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J. Biol. Chem., Vol. 277, Issue 52, 50463-50468, December 27, 2002

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Protein Kinase C Stimulates the Acid-sensing Ion Channel ASIC2a via the PDZ Domain-containing Protein PICK1^*

Anne Baron, Emmanuel Deval, Miguel Salinas, Eric Lingueglia, Nicolas Voilley, and Michel Lazdunski^§

From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR6097, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

Received for publication, August 29, 2002, and in revised form, October 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in central and sensory neurons where they are involved in neuromodulation and in pain perception. Recently, the PDZ domain-containing protein PICK1 (protein interacting with C-kinase) has been shown to interact with ASIC1a and ASIC2a, raising the possibility that protein kinase C (PKC) could regulate ASICs. We now show that the amplitude of the ASIC2a current, which was only modestly increased (~+30%) by the PKC activator 1-oleyl-2-acetyl-sn-glycerol (OAG, 50 µM) in the absence of PICK1, was strongly potentiated (~+300%) in the presence of PICK1. This PICK1-dependent regulatory effect was inhibited in the presence of a PKC inhibitory peptide and required the PDZ domain of PICK1 as well as the PDZ-binding domain of ASIC2a. We have also shown the direct PICK1-dependent phosphorylation of ASIC2a by [³²P]phosphate labeling and immunoprecipitation and identified a major phosphorylation site, ³⁹TIR, on the N terminus part of ASIC2a. The OAG-induced increase in ASIC2a current amplitude did not involve any change in the unitary conductance of the ASIC2a channel, whether co-expressed with PICK1 or not. These data provide the first demonstration of a regulation of ASICs by protein kinase phosphorylation and its potentiation by the partner protein PICK1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In sensory and central neurons, H⁺-activated cation currents flowing through acid-sensing ionic channels (ASICs)¹ have been widely recorded, showing functional and pharmacological properties depending on the homomeric or heteromeric association of different ASIC subunits (1, 2). Some ASIC subunits are specifically expressed in sensory neurons, such as ASIC1b (3) and ASIC3 (4, 5), whereas ASIC1a (5-8), ASIC2a (6, 8-11), ASIC2b (10), and ASIC4 (8, 12, 13) are also found in central neurons.

In sensory neurons, ASIC-like currents are thought to play an important role in nociception during tissue acidosis and inflammation (2, 5, 7, 14-17), and ASIC2a has also been proposed to participate in touch sensation (11). In the central nervous system, an important role for ASICs in neuromodulation of the synaptic transmission has been proposed (18). In hippocampal neurons, we have recently shown that small pH changes, compatible with local transient acidifications reported in the central nervous system during normal neuronal activity (18-21), could activate an ASIC-like current and trigger a membrane depolarization, leading to trains of action potentials (22). This hippocampal ASIC-like current was co-activated by zinc acting on ASIC2a-containing channels (22, 23). A role for ASICs in hippocampal synaptic plasticity and memory processes has been proposed recently (24). ASICs could also be involved in pathological situations such as brain ischemia and epilepsy that both produce significant extracellular acidification (25-28).

Although ASIC currents can be recorded in virtually all types of neuronal cells, their physiological regulation by mediators, transduction pathways, or protein kinases is unknown, although several putative phosphorylation sites can be predicted from the ASIC protein sequences. Recently, two PDZ domain-containing partner proteins have been isolated by yeast two-hybrid cloning: CIPP (channel-interacting PDZ domain-containing protein), which interacts with the ASIC3 C terminus (29), and PICK1 (protein interacting with C-kinase), which interacts with the ASIC1a and ASIC2a C terminus (30, 31). CIPP was shown to increase the ASIC3 current membrane density (29), but no functional effect of PICK1 on ASIC currents has been reported so far.

PICK1 was originally isolated by its ability to bind the C terminus of protein kinase C (PKC) via its single PDZ domain (32-34). Although PICK1 has only one PDZ domain, it can multimerize through its coiled-coil region and therefore cross-link two different partners (32, 33, 35), thus providing a molecular mechanism for the targeting of PKC to specific proteins regulated by phosphorylation. ASIC1a and ASIC2a co-localize with PICK1 at the peripheral sensory endings of dorsal root ganglia neurons as well as at the synapses and cell bodies of some central neurons, such as pyramidal hippocampal neurons (30, 31).

This work analyzes the regulation of ASIC2a by PKC. It shows that the ASIC2a current amplitude is increased by PKC phosphorylation and that PICK1 potentialized this regulation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Constructions and Mutagenesis-- Mouse PICK1 coding sequence (GenBank^TM accession number NM008837; 96% amino acid identity with rat PICK1 and identical PDZ domain) was amplified by PCR from mouse brain cDNA, sequenced, and subcloned into the bicistronic vector pCI-IRES-CD8 (36) for expression in COS cells. The PICK1PDZ mutant was obtained by replacing the ²⁷KD²⁸ residues by two alanines in the PDZ domain (35). The rat ASIC2a coding sequence (GenBank^TM accession number U53211) was subcloned into pCI vector (Promega) for expression in COS cells. The potential PKC phosphorylation site, ³⁹TIR, was suppressed by the introduction of the T39G or T39D point mutations. In another mutant, named ASIC2aC, the C-terminal PDZ-binding domain was suppressed by deletion of the last three residues (30, 31). Point mutations and deletions of rASIC2a and mPICK1 were performed by PCR strategies and entirely sequenced. ASIC2a and PICK1 wild-type and mutant proteins corresponding to the cDNAs used in the present work are schematically described in Fig. 1.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Schematic representation of ASIC2a and PICK1 wild-type and mutant proteins. In A, the ASIC2a protein displays two transmembrane domains (TM1 and TM2) with a cytoplasmic N and C terminus and a large extracellular loop. The last three amino acids of the protein correspond to the PDZ-interacting domain, which have been deleted in the mutant ASIC2aC. A PKC phosphorylation site, 39TIR, was predicted in the N-terminal domain of ASIC2a and mutated in ASIC2aT39G and ASIC2aT39D mutants. As shown in B, PICK1 contains a large PDZ domain implicated in the interaction with the C terminus of ASIC2a and a coiled-coil domain that is responsible for the protein dimerization. In the mutant PICK1PDZ, the two amino acids Lys-27 and Asp-28, which are critical in the PDZ domain, have been mutated to Ala.

Expression of PICK1 and ASIC2a in COS Cells-- COS cells, at a density of 20,000 cells/35-mm diameter Petri dish, were transfected using the DEAE-dextran method with a mix of pCI-rASIC2a, pCI-rASIC2aC, pCI-rASIC2aT39G, pCI-rASIC2aT39D with pCI-IRES-CD8, pCI-PICK1-IRES-CD8, pCI-PICK1PDZ-IRES-CD8 (1:10 ratio). Cells were used for electrophysiological measurements for 1-3 days after transfection. Successfully transfected cells were recognized by their ability to fix CD8 antibody-coated beads (Dynal A. S., Oslo, Norway). Ion currents were recorded using the whole cell and the outside-out modes of the patch clamp technique (37). Data were sampled at either 500 Hz or 10 kHz for whole cell and outside-out recordings, respectively, and low pass-filtered at 3 kHz using the pClamp8 software (Axon Instruments, Foster City, CA). Off-line low pass 1-kHz gaussian filtering and analysis of currents were performed using pClamp (Axon Instruments, Foster City, CA). The statistical significance of differences between sets of data was estimated by the Student's t test. The pipette solution contained 140 mM KCl, 5 mM NaCl, 2 mM MgCl₂, 5 mM EGTA, 10 mM HEPES (pH 7.3), and the bath solution contained 150 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES (pH 7.4). MES or acetate was used instead of HEPES to buffer the bath solution pH ranging from 6 to 5 and from 4.5 to 3, respectively. Changes in extracellular pH were induced by shifting one out of eight outlets of a microperfusion system in front of the cell. Experiments were carried out at room temperature (20-22 °C).

Chemicals-- Phorbol 12,13-dibutyrate (PDBu), the cell-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), and protein kinase C 19-36 inhibitory peptide (PKC-I) were all from Sigma.

In Vivo Phosphorylation of ASIC2a-- P100 dishes containing 3 million COS cells were transfected with 0.5 µg of pCI-rASIC2a with or without 5 µg of pCI-PICK1-IRES-CD8 using Exgen500 (Euromedex) following the supplier's protocol. Cells were serum-deprived for 24 h, phosphate-deprived for 2 h, and labeled with [³²P]orthophosphate (100 µCi/ml) for 3 h. PDBu stimulation was carried out for 20 min (2 µM). Cell numbers in the different conditions were checked to be equivalent. After phosphate-buffered saline wash, confluent cell layers were scraped in TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, protease inhibitor mixture (Roche Applied Science), phosphatase inhibitor mixture (Sigma)). After centrifugation, the membrane pellets were resuspended in TNEN buffer (TNE containing 1% Nonidet P-40) and sonicated. After a 30-min centrifugation at 100,000 × g, the supernatants were precleared with protein-A-Sepharose and incubated overnight at 4 °C with 1 µg of rabbit anti-rat ASIC2a antibody (Alomone Laboratories). Protein-A-Sepharose was added for 1 h at 4 °C and then washed five times with TNEN. Immunoprecipitated proteins were boiled in SDS buffer and separated by SDS-PAGE (8% polyacrylamide). The gel was vacuum-dried and analyzed with a BAS-1500 Fujifilm phosphorimaging device and Tina 2.0.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of PICK1 on Functional Properties and Density of ASIC2a Current-- When co-expressed with ASIC2a, PICK1 did not significantly modify the pH dependence of the current (Fig. 2A), the inactivation time constant, nor the time-to-peak of the current (Table I, lines 1 and 2). Also, the ASIC2a current density was not increased in the presence of PICK1 (Fig. 2B), unlike the situation observed with ASIC3 after its interaction with another PDZ domain-containing partner protein CIPP (29). A slight decrease of the ASIC2a current density was observed in the presence of PICK1 (27 ± 3 pA/pF versus 33 ± 5 pA/pF with and without PICK1, respectively), but it was not significant (p = 0.30).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of PICK1 on ASIC2a pH dependence and current density. A, pH dependence of ASIC2a currents recorded from COS-transfected cells at 50 mV in the presence () or the absence () of PICK1. Sigmoidal dose-response curves were used to fit the data. The slope factor was 1.65, pH0.5 = 4.44 ± 0.08 and pH0.5 = 4.36 ± 0.01, with and without PICK1, respectively (n = 6-27). B, effect of PICK1 expression on pH 5-evoked ASIC2a current densities measured at 50 mV (n = 59 and 20 with () and without () PICK1, respectively. n.s., not significant; p = 0.30, Student's t test).

View this table: [in this window] [in a new window]   Table I Effects of PICK1 and OAG treatment on ASIC2a current kinetics Time-to-peak values were measured, and inactivation time constant () values were determined by mono-exponential fits of the currents in control and after OAG treatment (10 min, 50 µM). The statistical significance of differences between control and OAG-treated data was estimated by the Student t-test, and p values were given.

PICK1 Potentiation of the OAG-induced Stimulation of ASIC2a Current-- When 50 µM OAG, the cell-permeable analog of the endogenous PKC activator diacylglycerol, was applied extracellularly, it induced a 1.35 ± 0.08-fold (n = 12) increase of ASIC2a current amplitude (Fig. 3, A () and B (bar 2)), significantly different from the basal variation of ASIC2a current amplitude (0.98 ± 0.08, n = 5, Fig. 3B, bar 1, ; p < 0.05). This effect was strongly potentiated in the presence of PICK1 with a 3.90 ± 0.54-fold increase (n = 20) of the ASIC2a current induced by OAG (50 µM) (Fig. 3, A () and B (bar 3, *; p < 0.05)). Similar results were obtained when the phorbol ester PDBu (2 µM) was used instead of OAG to stimulate PKC (not shown). Fig. 3C shows that the OAG-induced increase of the ASIC2a current in the presence of PICK1 was dependent on the initial ASIC2a current density. The lower the current density, the greater the OAG-induced increase factor. The OAG treatment did not significantly modify the ASIC2a current kinetics, whether co-expressed with PICK1 or not (Table I, lines 1 and 2).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   PKC-induced stimulation of ASIC2a current via PICK1 interaction. A, effect of 50 µM OAG on pH 5-evoked ASIC2a current amplitudes recorded at 50 mV from two different COS cells expressing () or not expressing () PICK1. Currents were elicited every 2 min, and their amplitudes were normalized to those measured before the application of OAG (I/I control). Increase factors were plotted as a function of time. The application of OAG is indicated by the black bar, and the dashed line represents the no-effect level. The current traces represented above correspond to ASIC2a currents recorded before (a and c), and after (b and d) the application of OAG, in the presence (a and b) and in the absence (c and d) of PICK1. The dotted lines represent the zero current level. B, basal pH 5-evoked ASIC2a current amplitude variation over a 10-min time lapse (bar 1) and increase factors of pH 5-evoked ASIC2a current amplitudes induced by a 10-12-min exposure of the cells to OAG 50 µM (bars 2-7). Each bar corresponds to a specific condition of transfection: bar 1, ASIC2a; bar 2, ASIC2a; bar 3, ASIC2a + PICK1; bar 4, ASIC2aC+ PICK1; bar 5, ASIC2a + PICK1PDZ; bar 6, ASIC2a + PICK1 in presence of the PKC-I; bar 7, ASIC2aT39G + PICK1; bar 8, ASIC2aT39D + PICK1 (n = 5-20, ; p < 0.05 ,as compared with bar 1, *; p < 0.05, as compared with bar 2, Student's t test). In C, the OAG-induced increase factor of pH 5-evoked ASIC2a current amplitude, measured in the presence () and in the absence () of PICK1, is represented as a function of initial ASIC2a current density (before the application of OAG). D, PICK1-dependent in vivo phosphorylation of ASIC2a. The arrow on the left side of the presented SDS-PAGE gel indicates the expected size of ASIC2a (80 kDa, according to the Western blotting of rat brain membranes shown on the supplier's technical note and Alvarez de la Rosa et al. (72)). ASIC2a phosphorylation level was tested with (lanes 1-2) or without (lanes 3-4) PICK1 co-expression and with (lanes 2 and 4) or without (lanes 1 and 3) a 20-min treatment by PDBu (2 µM). An equivalent protein amount was loaded for all conditions.

PICK1 Interaction with ASIC2a through Its PDZ Domain-- The interaction between PICK1 and ASIC2a was found to require the PDZ domain of PICK1 and the last four amino acids of ASIC2a (30, 31). When PICK1 was co-expressed with ASIC2aC or when PICK1PDZ was co-expressed with ASIC2a, the OAG-induced increase of ASIC2a current (1.46 ± 0.17-fold, n = 5, and 1.55 ± 0.20-fold, n = 6, Fig. 3B, bars 4 and 5, respectively) was not significantly different from the OAG effect on ASIC2a without PICK1 (1.35 ± 0.08, n = 12, Fig. 3B, bar 2) but still significantly different from the basal variations of ASIC2a current level (; p < 0.05). This suggests that the direct association of the C terminus of ASIC2a with the PDZ domain of PICK1 is necessary for the full OAG-induced increase of ASIC2a current, even if a small part of the OAG effect remains when the association between PICK1 and ASIC2a is disrupted. Table I (lines 3 and 4) shows that the kinetics of the ASIC2aC + PICK1 and of the ASIC2a + PICK1PDZ currents are similar to those of the ASIC2a current and are not modified by the OAG treatment.

PICK1-dependent PKC-induced Phosphorylation of ASIC2a-- To specifically inhibit PKC, we used the 19-36 PKC peptide (PKC-I), acting as a pseudosubstrate on the active site of PKC (38). In the presence of this PKC-I (50 µM), in the pipette solution, the OAG-induced increase of ASIC2a current in the presence of PICK1 was reduced to a 1.56 ± 0.23-fold increase (n = 8, Fig. 3B, bar 6, not significantly different from the basal variations of ASIC2a current, p = 0.08), thus showing the specific involvement of PKC in the effect of PICK1-dependent OAG-induced stimulation of ASIC2a current. An analysis of the ASIC2a sequence revealed a putative PKC phosphorylation site, ³⁹TIR, matching the conventional consensus (S/T)*X(K/R) motif (single amino acid codes with X corresponding to any amino acid and * corresponding to the phosphorylated amino acid) (39) in the cytoplasmic N terminus fragment, just before the first transmembrane domain. The ASIC2aT39G and ASIC2aT39D mutants produced currents similar to wild-type ASIC2a, but the OAG effect in the presence of PICK1 was greatly reduced to 1.68 ± 0.30-fold (n = 11) and 1.18 ± 0.90-fold (n = 6), respectively (Fig. 3B, bars 7 and 8), which was not significantly different from the OAG-induced increase of ASIC2a current in the absence of PICK1 (1.35 ± 0.08, Fig. 3B, bar 2; p = 0.27 and p = 0.23, respectively). Neither was significantly different from the basal variations of ASIC2a current (bar 1; p = 0.14 and p = 0.41, respectively). The OAG-induced increases in ASIC2aT39G and ASIC2aT39D current amplitudes were not significantly different (p = 0.4). The kinetics of the two ASIC2a mutants co-expressed with PICK1 were not significantly different (p > 0.05) from those of ASIC2a and were not affected by the OAG treatment (Table I, lines 5 and 6).

In the ASIC2aT39D mutant, one could expect that the aspartic residue mimics a phosphorylated threonine, thus constitutively stimulating the current. The mean current density of the ASIC2aT39D mutant expressed with PICK1 (40.93 ± 12.49 pA/pF, n = 14) was slightly higher but not significantly different from the mean current density of the ASIC2a current expressed with PICK1 (26.53 ± 3.37 pA/pF, n = 59, p = 0.12). As we do not know whether the cellular expressions are similar for the wild-type and the mutated channel, it is thus difficult to postulate upon the fact that the T39D mutation mimics a phosphorylated residue. The inhibition of the PICK1-dependent OAG-induced increase of the ASIC2a current by both the T39D and the T39G mutations shows that the ³⁹TIR site is necessary for the OAG-induced increase of ASIC2a current and strongly suggests a direct phosphorylation of ASIC2a by PKC.

To determine whether ASIC2a was directly phosphorylated by PKC, we performed an in vivo [³²P]phosphate labeling and immunoprecipitation on ASIC2a-transfected cells with or without PICK1 after treatment with PDBu (2 µM, 20 min). A typical experiment is shown in Fig. 3D. There is a small basal phosphorylation of ASIC2a (Fig. 3D, lane 3), which is unchanged by the PKC activator PDBu (Fig. 3D, lane 4). When ASIC2a is co-transfected with PICK1 cDNA, ASIC2a phosphorylation markedly increases when cells are treated with PDBu (~4-fold increase, Fig. 3D, lanes 1-2).

Effect of PICK1 and OAG Treatment on ASIC2a Unitary Channel Conductance-- We recorded ASIC2a channels in outside-out patches from COS cells expressing ASIC2a channels (Fig. 4A), ASIC2a channels co-expressed with PICK1 (Fig. 4B), and ASIC2a channels co-expressed with PICK1 after a 10-min treatment by 50 µM OAG (Fig. 4C). The outside-out mode recording was necessary to activate the channels by an extracellular pH drop but prevented the comparison of the channel activity before and after OAG-induced PKC activation on the same membrane patch. ASIC2a channels were activated by a small pH drop to pH 6 or pH 5.5 to limit the channel activity and allow the measurement of single openings. Fig. 4 shows that neither the PICK1 co-expression nor the OAG treatment in the presence of PICK1 increases the channel conductance (10.93 ± 1.22 picosiemens between 80 and 0 mV). Taken together with results in the Fig. 2B that do not show any PICK1-induced increase in cellular current density, these data suggest that the PICK1-dependent PKC-induced phosphorylation most probably increases the ASIC2a open probability rather than the number of functional channels.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effect of PICK1 and OAG treatment on ASIC2a unitary channel conductance. A-C, ASIC2a channels recorded at 50 mV in three different outside-out patches from COS cells expressing ASIC2a alone (A), ASIC2a co-expressed with PICK1 (B), and ASIC2a co-expressed with PICK1 after a 10-min treatment by 50 µM OAG (C). ASIC2a channels were activated by a small pH drop to pH 6 or pH 5.5 to limit the channel activity and allow the measurement of single openings. The whole response to pH drop is shown as a top trace (16-s recording), and enlargements are represented below to show single channel openings (time intervals corresponding to arrowheads on the whole trace). D, unitary current amplitude expressed as a function of membrane potential for ASIC2a (, seven patches), ASIC2a + PICK1 (, five patches), and ASIC2a + PICK1 after OAG treatment (, five patches). The ASIC2a channel conductance was estimated to 10.93 ± 1.22 picosiemens between 80 and 0 mV with a reversal potential near +50 mV (P/P = 10), corresponding to what was reported previously (6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study provides the first evidence for a regulation of an ASIC current by a protein kinase and for a potentiation of this regulation by a partner protein. It shows that the amplitude of the ASIC2a current is increased by PKC phosphorylation and that PICK1 is required for a potent activation (a factor of 3.9-fold), probably by allowing an optimal interaction between the kinase and its target channel. We demonstrate the involvement of the PDZ domain of PICK1 and of the PDZ-binding domain of ASIC2a in this regulatory effect and identify ³⁹TIR as a major phosphorylation site. Indeed, the PICK1-dependent OAG-induced increase of the ASIC2a current was highly reduced by the T39D and T39G mutations. However, it is not clear whether the T39D mutation mimics a phosphorylation of the threonine residue, thus constitutively stimulating the current, as the mean current density of the ASIC2aT39D mutant is not significantly different from that of ASIC2a. In vivo phosphorylation experiments confirm that PKC activation induces phosphorylation of ASIC2a in the presence of PICK1 (Fig. 3D) and correlates well with patch clamp experiments. The PICK1-dependent PKC-induced phosphorylation does not change the kinetics, pH dependence, and unitary conductance of ASIC2a channels but probably induces an increase of the channel open probability.

The other known partner protein for ASICs isolated by yeast two-hybrid experiments is CIPP, another PDZ domain-containing protein (29). Unlike PICK1 (30, 31) for ASIC2a, cotransfection with CIPP increases the ASIC3 current membrane density and produces a shift in the pH-dependent activation. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been shown to regulate ASICs; however, the physiological relevance of this regulation is not known (40).

ASIC2a was described to co-localize with PICK1 both at peripheral sensory endings of dorsal root ganglia neurons as well as at synapses and cell bodies of some central neurons (30, 31). On both central and sensory neurons, native ASIC-like currents are now well characterized, particularly their pH sensitivities and their effect on membrane excitability (5, 22, 41-45). There is evidence concerning their physiological involvement in hippocampal synaptic plasticity (24), inflammatory pain (5, 46), and hypoxia-induced cardiac pain (16, 17). The PICK1-dependent regulation of ASICs by PKC would thus further support the involvement of these H⁺-gated channels in synaptic plasticity and peripheral sensitization processes.

There is considerable evidence for the PKC-dependent central modulation of neurotransmission and synaptic plasticity processes (47-50). PKC activation causes an increase in both the amplitude and the frequency of miniature excitatory postsynaptic elements in hippocampal CA1 neurons (51, 52) and potentiates synaptic transmission in spinal neurons (53). In peripheral sensory neurons, PKC also plays a key role in peripheral sensitization of nociceptors induced by neuropeptides and inflammatory mediators (54-56). The PDZ domain-containing protein PICK1 is expressed at central synapses where it regulates the localization of channels, receptors, and transporters. The GluR2 subunit from the -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor (34, 35, 57-63), the GluR3 subunit (64), the metabotropic glutamate receptor mGluR7a (65-68), the dopamine transporter (DAT) (69), the ERBB2/HER2 receptor (70), and the mitogen-stimulated TIS21 protein (71) were identified previously as partners of PICK1. There is also evidence for the involvement of PICK1 in neurotransmission and synaptic plasticity processes. In hippocampal CA1 neurons, disruption of the interaction between -Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors and PICK1 causes an increase in basal synaptic transmission and blocks the generation of long term depression (LTD) on a PKC-dependent manner (59).

We have shown that ASIC2a is regulated by PKC through interaction with its partner protein PICK1. This result is an important step toward a better understanding of the physiological involvement of ASICs in pain sensing and neurotransmission.

	ACKNOWLEDGEMENTS

We thank M. Jodar, N. Leroudier, and V. Friend for excellent technical assistance and V. Lopez for secretarial assistance.

	FOOTNOTES

^* This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Ministère de la Recherche (ACI, Molécules et Cibles Thérapeutiques), the Association Française contre les Myopathies (AFM), the Association pour la Recherche sur le Cancer (ARC) and the AstraZenecaAB Research Area Central Nervous System/Pain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^§ To whom correspondence should be addressed. Tel.: 33-4-93-95-77-02 or 03; Fax: 33-4-93-95-77-04; E-mail: ipmc@ipmc.cnrs.fr.

Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208848200

	ABBREVIATIONS

The abbreviations used are: ASIC, acid sensing ion channel; PICK1, protein interacting with C-kinase; MES, 4-morpholinoethanesulfonic acid; OAG, 1-oleyl-2-acetyl-sn-glycerol; PDBu, phorbol 12, 13-dibutyrate; PKC, protein kinase C; PKC-I, PKC inhibitory peptide; CIPP, channel-interacting PDZ domain-containing protein; pF, picofarads.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				J. Yagi, H. N. Wenk, L. A. Naves, and E. W. McCleskey Sustained Currents Through ASIC3 Ion Channels at the Modest pH Changes That Occur During Myocardial Ischemia Circ. Res., September 1, 2006; 99(5): 501 - 509. [Abstract] [Full Text] [PDF]

				W. Wang, B. Duan, H. Xu, L. Xu, and T.-L. Xu Calcium-permeable Acid-sensing Ion Channel Is a Molecular Target of the Neurotoxic Metal Ion Lead J. Biol. Chem., February 3, 2006; 281(5): 2497 - 2505. [Abstract] [Full Text] [PDF]

				M. Salinas, L. D. Rash, A. Baron, G. Lambeau, P. Escoubas, and M. Lazdunski The receptor site of the spider toxin PcTx1 on the proton-gated cation channel ASIC1a J. Physiol., January 15, 2006; 570(2): 339 - 354. [Abstract] [Full Text] [PDF]