatroom temperature endogenous mechanisms that control ASIC1a activity. Thus in Neurobasal (Invitrogen, Carlsbad, CA) medium supplemented far, only the NMDA receptor, acting via a calcium/calmodu- with B27 (Invitrogen) and 0.5 mM L- or in physiological lin-dependent protein kinase II, has been shown to modulate saline solution (PSS) consisting of: 140 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl , 1.0 mM MgCl , 20 mM glucose, and 25 mM HEPES (pH ASIC1a (Gao et al., 2005). Activation of NMDA receptors 2 2 jpet.aspetjournals.org enhances ASIC1a-mediated currents, which consequently ex- to 7.4 with NaOH), 330 Ϯ 10 mOsm. Both solutions contained 3 ␮M acerbates acidotoxicity during ischemia (Gao et al., 2005). acetoxymethyl ester fura-2 and 0.3% dimethyl sulfoxide. The cover- slips were washed in PSS (fura-2 free) before experiments were ␴ Receptor activation has been shown to modulate mul- ␴ carried out. tiple cell membrane ion channels in neurons. -1 Receptors Electrophysiology Recordings. Neurons plated on glass cover- regulate ionotropic glutamate receptors and voltage-gated ϩ slips (as described above) were transferred to a recording chamber K channels, whereas ␴-2 receptors modulate voltage- mounted on a Zeiss Axiovert 200 and visualized at 400ϫ. Membrane 2ϩ gated Ca channels (Hayashi et al., 1995; Aydar et al., currents were amplified using an Axopatch 200B (Molecular Devices, at ASPET Journals on January 27, 2020 2002; Zhang and Cuevas, 2002, 2005). The inhibition of Sunnyvale, CA), filtered at 1 kHz, digitized at 5 kHz with a Digidata ionotropic receptors by ␴ receptors prevents 1322A (Molecular Devices), and acquired using Clampex 8 (Axon 2ϩ Instruments Inc.). Electrical access was achieved using the ampho- elevations in [Ca ]i associated with glutamate-induced excitotoxicity (Klette et al., 1995). All of these voltage- tericin B perforated-patch method to preserve intracellular integrity gated ion channels and NMDA receptors have been shown of neurons (Rae et al., 1991). An amphotericin B stock solution (60 mg/ml in dimethyl sulfoxide) was made daily, kept on ice, light to contribute to the demise of neurons during an ischemic ␮ ␴ protected, and diluted to 240 g/ml (0.4% dimethyl sulfoxide) in insult. Our laboratory has recently shown that -1 recep- control pipette solution immediately before patch-clamp experi- tors inhibit Ca2ϩ dysregulation evoked by ischemia and ments. The pipette solution consisted of the following: 75 mM K2SO4, that activation of ␴ receptors is neuroprotective at delayed 55 mM KCl, 5 mM MgSO4, and 25 mM HEPES (titrated to pH 7.2 time points in a rat model of ischemic stroke (Ajmo et al., with N-methyl-D-glucamine, 300 Ϯ 5 mOsm). Patch electrodes were 2006; Katnik et al., 2006). Interstitial pH in the brain pulled from thin-walled borosilicate glass (World Precision Instru- remains low several hours after an ischemic event (Ned- ments, Inc., Sarasota, FL) using a Sutter Instruments P-87 pipette ergaard et al., 1991), and pharmacological blockade of puller (Sutter Instrument Company, Novato, CA) and had resis- ⍀ ASIC1a by amiloride or psalmotoxin1 administered even tances of 1.0 to 1.5 M . Access resistance (Rs) was monitored Յ ⍀ 5 h after middle cerebral artery occlusion has been shown throughout experiments for stable values 20 M and were always compensated at 40% (lag, 10 ␮s). All cells were voltage-clamped at to diminish stroke injury (Simon, 2006). These observa- Ϫ ␴ 70 mV. tions raise the possibility that receptors may regulate Solutions and Reagents. The control bath solution for all exper- ASIC1a function and ASIC1a-induced intracellular cal- iments was PSS. In one series of experiments requiring high extra- cium transients and provide neuroprotection when stimu- cellular Kϩ, an additional 34.6 mM KCl was isosmotically substi- lated at delayed time points after an ischemic insult. tuted for NaCl. All drugs were applied in PSS (or high-Kϩ PSS) using Experiments were conducted to determine the effects of ␴ a rapid application system identical to that described previously receptors on ASIC-mediated membrane currents and tran- (Cuevas and Berg, 1998). ASIC activation was induced by applying 2ϩ ␴ PSS with a pH of 6.0 (Ϯdrug) to specifically target ASIC1a (Askwith sient [Ca ]i elevations. It was determined that receptor 2ϩ et al., 2004). Individual cells were exposed to no more than four low agonists inhibit acidosis-induced increases in [Ca ]i and peak membrane currents in cells expressing homomeric pH applications, and no rundown of the responses was observed with this protocol. All chemicals used in this investigation were of analytic ASIC1a channels. Pharmacological studies demonstrated ␴ grade. The following drugs were used: DTG, opipramol, ibogaine, that the -1 receptor subtype was responsible for these ef- metaphit, nifedipine, AP5, and PB28 (Sigma-Aldrich, St. Louis, MO); fects. Moreover, acidosis was also shown to activate down- carbetapentane, BD1063, CNQX, and PRE-084 (Tocris Bioscience, 2ϩ stream Ca influx pathways [e.g., NMDA and AMPA/kai- Ellisville, MO); dextromethorphan (MP Biomedicals, Irvine, CA); 2ϩ nate receptors and voltage-gated Ca channels (VGCC)], psalmotoxin1 (Spider Pharm, Yarnelle, AZ); tetrodotoxin and thap- ϩ and activation of ␴-1 receptors also diminished Ca2 entry sigargin (Alomone Labs, Jerusalem, Israel); amiloride (Alexis Cor- ␴-1 Receptors and ASIC1a Function 493

poration, Lausen, Switzerland); cadmium (Thermo Fisher Scientific, tula Psalmopoeus cambridgei has been shown to be a selec- Waltham, MA); and fura-2 acetoxymethyl ester (Invitrogen). tive blocker of homomultimeric ASIC1a channels (Diochot et ϩ Data Analysis. Analyses of measured intracellular Ca2 and 2ϩ al., 2007). Figure 1C shows representative traces of [Ca ]i membrane current responses were conducted using Clampfit 9 (Axon as a function of time recorded from a neuron before (Control), Instruments Inc.). Fluorescence intensities were recorded from fura- after a 10 to 20-min preincubation in bath applied PcTx1 (500 2-loaded neuronal cell bodies. Time-lapse imaging data files collected with SlideBook 4.02 (Intelligent Imaging Innovations, Inc., Denver, ng/ml venom protein) and after washout of the toxin (Wash). CO) were converted to a text format and imported into Clampfit for In identical experiments, PcTx1 produced a statistically sig- subsequent analysis. Statistical analysis was conducted using nificant reversible decrease in ASIC1a-mediated elevations 2ϩ Ϯ SigmaPlot 9 and SigmaStat 3 software (Systat Software, Inc., San in [Ca ]i (57 2%) (Fig. 1D). The effects produced by this Jose, CA). Statistical differences were determined using paired and concentration of PcTx1 are consistent with results obtained unpaired Student’s t tests for within- and between-group experi- for ASIC1a responses in mouse cortical neurons (Xiong et al., Ͻ ments, respectively, and were considered significant if p 0.05. For 2004). These data indicate that, in cultured cortical neurons multiple group comparisons, either a one- or a two-way analysis of from embryonic rats, acidosis results in elevations in [Ca2ϩ] variance, with or without repeat measures, were used, as appropri- i ate. When significant differences were determined with an analysis that are mediated via the activation of homomultimeric of variance, post hoc analysis was conducted using a Tukey test to ASIC1a channels, as reported for cultured cortical neurons determine differences between individual groups. For the generation from embryonic mice (Xiong et al., 2004). of concentration response curves, data were best fit using a single- Activation of ␴ receptors has been shown to inhibit numer- site Langmuir-Hill equation. ous plasma membrane ion channels in neurons (Hayashi et Downloaded from al., 1995; Zhang and Cuevas, 2002, 2005). Therefore, exper- Results iments were carried out to study the effects of ␴ receptor ␴ ASIC subtypes are distinguishable by pH sensitivity and activation on ASIC1a function using the pan-selective re- ion selectivity, with homomultimeric ASIC1a being the only ceptor agonist, DTG. Figure 2A shows representative traces 2ϩ 2ϩ subtype that is Ca -permeable (Yermolaieva et al., 2004). of [Ca ]i recorded from a single neuron during acidosis in ␮ jpet.aspetjournals.org Experiments were carried out to determine the effects of the absence (Control) and presence of 100 M DTG, a con- ASIC activation on intracellular Ca2ϩ transients and to iden- centration previously shown to effectively regulate other ion 2ϩ channels (Zhang and Cuevas, 2002, 2005) and after 10-min tify the specific ASIC subtype(s) affecting [Ca ]i in our rat cortical neuron model. Figure 1A shows representative traces washout of the drug (Wash). DTG rapidly and reversibly 2ϩ inhibited the low pH-induced transient increases in [Ca2ϩ] . of [Ca ]i as a function of time recorded from a single neuron i during acidosis (pH 6.0) in the absence (Control) and pres- In identical experiments, 100 ␮M DTG produced a statisti- ence of amiloride (100 ␮M) and after a 10-min washout of cally significant decrease (46 Ϯ 3%) in ASIC1a-mediated 2ϩ ␮ at ASPET Journals on January 27, 2020 drug (Wash). The general ASIC inhibitor, amiloride, revers- elevations in [Ca ]i (Fig. 2B). Opipramol (10 M), another 2ϩ Ϯ ␴ ␴ ibly blocked ASIC-mediated increases in [Ca ]i by 88 1% pan-selective -1/ -2 agonist, also inhibited ASIC1a-medi- 2ϩ Ϯ (Fig. 1B). Psalmotoxin1 (PcTx1) from the venom of the taran- ated increases in [Ca ]i by 57 1% (Fig. 2B). The concen-

Fig. 1. ASIC1a blockers inhibit proton-evoked in- 2ϩ creases in [Ca ]i in cultured cortical neurons from embryonic (E18) rats. A, representative traces of 2ϩ [Ca ]i as a function of time recorded from a single cell during acidosis in the absence (Control, Wash) and presence of 100 ␮M Amiloride. B, mean change in peak 2ϩ Ϯ [Ca ]i ( S.E.M.) measured in response to low pH so- lution (pH 6.0) under the indicated conditions (n ϭ 69). 2ϩ C, representative traces of [Ca ]i as a function of time recorded from a neuron during acidosis (Control, Wash) and after 20-min preincubation in 500 ng/ml PcTx1 venom. Psalmotoxin1 venom was only present in the pH 7.4 conditioning solution. D, mean change in peak 2ϩ Ϯ [Ca ]i ( S.E.M.) measured during acidosis under the indicated conditions (n ϭ 158). Asterisks, significant difference from Control and Wash groups in B and D (p Ͻ 0.001). 494 Herrera et al.

Fig. 2. Pan-selective ␴ agonists inhibit proton-evoked tran- 2ϩ sient increases in [Ca ]i. A, representative traces of 2ϩ [Ca ]i as a function of time recorded from a single neuron during acidosis in the absence (Control, Wash) and pres- ␮ 2ϩ ence of 100 M DTG. B, mean change in peak [Ca ]i (ϮS.E.M.) measured during acidosis in the absence (Con- trol; n ϭ 119) and presence of 100 ␮M DTG (n ϭ 99) or 10 significant difference from ,ء .(␮M opipramol (OPI; n ϭ 119 the control group (p Ͻ 0.001); #, significant difference from Ͻ 2ϩ DTG group (p 0.05). C, representative traces of [Ca ]i as a function of time recorded from a neuron during acidosis in the absence (Control) and presence of 100 and 300 ␮M DTG. D, concentration-response relationship for DTG in- 2ϩ Ϯ hibition of mean change in peak [Ca ]i ( S.E.M.). Values obtained for each cell were normalized to their respective Downloaded from controls (no DTG) (n ϭ 117–124). Line represents a best fit to the data using a single-site Langmuir-Hill equation. jpet.aspetjournals.org

tration-response relationship for DTG inhibition of ASIC1a To provide further evidence that ␴-1 receptors modulate was determined to confirm that the actions of DTG on ASIC1a-induced Ca2ϩ elevations, experiments were con- ASIC1a are consistent with ␴ receptor activation. Increasing ducted using the irreversible ␴ antagonist, metaphit, and a ␴

DTG concentrations resulted in further depression of selective -1 antagonist, BD1063, in combination with the at ASPET Journals on January 27, 2020 2ϩ ␴ ASIC1a-mediated increases in [Ca ]i (Fig. 2C). A plot of the -1 agonist, CBP. Cells were exposed to acidosis in the ab- concentration-response relationship obtained from measure- sence and presence of CBP (30 ␮M), with or without prein- ments made in multiple cells is shown in Fig. 2D. The data cubation in metaphit (50 ␮M; 30 min to 1 h, 23°C). CBP 2ϩ were best fit using a single-site Langmuir-Hill equation with decreased the acid-induced elevations in [Ca ]i in control ␮ an IC50 value of 109 M and a Hill coefficient of 0.9. These cells, and this inhibition was lessened by preincubation with values are consistent with the effects of DTG being mediated metaphit (Fig. 4A). Figure 4B shows results from several via activation of ␴ receptors (Zhang and Cuevas, 2002, 2005; similar experiments determining percentage inhibition of 2ϩ Katnik et al., 2006). Taken together, these results suggest acid-induced increases in [Ca ]i observed in the presence of ␴ 2ϩ that receptors depress acid-induced increases in [Ca ]i CBP in control neurons (Control) and neurons preincubated and modulate ASIC1a function. with metaphit (MET). Although CBP decreased the eleva- ␴ 2ϩ Ϯ Receptor subtype-selective agonists were used to identify tions in [Ca ]i evoked by acidosis in control cells by 52 2%, the specific ␴ receptor subtype(s) mediating these effects. the ␴-1 receptor agonist only reduced the response by 30 Ϯ 2ϩ ␴ Representative traces of [Ca ]i as a function of time re- 3% in cells preincubated in the irreversible receptor antag- corded from two cells during acidosis in the absence (Control) onist. This Ͼ40% decrease in the effects of CBP after and presence of the ␴-1 selective agonists carbetapentane metaphit preincubation was statistically significant (p Ͻ (CBP) and dextromethorphan (DEX) at the indicated concen- 0.001). The selective ␴-1 antagonist, BD1063, showed more trations are shown in Fig. 3, A and B, respectively. ␴-1 pronounced effects. Figure 4C shows representative traces of 2ϩ Selective agonists blocked the low pH-induced elevations in [Ca ]i as a function of time recorded from two neurons 2ϩ [Ca ]i in a concentration-dependent and reversible manner. during acidosis in absence (Control) and presence of CBP (30 Concentration-response plots for mean changes in peak ␮M), without (PSS) and with coapplication in BD1063 (10 2ϩ ␴ ␴ [Ca ]i recorded in identical experiments using the -1-se- nM). In identical experiments, the agonist CBP completely lective ligands CBP, DEX, and PRE-084 are shown in Fig. loses its ability to block the low pH-induced elevations in 2ϩ ␴ 3C. The data were best fit using the Langmuir-Hill equation, [Ca ]i in the presence of the antagonist BD1063 compared ␴ and values obtained for IC50 and the Hill coefficients were with a 40% block in control cells (Fig. 4D). Inhibition of -1 13.8 and 0.7 ␮M (CBP), 22 and 0.8 ␮M (DEX), and 13.7 and receptors by BD1063 significantly blocked the effects of CBP ␮ 2ϩ 0.6 M (PRE-084), respectively. Presented for comparison is on ASIC1a-mediated [Ca ]i elevations compared with con- the best fit to the data obtained for CBP inhibition, via ␴-1 trol cells (p Ͻ 0.001). These data confirm the effects of ␴-1 2ϩ 2ϩ receptors, of chemical ischemia-induced increases in [Ca ]i agonists on ASIC1a-mediated increases in [Ca ]i are the ϭ ␮ ␴ in cortical neurons (dotted line, IC50 18.7 M; Hill coeffi- result of these compounds acting on -1 receptors. cient, 0.8) (Katnik et al., 2006). This curve superimposes on Further experiments were carried out using the ␴-2-selec- the responses to CBP observed in the current study. tive ligands ibogaine and PB28 to determine whether the ␴-2 ␴-1 Receptors and ASIC1a Function 495

Fig. 3. Activation of ␴-1 receptors inhibits ASIC1a-induced 2ϩ 2ϩ increases in [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 and 100 ␮M CBP (A) or in the absence (Control) and presence of 10 and 100 ␮M DEX (B). C, concentration-response relationships for 2ϩ Ϯ mean change in peak [Ca ]i ( S.E.M.) measured during acidosis in the presence of the indicated ␴-1 selective li- gands DEX, PRE-084, and CBP. Values were normalized to control (absence of ␴ ligand). Solid and dashed lines, best fits to the data using single-site Langmuir-Hill equations. Dotted line, best fit to the data obtained for CBP inhibition of chemical ischemia-induced (4 mM azide) increases in 2ϩ [Ca ]i and is shown for comparison (Katnik et al., 2006). For each condition, n Ͼ 73. Downloaded from jpet.aspetjournals.org

Fig. 4. Inhibition of ␴-1 receptors blocks CBP-mediated suppression of

2ϩ at ASPET Journals on January 27, 2020 proton-evoked increases in [Ca ]i.A, 2ϩ representative traces of [Ca ]i as a function of time recorded from two dif- ferent neurons during acidosis in the absence (Control) and presence of 30 ␮M CBP, without (PSS, left traces) and with 1-h preincubation in 50 ␮M metaphit (right traces). B, percentage 2ϩ inhibition of [Ca ]i increases (ϮS.E.M.) by CBP measured during acidosis under control conditions (Control; n ϭ 135) and after metaphit preincubation (MET; n ϭ 113). C, rep- resentative traces of acid-induced in- 2ϩ creases in [Ca ]i as a function of time recorded from two different neurons in the absence (Control) and presence of 30 ␮M CBP, without (PSS, left traces) and with coapplication of 10 nM BD1063 (right traces). D, percent- 2ϩ age inhibition of [Ca ]i increases (ϮS.E.M.) by CBP under control con- ditions (Control; n ϭ 178) and in the presence of BD1063 (n ϭ 116). B and -significant difference from con ,ء ,D trol groups (p Ͻ 0.001).

receptor subtype also affects ASIC1a function. Figure 5A PB28 resulted in inhibition of ASIC1a-mediated increases in 2ϩ 2ϩ ␮ shows representative traces of [Ca ]i as a function of time [Ca ]i with IC50 values of 69 and 11 M and Hill coefficients recorded from two cells during acidosis in the absence (Con- of 0.86 and 0.85, respectively. For comparison, the best fit to trol) and presence of ibogaine (IBO; left traces) and PB28 the data obtained for ibogaine inhibition of ICa-induced in- ␴ 2ϩ (right traces) at the indicated concentrations. The -2 ligands creases in [Ca ]i is presented, which has been shown to be ␴ ϭ ␮ inhibited acidosis-evoked increases in mean peak changes in mediated by -2 receptors (dashed line; IC50 31 M; Hill 2ϩ [Ca ]i in a concentration-dependent manner (Fig. 5B). Best coefficient, 1.1) (Zhang and Cuevas, 2002). Unlike the similar fits to the data demonstrated that application of ibogaine and concentration-response relationship observed for CBP inhi- 496 Herrera et al.

Fig. 5. ␴-2 Receptor ligands inhibit 2ϩ ASIC1a-mediated elevations in [Ca ]i at concentrations inconsistent with ␴-2- mediated effects and in a metaphit-in- sensitive manner. A, representative 2ϩ traces of [Ca ]i as a function of time recorded from two neurons during aci- dosis in the absence (Control) and pres- ence of 10 and 100 ␮M (IBO (left traces), or in the absence (Control) and presence of 1 and 10 ␮M PB28 (right 2ϩ traces). B, mean change in peak [Ca ]i (ϮS.E.M.) measured during acidosis in the presence of the ␴-2 selective ligands PB28 and IBO. Solid lines, best fits to the data using single-site Langmuir- Hill equations. Dashed line, best fit to the data obtained for IBO inhibition

of ICa, shown for comparison (Zhang and Cuevas, 2002). For each condition, n Ͼ108 cells. C, [Ca2ϩ] as a function i Downloaded from of time recorded from two different neurons during acidosis in the absence (Control) and presence of 20 ␮M PB28, without (PSS) and with (ϩMET) 1-h preincubation in 50 ␮M metaphit. D, 2ϩ percentage inhibition of [Ca ]i (ϮS.E.M.) by PB28 measured during acidosis under control conditions (Con- jpet.aspetjournals.org trol; n ϭ 324) and after metaphit pre- incubation (ϩMET; n ϭ 276). There is no significant difference between Con- trol and ϩMET groups, (p ϭ 0.64). bition of ASIC1a and ischemia responses, there is a discrep- preincubation (1 h, 23°C) in 10 ␮M thapsigargin (THAP and ϩ ancy between the ibogaine block of responses mediated by THAP DTG, respectively). Thapsigargin alone did not de- at ASPET Journals on January 27, 2020 2ϩ ASIC1a and by VGCCs. To determine whether the effects of crease the elevations in [Ca ]i produced by acidosis, and ␴ ␴ 2ϩ these -2 ligands were mediated by activation of -2 recep- DTG depressed the increases in [Ca ]i under both condi- tors, experiments using metaphit were carried out. PB28 (20 tions (Ϯthapsigargin preincubation). Analysis of the data ␮M) produced an inhibition of ASIC1a-mediated increases in collected in identical experiments indicates that preincuba- 2ϩ ϩ [Ca ]i in both control (PSS) and metaphit ( MET)-treated tion with thapsigargin does not significantly alter the effects 2ϩ cells (Fig. 5C). PB28 blocked ASIC1a-mediated increases in of DTG on acid-mediated increases in [Ca ]i (Fig. 6C). Thus, 2ϩ Ϯ Ϯ [Ca ]i by 67 10 and 60 2% in the absence and presence DTG does not decrease the low pH-induced elevations in ϳ 2ϩ of metaphit preincubation, respectively (Fig. 5D). The 10% [Ca ]i by affecting release of calcium from intracellular reduction in the effects of PB28 produced by metaphit was stores. The fact that depletion of intracellular stores fails to ϭ 2ϩ not statistically significant (p 0.64). Furthermore, BD1063 depress increases in [Ca ]i evoked by acidosis suggests that (10 nM) failed to block the effects of PB28 (20 ␮M) on Ca2ϩ influx through the plasma membrane accounts for 2ϩ 2ϩ ASIC1a-induced elevations in [Ca ]i (data not shown). most, if not all, of the increases in [Ca ]i evoked by ASIC1a These results demonstrate that the effects of PB28 on activation. 2ϩ 2ϩ ASIC1a-mediated increases in [Ca ]i are not mediated by Simultaneous Ca fluorometry and whole-cell patch-clamp ␴-2 receptors because the inhibition of these responses to recordings were performed to study ASIC1a-mediated mem- acidosis by PB28 is metaphit-insensitive and occurs at con- brane currents and to determine how much of the observed centrations inconsistent with ␴-2 receptor activation. Ca2ϩ influx is due to Ca2ϩ entry through ASIC1a channels. The activation of ␴ receptors has been shown previously to Cells were voltage-clamped at Ϫ70 mV to minimize NMDA directly affect Ca2ϩ release from intracellular stores (Cas- receptor and VGCC activation and, thus, isolate ASIC1a cur- sano et al., 2006). Thus, experiments were conducted to re- rents. Figure 7A shows that ASIC1a stimulation by low pH solve whether ␴ receptor activation reduces acid-induced solution (pH 6.0) resulted in a small intracellular Ca2ϩ tran- 2ϩ 2ϩ increases in [Ca ]i, in part via the inhibition of calcium- sient measured in a patched cell ([Ca ]i V-Clamp). In contrast, 2ϩ induced calcium release from the endoplasmic reticulum, a second cell ([Ca ]i Control) in the same field of view, which triggered by Ca2ϩ influx through the plasma membrane. For was not voltage clamped, had a significantly larger increase in 2ϩ these experiments, thapsigargin was used to block the sar- [Ca ]i. Acidosis also resulted in a large inward current in the coplasmic/endoplasmic reticulum Ca2ϩ-ATPase, which re- patched cell (Fig. 7A, inset). The cumulative results from sev- sults in depletion of both ryanodine- and IP3-sensitive stores. eral similar experiments demonstrate that ASIC1a activation 2ϩ Ϫ Figure 6A shows representative traces of [Ca ]i as a func- of neurons voltage clamped at 70 mV resulted in elevations in 2ϩ tion of time recorded from a neuron during acidosis in the [Ca ]i that were an order of magnitude smaller than changes absence (Control) and presence of 100 ␮M DTG, whereas Fig. evoked in cells that were not voltage-clamped (Fig. 7B). These 6B shows traces in the absence and presence of DTG after data confirm ASIC1a activation results in minimal Ca2ϩ influx ␴-1 Receptors and ASIC1a Function 497

Fig. 6. Intracellular Ca2ϩ stores are not involved in DTG modulation of ASIC1a- 2ϩ induced increases in [Ca ]i. A, represen- 2ϩ tative traces of [Ca ]i as a function of time recorded from a neuron during aci- dosis in the absence (Control) and pres- ence of 100 ␮M DTG. B, representative 2ϩ traces of [Ca ]i as a function of time recorded from a neuron during acidosis after preincubation in 10 ␮M thapsigar- gin (1 h, 23°C) in the absence (THAP) and presence of 100 ␮M DTG (THAP ϩ DTG). 2ϩ C, mean change in peak [Ca ]i (ϮS.E.M.) measured in response to acido- sis with (THAP; n ϭ 346) or without (PSS; n ϭ 216) thapsigargin preincuba- tion in the absence (Control) and pres- Downloaded from -significant differ ,ء .ence of 100 ␮M DTG ence from the control group (p Ͻ 0.001). There was no significant difference be- tween PSS and THAP groups (p ϭ 0.48). jpet.aspetjournals.org

2ϩ at ASPET Journals on January 27, 2020 Fig. 7. ASIC1a-mediated [Ca ]i in- creases are membrane potential de- pendent. A, representative traces of 2ϩ [Ca ]i recorded in response to ASIC1a activation from a neuron elec- trically accessed using the perforated patch whole-cell configuration and Ϫ 2ϩ held at 70 mV ([Ca ]i V-Clamp) and from a second neuron in the same field of view that was not electrically 2ϩ accessed ([Ca ]i; Control). Inset, whole-cell current trace recorded si- multaneously from the voltage- clamped neuron. Lines above traces indicate application of pH 6.0 solu- tion. Scale bars, 500 pA, 5 s. B, mean 2ϩ Ϯ change in peak [Ca ]i ( S.E.M.) measured during acidosis in nonvolt- age-clamped neurons (Control; n ϭ 37) and in voltage-clamped neurons -significant differ ,ء .(V-Clamp; n ϭ 4) ence between groups (p Ͻ 0.001). C, 2ϩ representative traces of [Ca ]i re- corded in response to application of extracellular high Kϩ (40 mM) solu- tion in the absence (Control) and pres- ence of 100 ␮M CBP. D, bar graph of 2ϩ mean change in peak [Ca ]i (ϮS.E.M.) measured in experiments identical to those shown in (C) (n ϭ significant difference between ,ء .(59 groups (p Ͻ 0.001).

through the ASIC1a channel itself and that the majority of the It was noted that in cortical neurons held under current- 2ϩ acid-induced [Ca ]i increases are mediated by downstream clamp mode, application of protons evokes a rapid depolar- Ca2ϩ influx pathways. ization that, unlike the case in voltage-clamped neurons, was 498 Herrera et al.

2ϩ associated with pronounced elevations in [Ca ]i (data not shown). Thus, ASIC1a channels are probably promoting sig- nificant Ca2ϩ influx into the neurons by depolarizing the cells. To mimic this change in membrane potential evoked upon ASIC1a activation, cells were exposed to high-Kϩ (40 mM) extracellular solution. Figure 7C shows representative 2ϩ ϩ [Ca ]i traces recorded in response to high-Ko application in the absence (Control) and presence of CBP (100 ␮M, ϩCBP). Depolarizing the neurons in this manner evoked robust 2ϩ [Ca ]i elevations that were blocked by addition of CBP. In ϩ identical experiments, CBP reduced the high Ko -evoked in- 2ϩ Ϯ creases in [Ca ]i by 83 1%, and this decrease was statis- tically significant (p Ͻ 0.001) (Fig. 7D). Thus, ␴ receptor activation inhibits Ca2ϩ channels downstream of ASIC1a in addition to the proton-gated channels themselves. To determine the specific ion channels contributing to the 2ϩ ASIC1a-induced [Ca ]i influxes, several inhibitors of

plasma membrane ion channels were used. Activation of Downloaded from ASIC1a depolarizes these neurons, which could stimulate action potential firing and, consequently, synaptic transmis- 2ϩ sion, both of which may elevate [Ca ]i. Thus, tetrodotoxin (TTX; 500 nM) was used to inhibit voltage-gated Naϩ chan- nels to prevent the genesis of action potentials. Figure 8A ϳ shows that application of TTX inhibited 10% of the acid- jpet.aspetjournals.org 2ϩ induced increases in [Ca ]i but did not significantly affect CBP (50 ␮M) modulation of the Ca2ϩ responses, compared with the PSS group. Because TTX had no effect on the ␴-1/ ASIC1a interaction, all subsequent experiments included 500 nM TTX in the bath solutions to prevent spontaneous action potentials from contributing to the measured in- 2ϩ

creases in [Ca ]i. Inhibition of NMDA receptors by AP5 (100 at ASPET Journals on January 27, 2020 ␮ Fig. 8. Multiple plasma membrane ion channels downstream of ASIC1a M) significantly reduced the acid-induced elevations in activation contribute to acidosis-evoked [Ca2ϩ] increases. A, relative 2ϩ i ␮ 2ϩ Ϯ [Ca ]i, and coapplication with CBP (50 M) resulted in a changes in [Ca ]i ( S.E.M.) during acidosis in the absence (PSS) and 2ϩ presence of 500 nM TTX alone or TTX (500 nM) coapplied with 100 ␮M further decrease in [Ca ]i that was statistically significant ϳ AP5, 10 ␮M nifedipine, or 100 ␮M cadmium. All combinations were done ء Fig. 8A). These results show that 30% of ASIC1a-induced ␮ Ͼ) 2ϩ without (Control) and with 50 M CBP. For each condition, n 101. , [Ca ]i increases are dependent on NMDA receptor activa- significant differences from respective control groups (p Ͻ 0.001); #, tion. Furthermore, because the effects of AP5 and CBP are significant differences from PSS group (p Ͻ 0.01 for TTX versus PSS, p Ͻ less than additive, activation of ␴-1 receptors depresses 0.001 for all others); †, significant differences from TTX group (p Ͻ 0.001). 2ϩ Ϯ 2ϩ B, relative changes in [Ca ]i ( S.E.M.) during acidosis in the absence ASIC1a-evoked increases in [Ca ]i, in part by blocking this (Control) and presence of 10 ␮M CNQX without CBP (PSS) or with CBP significant ,ء .NMDA receptor-dependent component. Blockade of L-type at the indicated concentrations. For each condition, n Ͼ 95 VGCCs by nifedipine (10 ␮M) significantly inhibited (Ͼ75%) differences from PSS group within Control or CNQX groups (p Ͻ 0.001); ␮ acid-induced [Ca2ϩ] elevations, and CBP (50 ␮M) continued #, significance differences between 50 and 300 M CBP groups within i Control or CNQX groups (p Ͻ 0.001); †, significant differences between to provide further blockade of the residual increases in control and CNQX within PSS (p Ͻ 0.001) and 300 ␮M CBP (p Ͻ 0.05) 2ϩ ␮ [Ca ]i (Fig. 8A). Cadmium (100 M), a broad-spectrum groups. blocker of plasma membrane calcium channels, inhibited ϳ 2ϩ 2ϩ 90% of the acid-induced increases in [Ca ]i, and CBP (50 did produce an additional block of the Ca response on top ␮M) had no effect on the remaining Ca2ϩ influx (Fig. 8A). of the effects of CNQX. Figure 8B shows that coapplication of Thus, voltage-gated Ca2ϩ channels either directly (influx 300 ␮M CBP and CNQX blocked Ͼ80% of the acid-induced 2ϩ through the channel) or indirectly (facilitating glutamate increases in [Ca ]i, and this result was statistically signifi- 2ϩ 2ϩ release) account for most of the [Ca ]i increases resulting cant compared with CNQX alone. Thus, [Ca ]i elevations from ASIC1a activation, and ␴-1 receptor activation provides elicited upon ASIC1a activation involve Ca2ϩ influx through an inhibition similar to that observed with Cd2ϩ. both ASIC1a and other Ca2ϩ-permeable plasma membrane Further experiments were conducted to determine ion channels expressed in cortical neurons. More impor- 2ϩ ␴ whether AMPA/kainate receptors are involved in the [Ca ]i tantly, it is also evident that activation of -1 receptors elevations elicited upon ASIC1a activation. Figure 8B shows results in depression of Ca2ϩ influx through all of these 2ϩ relative changes in [Ca ]i in the absence (Control) and pres- sources, either by inhibiting ASIC1a or the downstream ence of the AMPA/ blocker CNQX (10 ␮M) channels themselves. and the effects of CBP at two different concentrations (50 and Two ASIC blockers, amiloride and PcTx1, were used to 300 ␮M). Maximal blockade of AMPA/kainate receptors alone confirm that the acid-activated inward currents observed in ϳ 2ϩ produced 40% reduction of the elevations of [Ca ]i, and 50 our cortical neuron model were mediated by ASIC1 channels. ␮M CBP did not provide additional, statistically significant Figure 9A shows representative traces of membrane currents block. However, increasing the CBP concentration to 300 ␮M as a function of time recorded from a single cell in the ab- ␴-1 Receptors and ASIC1a Function 499

Fig. 9. 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 Ϫ70 mV in the absence (Control) and presence of 100 ␮M amiloride (i) or 500 ng/ml PcTx1 venom (ii). B, relative mean peak proton-gated currents (ϮS.E.M.) re- corded from neurons in normal PSS (Con- trol) or in PSS containing either 100 ␮M amiloride (n ϭ 6) or 500 ng/ml PcTx1 venom (n ϭ 4). Values were normalized to the maximal proton-evoked response re- corded for control conditions (no drug) in ء

each cell (I/Imax). , significant difference Downloaded from from the control group (p Ͻ 0.01). C, rel-

ative peak proton-gated currents (I/Imax, ϮS.E.M.) recorded in the absence (PSS) and presence of 500 nM TTX (n ϭ 4) alone and TTX with 100 ␮M AP-5 (n ϭ 3), 10 ␮M CNQX (n ϭ 4), 10 ␮M nifedipine (Nif; n ϭ 6), or 100 ␮M cadmium (Cd; n ϭ 3) in

the bath solution. Cells were voltage- jpet.aspetjournals.org clamped at Ϫ70 mV. No significant differ- ences between the groups were noted (p ϭ 0.826). at ASPET Journals on January 27, 2020

sence (Control) and presence of amiloride (100 ␮M, top show that activation of ␴ receptors with DTG inhibited traces, i), and from a different neuron in the absence (Con- ASIC1a-mediated currents by 53 Ϯ 9%, and this decrease trol) and presence of PcTx1 (500 ng/ml venom, bottom traces, was statistically significant (Fig. 10B). To determine whether ii). Figure 9B summarizes the normalized peak proton-gated the effects of DTG on ASIC1a currents were mediated by ␴-1 whole-cell currents recorded from several cells in the absence or ␴-2 receptors, the effects of the ␴-1 selective agonist CBP, (Control) and presence of amiloride and PcTx1. Amiloride on whole-cell currents were measured. Figure 10C shows produced a 79 Ϯ 6% inhibition of acid-activated currents, representative membrane current traces as a function of time whereas 500 ng/ml PcTx1 containing venom produced a 42 Ϯ recorded in the absence (Control) and presence of CBP (50 11% reduction in this current. Both reductions were signifi- ␮M). After multiple experiments, analysis of the measured cantly different from control (p Ͻ 0.01). Experiments were current densities showed that control cells had statistically also carried out to confirm that ASIC1a currents were not significant larger current densities than CBP-treated cells affected by the channel blockers used in the imaging studies (Fig. 10D). In the presence of 50 ␮M CBP, ASIC1a-mediated described above. Figure 9C shows relative peak proton-gated currents were decreased by 30 Ϯ 5% relative to control. In currents, normalized to control (PSS), during acidosis in the contrast, PB28 (20 ␮M) failed to inhibit ASIC1a-mediated absence (PSS) and presence of the indicated drugs. These membrane currents, suggesting ␴-2 receptors do not regulate data demonstrate that TTX, AP5, CNQX, nifedipine, or cad- ASIC1a function (data not shown). Therefore, these results mium do not have any direct effects on ASIC1a channels. demonstrate that ␴-1 receptors functionally couple to ␴ Receptor activation has been shown to modulate multiple ASIC1a channels in cortical neurons and to targets down- cell membrane ion channels in neurons that are activated stream of ASIC1a activation. after ASIC1a stimulation (Hayashi et al., 1995; Zhang and ␴ Cuevas, 2002). This fact raises the possibility that -1 recep- Discussion tors only couple to the secondary events that result from ASIC1a activation and not to ASIC1a itself. To investigate The principal finding reported here is that activation of ␴ whether ␴ receptors affect ASIC1a function, whole-cell patch- receptors depresses membrane currents and elevations in ␴ 2ϩ clamp recordings were performed in the presence of ago- [Ca ]i mediated by ASIC1a channels in cortical neurons. nists. Figure 10A shows representative membrane current The pharmacological properties of the receptor involved are traces as a function of time recorded from a cell in the consistent with the effects being specifically mediated by the absence (Control) and presence of the pan-selective ␴ agonist ␴-1 receptor subtype. Furthermore, most of the elevations in ␮ 2ϩ 2ϩ DTG (100 M). Results from several similar experiments [Ca ]i triggered by acidosis are the result of Ca channels 500 Herrera et al.

Fig. 10. ␴ 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 perfor- ated-patched neuron held at Ϫ70 mV in the ab- sence (Control) and presence of 100 ␮M DTG. B, mean peak proton-gated current densities (ϮS.E.M.) measured from neurons held at Ϫ70 mV without (Control) or with 100 ␮M DTG in the bath solution (n ϭ 6). C, representative traces of ASIC1a-mediated currents as a func- tion of time recorded from a voltage-clamped neuron (Ϫ70 mV) in the absence (Control) and presence of 50 ␮M CBP. D, mean peak proton- Downloaded from gated current densities (ϮS.E.M.) recorded un- der identical conditions as (C) (n ϭ 10). B and C, significant differences from respective control ,ء groups (p Ͻ 0.001). jpet.aspetjournals.org

opening downstream of ASIC1a activation. Stimulation of responses to ASIC1a activation. Studies have shown that the at ASPET Journals on January 27, 2020 ␴-1 receptors effectively suppressed these secondary Ca2ϩ affinity of carbetapentane for ␴-1 receptors is Ͼ50-fold fluxes both by inhibiting ASIC1a and the other channels greater than for ␴-2 receptors (Rothman et al., 1991; Vilner

directly. and Bowen, 2000). The calculated IC50 for carbetapentane 2ϩ ␴ ASIC are regulated by various factors such as pH, mem- inhibition of ischemia-evoked increases in [Ca ]i via -1 brane distention, and arachidonic acid and, therefore, func- receptor activation is 18.7 ␮M (Katnik et al., 2006), which is ␮ tion as signal integrators in the CNS (Allen and Attwell, comparable with the 13.8 MIC50 for CBP inhibition of 2ϩ 2002; Lopez, 2002). All of these factors elicit or potentiate ASIC1a-induced [Ca ]i increases. Carbetapentane also in- ASIC-mediated responses. Information on endogenous mech- hibits epileptiform activity in rat hippocampal slices via ␴-1 ␮ anisms that inhibit ASIC function is lacking. It has been receptors with an IC50 value of 38 M (Thurgur and Church, shown that NMDA receptors modulate ASIC1a function via 1998). Likewise, we show that the ␴-1 agonists dextrome- ϭ ␮ ϭ ␮ the activation of a calcium/calmodulin-dependent protein ki- thorphan (IC50 22 M) and PRE-084 (IC50 13.7 M), nase II signaling cascade, but activation of this pathway both of which have Ͼ100-fold greater affinities for ␴-1 than results in an increase in currents through ASIC1a (Gao et al., ␴-2 receptors, block ASIC1a-mediated responses at concen- 2005). Thus, our finding that activation of ␴ receptors de- trations consistent with those reported in the literature. Dex- presses ASIC1a-mediated responses is novel. Our conclusion tromethorphan inhibits spreading depression in rat neocor- ϳ ␮ that the responses observed are mediated specifically by tical brain slices with an IC50 30 M (Anderson and ASIC1a is supported by the inhibition produced with the Andrew, 2002), whereas PRE-084 protects human retinal ϳ ␮ selective ASIC1a channel blocker, PcTx1 (Diochot et al., cells against oxidative stress with an IC50 10 M (Bucolo et 2007), and that cultured cortical neurons from embryonic al., 2006). The fact that IC50 values determined here for mice deficient in the ASIC1a subunit fail to show increases in carbetapentane, dextromethorphan, and PRE-084 are in the 2ϩ [Ca ]i or membrane currents at the proton concentrations low micromolar range suggests that it is unlikely these ago- used here (Xiong et al., 2004). ASIC2a and ASIC2b subunits nists are affecting ASIC1a activity via ␴-2 receptors because are also expressed in the CNS, but homomeric ASIC2a chan- high micromolar to millimolar concentrations of these com- nels are activated below pH 5.5, and ASIC2b does not gen- pounds are required to stimulate ␴-2 receptors. Moreover, erate currents in response to low pH (Lingueglia et al., 1997). ␴-2 selective agonists failed to inhibit ASIC1a-mediated re- Furthermore, neither homomeric ASIC2a nor heteromulti- sponses at concentrations consistent with ␴-2-specific effects. meric ASIC1a/ASIC2a channels conduct Ca2ϩ and thus could The strongest evidence that ␴-1 receptor activation modu- 2ϩ ␴ not account for the changes in [Ca ]i observed here (Yermo- lates ASIC1a comes from experiments using the antago- laieva et al., 2004). nists, metaphit and BD1063. Metaphit has been shown to 2ϩ ␴ ␮ Results from Ca imaging experiments suggest that it is bind irreversibly to -1 receptors with an IC50 value of 50 M specifically the ␴-1 receptor subtype that modulates neuronal (Wu et al., 2003). Preincubation in metaphit blocks ␴-1 re- ␴-1 Receptors and ASIC1a Function 501 ceptor-mediated modulation of voltage-gated Kϩ channels in down-regulates all of these events. However, the fact that intracardiac neurons and depression of ischemia-induced el- activation of ␴-1 receptors depressed ASIC1a-mediated cur- 2ϩ Ϫ ␴ evations in [Ca ]i in cortical neurons (Zhang and Cuevas, rents in cells voltage-clamped at 70 mV indicates that -1 2005; Katnik et al., 2006). Preincubation of cortical neurons receptors are functionally coupled to ASIC1a and that the ␮ 2ϩ in 50 M metaphit antagonized CBP inhibition of ASIC1a by depression in acid-evoked increases in [Ca ]i is not exclu- ϳ40%. BD1063 has been shown to have a higher affinity for sively the result of ␴-1 receptors blocking channels down- ␴-1 than ␴-2 receptors and attenuates the dystonia produced stream of ASIC1a. by DTG in rats in a dose-dependent manner, suggesting this The finding that ␴-1 receptors can inhibit ASIC1a channels ligand acts as an antagonist at ␴ sites (Matsumoto et al., has significant physiological and pathophysiological implica- 1995). Here, we show that CBP is unable to block acid- tions. It has been proposed that ASIC1a activation may fa- 2ϩ induced increases in [Ca ]i when coapplied with BD1063. In cilitate neurotransmission by compensating for the decrease addition, we found that metaphit fails to inhibit the effects of in excitatory neurotransmission caused by direct inhibition the ␴-2 agonist, PB28, on ASIC1a-mediated responses. of postsynaptic Naϩ and Ca2ϩ channels by protons that are 2ϩ Taken together, these data show that increases in [Ca ]i in released during exocytosis (Krishtal et al., 1987; Zha et al., response to ASIC1a activation are modulated only by ␴-1 2006). Furthermore, the expression levels of ASIC1a have receptors. direct effects on the density of dendritic spines in hippocam- Several studies have suggested that Ca2ϩ influx through pal neurons (Zha et al., 2006). Thus, ␴-1 receptors may in-

ASIC1a channels is a key mechanism leading to cell death fluence cell-to-cell signaling in the CNS by affecting ASIC1a Downloaded from (Xiong et al., 2004; Yermolaieva et al., 2004). Depletion of activity. One of the consequences of ASIC1a overexpression Ca2ϩ from intracellular stores indicates that most, if not all, in mice is enhanced fear conditioning (Wemmie et al., 2004), 2ϩ ␴ of the acid-induced increases in [Ca ]i is due to plasma whereas stimulation of -1 receptors is known to ameliorate membrane influx. However, our results show that multiple conditioned fear stress (Kamei et al., 1997). These observa- ion channels downstream of ASIC1a activation contribute to tions, coupled with our current report, suggest that ␴-1 re- 2ϩ acidosis-induced elevations in [Ca ]i, including NMDA and ceptor activation may produce anxiolytic effects via the inhi- jpet.aspetjournals.org AMPA/kainate receptors and VGCCs. The activation of bition of ASIC1a channels. NMDA and AMPA/kainate receptors after ASIC1a stimula- The inhibition of ASIC1a by ␴-1 receptors is a potential tion was observed even when neuronal conduction was inhib- component of the neuroprotective properties of ␴ receptors ited with tetrodotoxin. This observation suggests a presyn- because activation of ASIC1a has been shown to contribute to aptic localization of ASIC1a, whereby activation of the stroke injury (Xiong et al., 2004). More importantly, inhibi- channel by protons results in synaptic transmission and sub- tion of ASIC1a has been shown to be neuroprotective at

sequent activation of postsynaptic glutamatergic receptors. delayed time points after ischemic stroke (Simon, 2006). at ASPET Journals on January 27, 2020 Consistent with this hypothesis, ASIC1a has been found to Thus, ␴-1 receptor-mediated inhibition of ASIC1a may con- regulate neurotransmitter release probability in mouse hip- tribute to the enhanced neuronal survival after ␴ receptor pocampal neurons (Cho and Askwith, 2008). activation 24 h poststroke in rats (Ajmo et al., 2006). Fur- ␴ Receptors have been identified in both presynaptic and thermore, our data suggest that activation of ASIC1a stim- postsynaptic sites (Gonzalez-Alvear and Werling, 1995; ulates the activity of NMDA and AMPA/kainate receptors Alonso et al., 2000) and thus may modulate channels in both and VGCCs, all of which have been linked to ischemia-in- regions. In the presence of specific inhibitors of ionotropic duced brain injury. Thus, ␴-1 receptor activation may provide glutamate receptors, activation of ␴-1 receptors with CBP further neuroprotection by reducing the activity of these 2ϩ further decreased proton-evoked increases in [Ca ]i, but the channels, which occurs subsequent to ASIC1a stimulation. effects of CBP and the glutamate channel inhibitors were less Consistent with this pleiotropic effect of ␴-1 receptors is our than additive. Thus, ␴-1 receptors also inhibit Ca2ϩ entry via observation that ␴-1 receptor activation suppressed extracel- ϩ 2ϩ NMDA and AMPA/kainate receptors directly by inhibiting lular high-K -induced increases in [Ca ]i, which would also these channels and indirectly by depressing ASIC1a activa- activate these downstream effectors. In conclusion, ␴-1 re- tion. Application of the L-type VGCC inhibitor, nifedipine, ceptors inhibit ASIC1a channel function and blunt acidosis- 2ϩ 2ϩ ␴ and the broad-spectrum Ca channel inhibitor, cadmium, evoked ionic fluxes and increases in [Ca ]i. Thus, -1 2ϩ Ͼ blocked ASIC1a-induced increases in [Ca ]i by 70 and receptors can be targeted for therapeutic intervention in Ͼ90%, respectively. This observation indicates that most of pathophysiological conditions involving ASIC1a activation. 2ϩ the increases in [Ca ]i produced upon ASIC1a activation are 2ϩ dependent on Ca influx through VGCCs. Coapplication of Acknowledgments CBP with nifedipine, but not with Cd2ϩ, resulted in further 2ϩ We thank Nivia Cuevas for comments on a draft of the manu- reduction in the proton-evoked increases in [Ca ]i. The con- script. clusion that Ca2ϩ influx through ASIC1a channels itself con- 2ϩ tributed only a small fraction to the total observed [Ca ]i References 2ϩ increases was confirmed with simultaneous Ca fluorome- Ajmo CT Jr, Vernon DO, Collier L, Pennypacker KR, and Cuevas J (2006) Sigma try and whole-cell patch-clamp recordings. Cells voltage- receptor activation reduces infarct size at 24 hours after permanent middle cere- Ϫ bral artery occlusion in rats. Curr Neurovasc Res 3:89–98. clamped at 70 mV, which prevents NMDA receptor and Allen NJ and Attwell D (2002) Modulation of ASIC channels in rat cerebellar VGCC activation, demonstrated minimal acid-evoked eleva- Purkinje neurons by ischaemia-related signals. J Physiol 543:521–529. 2ϩ Alonso G, Phan V, Guillemain I, Saunier M, Legrand A, Anoal M, and Maurice T tions in [Ca ]i. Taken together, our results show that the (2000) Immunocytochemical localization of the sigma(1) receptor in the adult rat 2ϩ increases in [Ca ]i evoked by ASIC1a activation are the central nervous system. Neuroscience 97:155–170. Anderson TR and Andrew RD (2002) Spreading depression: imaging and blockade in result of synaptic transmission and subsequent opening of the rat neocortical brain slice. J Neurophysiol 88:2713–2725. 2ϩ multiple Ca channels and that stimulation of ␴-1 receptors Askwith CC, Wemmie JA, Price MP, Rokhlina T, and Welsh MJ (2004) Acid-sensing 502 Herrera et al.

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