Regulation of Sensory Neuron-specific Acid-sensing Ion Channel 3 by the Adaptor Protein Na+/H+ Exchanger Regulatory Factor-1
2005; Elsevier BV; Volume: 281; Issue: 3 Linguagem: Inglês
10.1074/jbc.m509669200
ISSN1083-351X
AutoresEmmanuel Deval, Valérie Friend, Cécile Thirant, Miguel Salinas, Martine Jodar, Michel Lazdunski, Éric Lingueglia,
Tópico(s)Ion channel regulation and function
ResumoAcid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation. Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation. Physiopathological conditions such as ischemia, inflammation, tumors, or injury are associated with a decrease in extracellular pH, and tissue acidosis has been linked to pain in human volunteers (1Steen K.H. Issberner U. Reeh P.W. Neurosci. Lett. 1995; 199: 29-32Crossref PubMed Scopus (104) Google Scholar, 2Jones N.G. Slater R. Cadiou H. McNaughton P. McMahon S.B. J. Neurosci. 2004; 24: 10974-10979Crossref PubMed Scopus (205) Google Scholar, 3Ugawa S. Ueda T. Ishida Y. Nishigaki M. Shibata Y. Shimada S. J. Clin. Invest. 2002; 110: 1185-1190Crossref PubMed Scopus (281) Google Scholar). Nociceptive neurons display voltage-independent H+-gated cationic currents, which are in part mediated by acid-sensing ion channels (ASICs) 2The abbreviations used are: ASICacid-sensing ion channelNHERFNa+/H+ exchanger regulatory factorNHE-3Na+/H+ exchanger 3PDZpostsynaptic density-95; Drosophila discs large; zonula occludens-1DRGdorsal root ganglionTRPtransient receptor potentialERMezrin-radixin-moesinPMAphorbol 12-myristate 13-acetateERendoplasmic reticulumPKCprotein kinase CHAhemagglutininMES4-morpholineethanesulfonic acid. 2The abbreviations used are: ASICacid-sensing ion channelNHERFNa+/H+ exchanger regulatory factorNHE-3Na+/H+ exchanger 3PDZpostsynaptic density-95; Drosophila discs large; zonula occludens-1DRGdorsal root ganglionTRPtransient receptor potentialERMezrin-radixin-moesinPMAphorbol 12-myristate 13-acetateERendoplasmic reticulumPKCprotein kinase CHAhemagglutininMES4-morpholineethanesulfonic acid. (4Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1130) Google Scholar, 5Reeh P.W. Kress M. Curr. Opin. Pharmacol. 2001; 1: 45-51Crossref PubMed Scopus (139) Google Scholar). The ASIC family comprises six isoforms encoded by four different genes and expressed in sensory and/or central neurons (6Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (177) Google Scholar, 7Lingueglia E. Deval E. Lazdunski M. Peptides. 2006; (in press)PubMed Google Scholar). Functional ASICs are homomeric or heteromeric channels, probably tetramers, with different kinetics, external pH sensitivities, and tissue distribution (6Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (177) Google Scholar, 7Lingueglia E. Deval E. Lazdunski M. Peptides. 2006; (in press)PubMed Google Scholar, 8Krishtal O. Trends Neurosci. 2003; 26: 477-483Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). ASIC3 is a sensory neuron-specific ASIC (9Waldmann R. Bassilana F. De Weille J.R. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar), which is principally found in the small and medium nociceptive neurons (10Voilley N. de Weille J. Mamet J. Lazdunski M. J. Neurosci. 2001; 21: 8026-8033Crossref PubMed Google Scholar). Its expression has been associated with a biphasic current comprising a transient, fast inactivating component followed by a sustained, noninactivating phase (9Waldmann R. Bassilana F. De Weille J.R. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). ASIC3 has therefore been proposed to participate in the nonadaptive pain caused by acids, and it has been clearly involved in sensing of tissue acidosis in muscle (11Sluka K.A. Price M.P. Breese N.M. Stucky C.L. Wemmie J.A. Welsh M.J. Pain. 2003; 106: 229-239Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), cardiac ischemia (12Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Crossref PubMed Scopus (328) Google Scholar), and inflammation (10Voilley N. de Weille J. Mamet J. Lazdunski M. J. Neurosci. 2001; 21: 8026-8033Crossref PubMed Google Scholar, 13Voilley N. Curr. Drug Targets Inflamm. Allergy. 2004; 3: 71-79Crossref PubMed Scopus (147) Google Scholar). ASIC3 gene inactivation in mice has confirmed a role in pain sensation and in some aspects of mechanosensation (11Sluka K.A. Price M.P. Breese N.M. Stucky C.L. Wemmie J.A. Welsh M.J. Pain. 2003; 106: 229-239Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 14Price M.P. McIlwrath S.L. Xie J. Cheng C. Qiao J. Tarr D.E. Sluka K.A. Brennan T.J. Lewin G.R. Welsh M.J. Neuron. 2001; 32: 1071-1083Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 15Chen C.C. Zimmer A. Sun W.H. Hall J. Brownstein M.J. Zimmer A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8992-8997Crossref PubMed Scopus (260) Google Scholar, 16Page A.J. Brierley S.M. Martin C.M. Price M.P. Symonds E. Butler R. Wemmie J.A. Blackshaw L.A. Gut. 2005; 54: 1408-1415Crossref PubMed Scopus (235) Google Scholar), although the latter issue is still discussed (17Drew L.J. Rohrer D.K. Price M.P. Blaver K.E. Cockayne D.A. Cesare P. Wood J.N. J. Physiol. 2004; 556: 691-710Crossref PubMed Scopus (215) Google Scholar). acid-sensing ion channel Na+/H+ exchanger regulatory factor Na+/H+ exchanger 3 postsynaptic density-95; Drosophila discs large; zonula occludens-1 dorsal root ganglion transient receptor potential ezrin-radixin-moesin phorbol 12-myristate 13-acetate endoplasmic reticulum protein kinase C hemagglutinin 4-morpholineethanesulfonic acid. acid-sensing ion channel Na+/H+ exchanger regulatory factor Na+/H+ exchanger 3 postsynaptic density-95; Drosophila discs large; zonula occludens-1 dorsal root ganglion transient receptor potential ezrin-radixin-moesin phorbol 12-myristate 13-acetate endoplasmic reticulum protein kinase C hemagglutinin 4-morpholineethanesulfonic acid. ASIC subunits have two transmembrane domains flanking a large extracellular cysteine-rich region. Both NH2 and COOH termini are therefore intracytoplasmic and might interact with other proteins involved, for instance, in the localization and the regulation of these channels. PSD-95 (postsynaptic density-95 protein)/Dlg/ZO-1 (PDZ) domain-containing proteins interacting with the COOH terminus of ASIC3 and able to modulate the H+-gated current have recently been identified. The first one was the scaffolding protein, CIPP (channel-interacting PDZ domain protein), which contains four PDZ domains and increases ASIC3 current density (18Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). PSD-95 and Lin-7b proteins also modulate ASIC3 levels in the plasma membrane (19Hruska-Hageman A.M. Benson C.J. Leonard A.S. Price M.P. Welsh M.J. J. Biol. Chem. 2004; 279: 46962-46968Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Other types of associations play a role in ASIC3 regulation. PICK1 (protein interacting with C kinase-1) participates in the protein kinase C (PKC) regulation of ASIC3 through its PDZ-dependent association with the ASIC2b subunit (20Deval E. Salinas M. Baron A. Lingueglia E. Lazdunski M. J. Biol. Chem. 2004; 279: 19531-19539Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), which itself can form a heteromeric channel with ASIC3. The integral membrane protein stomatin has also been shown to associate with ASIC3, as well as with ASIC1a and ASIC2a, and to alter the gating properties of ASIC2a and ASIC3 without altering channel surface expression (21Price M.P. Thompson R.J. Eshcol J.O. Wemmie J.A. Benson C.J. J. Biol. Chem. 2004; 279: 53886-53891Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This paper describes the adaptor protein NHERF-1 (Na+/H+ exchanger regulatory factor-1) as an accessory protein that teams up with ASIC3 to provide a new type of modulation of its function and of its subcellular localization in relation with the cytoskeleton. Yeast Two-hybrid Screening—An ASIC3 COOH-terminal bait corresponding to the last 68 amino acids of rat ASIC3 was used to screen a rat DRG cDNA library as previously described (18Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). For subsequent analysis, the full-length rat NHERF-1 and NHERF-2 cDNAs, encoding the 356- and 337-amino acid proteins, respectively, as well as the mutated forms of both ASIC3 and NHERFs were obtained by PCR (22Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6812) Google Scholar). Immunoprecipitation and Western Blot—COS-7 cells were transiently transfected with rat HA-NHERF-1 or HA-NHERF-2 (NH2-terminal HA tag), rat Myc-ASIC3 (NH2-terminal Myc tag), or both, by the DEAE-dextran method. F-11 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Cells were scrapped in lysis buffer (50 mm Tris/HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, and Roche Complete protease inhibitors (Roche Applied Science)) 24-48 h post-transfection and then centrifuged for 30 min at 100,000 × g. For phosphorylation experiments, cells were treated for 20 min with a 100 nm concentration of the PKC activator phorbol 12-myristate 13-acetate (PMA) or with a 10 μm concentration of the PKC inhibitor chelerythrine (Sigma) before lysis. Supernatant was collected and immunoprecipitated with anti-HA 3F10 antibody (Roche Applied Science), anti-Myc A14 antibody (Santa Cruz Biotechnology), or anti-NHERF-1 antibody (Affinity Bioreagents). After incubation with protein A or protein G affinity gel (Sigma), beads were washed four times with lysis buffer and boiled in SDS-PAGE buffer for 5 min. Immunoprecipitated or cell lysate proteins were separated on 12% polyacrylamide gels by SDS-PAGE and electroblotted onto polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp.). Membranes were blotted with the 3F10 (1:1,000), A14 (1:500), anti-NHERF-1 (1:800), or anti-NHERF-2 (1:1,000) antibody for 1 h at room temperature or overnight at 4 °C. Peroxidase-conjugated (for chemiluminescence revelation; 1:10,000) or alkaline phosphatase-conjugated (for fluorescence revelation; 1:10,000) secondary antibodies were used. Blots were revealed with the supersignal WestPico luminescent detection system (Pierce) or with ECF substrate (Amersham Biosciences) followed by fluorescence scanning using a 570-nm filter in a ProXpress system (PerkinElmer Life Sciences). Freshly isolated mouse dorsal root ganglia were homogenized on ice with a Dounce homogenizer in lysis buffer and then incubated for 30 min at 4 °C before centrifugation for 15 min at 10,000 × g. Supernatants were collected, and protein concentrations were determined by the Bradford method. Western blots were performed as previously described. Surface Biotinylation in COS-7 Cells—Surface biotinylation experiments were carried out according to standard protocols. Briefly, COS-7 cells were transiently transfected by the DEAE-dextran method and used 48 h post-transfection. Cells were incubated on ice for 30 min with 1 mg/ml Sulfo-NHS-Biotin reagent (Pierce) dissolved in PBS (pH 8.0) and then washed twice with ice-cold PBS complemented with 100 mm glycine and incubated for 20 min in the same buffer on ice. Cell lysis was performed as previously described for the immunoprecipitation experiments. After the 100,000 × g centrifugation step, supernatant was mixed with 30-50 μl of streptavidin-agarose beads (Sigma) and incubated for 2 h at 4 °C. Beads were washed four times with lysis buffer and then heated in SDS-PAGE loading buffer for 20 min at 65 °C. Proteins were next resolved on a 12% polyacrylamide gel, and subsequent steps (transfer and Western blot analysis) were carried out as described previously. Immunofluorescence and in Situ Hybridization—COS-7 cells were grown on 35-mm plates and transfected by the DEAE-dextran method. The next day, cells were dissociated with 1 mm EDTA in phosphate-buffered saline (PBS) and plated on glass coverslips in 24-well plates. One day later, cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, blocked with 10% horse serum, and incubated with primary antibodies anti-Myc A14 (1:800) or 9E10 (1:200; Santa Cruz Biotechnology, Inc.), anti-HA 3F10 (1:2,000), or anti-ezrin (1:700; gift of Dr. M. Arpin), followed by the secondary antibodies goat anti-rabbit Alexa 488 (1:1,500), donkey anti-rat Alexa 594 (1:1,500), or donkey anti-mouse Alexa 594 (1:1,500) (Molecular Probes, Inc., Eugene, OR). Staining was visualized using an Axioplan 2 microscope (Carl Zeiss). In situ hybridization was essentially performed as previously described (10Voilley N. de Weille J. Mamet J. Lazdunski M. J. Neurosci. 2001; 21: 8026-8033Crossref PubMed Google Scholar, 18Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) with an antisense synthetic oligonucleotide corresponding to the rat ASIC3 sequence (CAACATGTCCTCAAGGGAGTGGCCG) and a sense primer for control experiments corresponding to the sequence GGCACGATATTCGAGACATGCTGCTCTC. For fluorescent labeling, detection was performed with the ELF-97 mRNA in situ hybridization kit (Molecular Probes) based on the streptavidin-alkaline phosphatase interaction with the biotinylated probe and the formation of green fluorescent precipitates in the presence of the ELF-97 substrate. Detection of the NHERF-1 protein by immunohistochemistry was performed after the in situ hybridization with an anti-NHERF-1 rabbit polyclonal antibody (1:200; Affinity Bioreagents) followed by a goat anti-rabbit Texas Red secondary antibody (Jackson ImmunoResearch Laboratories). All experiments were done on at least two animals. Reverse Transcription-PCR Analysis—Experiments were performed from adult rat total RNA as previously described (18Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The following sense and antisense primers were used for PCR: ASIC3, CTGGCAACGGACTGGAGATTA and TGTAGTAGCGCACGGGTTGG (amplicon of 506 bp); NHERF-1, CGGCTCTGCACCATGAAGAA and CTCAGAGGTTGCTGAAGAGTTC (amplicon of 625 bp); and NHERF-2, ATACATCCGCTCTGTGGACCC and CTGCTGAGGCTTGGGGAGCT (amplicon of 576 bp). Twenty-five cycles of PCR (95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min) were performed, except for β-actin, for which only 22 cycles were done. One-third of the PCR was resolved on a 2% agarose gel. The PCR conditions have been designed to avoid overamplification and allow a better comparison between tissues. Transfection and Electrophysiology of COS-7 and F-11 Cells—COS cells were transfected with a mix of rat NHERF-1 and rat ASIC3 or ASIC3Δ3 cDNAs using the DEAE-dextran method. Cells were used for electrophysiological measurements 1-4 days after transfection. F-11 DRG cells cultured in Ham's F-12 medium (Invitrogen) supplemented with 15% fetal bovine serum (ICN Biomedicals), 1× HAT (sodium hypoxanthine, aminopterin, and thymidine), 200 μg/ml allo-4-hydroxy-l-proline (Sigma), and 1% antibiotics (penicillin and streptomycin; Invitrogen) were transfected with NHERF-1 or NHERF-1ΔPDZ1 and rat ASIC3 cDNAs using Lipofectamine 2000 (Invitrogen). Cells were used for electrophysiological measurements 2-4 days after transfection. We used the patch clamp technique to measure membrane currents in whole-cell configuration. Currents were amplified with an RK-400 amplifier (Bio-Logic Science Instruments), digitized with a 16-bit data acquisition system (Digidata 1322A; Axon Instruments), and recorded on a hard disk using pClamp software (version 9.2.0.11; Axon Instruments). The currents were sampled at 10 kHz and low pass-filtered at 3 kHz. Off-line analysis of currents was performed using pClamp (Axon Instruments). The pipette solution contained 140 mm KCl, 5 mm NaCl, 2 mm MgCl2, 5 mm EGTA, 10 mm HEPES (pH 7.25), and the bath solution contained 150 mm NaCl, 5 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 10 mm HEPES (pH 7.4). MES was used instead of HEPES to buffer bath solution pH ranging from 6 to 5. For F-11 DRG cells, the bath solution was supplemented with 10 mm glucose, and the pipette solution was modified to 140 mm KCl, 2.5 mm Na2-ATP, 2 mm MgCl2, 2 mm CaCl2, 5 mm EGTA, 10 mm HEPES (pH 7.25). Oocyte Injection and Electrophysiology—Oocytes were kept at 19 °C in ND96 solution (containing 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 2 mm MgCl2, and 5 mm HEPES) supplemented with penicillin (6 μg/ml) and streptomycin (5 μg/ml). Currents were recorded within 3-5 days of DNA injections. In a 0.3-ml perfusion chamber, a single oocyte was gently impaled with two standard glass microelectrodes (1-2.5 megaohms) filled with a 3 mm KCl solution and maintained under voltage clamp using a Dagan TEV 200 amplifier. Data acquisition and analysis were performed using pClamp software (Axon Instruments). All experiments were performed at 19 °C, and MES or acetic acid was used instead of HEPES to buffer ND96 solution pH ranging from 6 to 3. Statistical Analysis—Data points represent the mean ± S.E. of n independent experiments. The one-way analysis of variance test followed by a Tukey post hoc test was used for statistical analysis using Prism software (version 4.03; GraphPad). NHERF-1 and NHERF-2 Associate with the ASIC3 COOH Terminus in a Yeast Two-hybrid Assay—The ASIC3 COOH terminus shares homology with type I PDZ-binding motifs ((S/T)X(V/L)) (23Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1212) Google Scholar). The yeast two-hybrid system was used to identify putative ASIC3-interacting proteins. A rat DRG cDNA library (18Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) was screened with the last 68 amino acids of rat ASIC3 as a bait. Among the isolated clones, one clone encoded a full-length NHERF-1 protein, also named EBP50 (ezrin-radixin-moesin-binding phosphoprotein of 50 kDa), and another encoded a partial NHERF-2 protein, also identified as E3KARP (Na+/H+ exchanger 3 kinase A-regulatory protein), TKA-1 (tyrosine kinase activator-1), or SIP-1 (SRY-interacting protein-1). NHERF-1 and NHERF-2 are PDZ-containing proteins that mediate specific protein-protein interactions and thereby can serve as adaptors. They have been involved in the regulation of the targeting and trafficking of specific integral membrane proteins (for a review, see Ref. 24Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiology (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (135) Google Scholar). They both contain two tandem PDZ domains followed by a carboxyl-terminal sequence that binds to members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeleton adaptors. The NHERF-1 and NHERF-2 clones isolated from the rat DRG library both contained functional PDZ domains, strongly suggesting an interaction between these PDZ domains and the COOH-terminal region of ASIC3. NHERF-1 and NHERF-2 Bind to ASIC3 through Their PDZ-1 Domain in Transfected COS Cells—To confirm the two-hybrid results and test the interaction with the full-length proteins, binding between NHERFs and ASIC3 was assayed by coimmunoprecipitation in transfected COS-7 cells. Constructs encoding Myc-tagged ASIC3 and HA-tagged NHERF-1 and NHERF-2 or mutant variants were co-transfected in COS cells, and cell lysates were immunoprecipitated with anti-HA antibodies, followed by Western blot analysis. Wild-type ASIC3 interacted with NHERF-1 and NHERF-2 (Fig. 1, A (lane 1) and B (lane 7)), and association occurred regardless of which was initially precipitated (data not shown). This interaction was impaired by removing the last three amino acids of ASIC3, which form most of the PDZ binding motif (23Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1212) Google Scholar) (ASIC3 Δ531-533 mutant; Fig. 1, A (lane 2) and B (lane 8)). As expected, NHERF-1 did not co-precipitate with ASIC1a and ASIC2a (Fig. 1A, lanes 3 and 4), two ASIC subunits with a different COOH-terminal PDZ-binding motif. The two NHERF-1 PDZ domains have considerable structural homology but recognized distinct synthetic peptides matching the consensus sequence (S/T)(R/Y)L-COOH for PDZ-1 (similar to the COOH-terminal motif of ASIC3) and S(S/T)WL-COOH for PDZ-2 (25Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (249) Google Scholar). In good agreement with this, versions of NHERF-1 and NHERF-2 made with a deletion of the first or the second PDZ domain demonstrated that the PDZ-1 domain of NHERF-1 and NHERF-2, but apparently not the PDZ-2 domain, was necessary for the interaction (Fig. 1, A (lanes 5 and 6) and B (lanes 9 and 10)). However, surface plasmon resonance measurements have shown that both PDZ domains of NHERF-1 can interact with an immobilized peptide corresponding to the COOH terminus of CFTR (comprising the TRL motif) but that the PDZ-1/CFTR complex is formed much faster than the PDZ-2/CFTR interaction (26Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar). We cannot therefore exclude the possibility of some weak interaction between ASIC3 and the PDZ-2 domain of NHERF-1 in certain conditions. NHERF-1 mutants with a COOH-terminal deletion of the last 30 or 60 amino acids were still able to associate with ASIC3 (supplementary Fig. 1), indicating that the ERM domain of NHERF-1 was not involved in the interaction. Levels of wild-type and mutant proteins were comparable in all of these experiments (Fig. 1, A (lanes 1 and 2 and lanes 5 and 6) and B (lanes 7-10)). These results demonstrate that the ASIC3 COOH-terminal PDZ binding motif interacts with the PDZ-1 domain of NHERF-1 and NHERF-2. They also suggest a constitutive association of NHERFs and ASIC3 in transfected mammalian cells. Phosphorylation of the ASIC3 COOH-terminal Domain Increases NHERF-1 Binding—Phosphorylation-dependent modulation of the interaction between PDZ proteins and PDZ binding motifs has been well documented. ASIC3 contains a conserved consensus site for phosphorylation by PKC in its intracellular COOH-terminal domain (Ser-523; Fig. 2A). Previous work has suggested that this residue is phosphorylated by PKC (20Deval E. Salinas M. Baron A. Lingueglia E. Lazdunski M. J. Biol. Chem. 2004; 279: 19531-19539Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We have investigated the impact of phosphorylation at this position on the interaction between NHERF-1 and ASIC3. PKC stimulation by PMA increased ASIC3 co-precipitation with NHERF-1 by ∼50% in COS-7 cells co-transfected with Myc-ASIC3 and HA-NHERF-1 compared with a condition where PKC was inhibited with chelerythrine (Fig. 2, B (lanes 1 and 2) and C). A further indication that phosphorylation of serine 523 was likely to be involved in this regulation included the observation that the nonphosphorylatable glycine mutant (S523G) prevented the PMA effect (Fig. 2B, lane 3). In addition, a mutation mimicking phosphorylation in ASIC3 (S523D) mimicked the PMA effect despite the presence of chelerythrine (Fig. 2B, lanes 4). All of these data strongly suggest that PKC phosphorylation of serine 523 in the COOH-terminal region of ASIC3 positively modulates the interaction between ASIC3 and NHERF-1. NHERF-1 and NHERF-2 Increase ASIC3 Current in Xenopus Oocytes—We subsequently measured the effect of NHERF-1 and NHERF-2 on ASIC3 current in Xenopus oocytes. Both proteins were able to strongly potentiate the pH 5.0-evoked ASIC3 transient current amplitude when co-injected with the channel (∼6.4- and ∼7.7-fold increase, respectively; Fig. 3, A and B). The more dramatic effect was seen on the sustained current (∼30.9- and ∼42.4-fold increase, respectively, Fig. 3C). The pH dependence of the peak and sustained currents was not altered (Fig. 3D); nor was the selectivity of the sustained current (Fig. 3E). The kinetics of desensitization were only slightly modified (supplementary Fig. 2). The NHERF-1 PDZ-1 domain, but not the PDZ-2 domain, and the last COOH-terminal region of ASIC3 were required for the NHERF-1 effect to occur on both the peak and sustained current (Fig. 3, B and C), consistent with the biochemical data described in Fig. 1. Deletion of the NHERF-1 ERM-binding domain (NHERF-1Δ60) decreased the effect of NHERF-1 on the ASIC3 transient current (Fig. 3B). The amplitude measured in the presence of NHERF-1Δ60 was still higher than the amplitude observed with ASIC3 expressed alone (5.87 ± 0.70 μA (n = 18) versus 1.57 ± 0.24 μA (n = 90), respectively, p < 0.01). However, the amplitude was significantly lower than the one of ASIC3 co-expressed with wild type NHERF-1 (5.87 ± 0.70 μA (n = 18) versus 9.96 ± 0.73 μA (n = 79), respectively, p < 0.05). This suggests that the effect on the peak current is partially dependent on the interaction with ERM proteins and thereby on the association of the channel with the cytoskeleton. The NHERF-1Δ60 mutant had no significant effect on the sustained current (Fig. 3C), which may reflect differences in the regulation by NHERF-1 of the peak and the sustained components of the ASIC3 current. NHERF-1 Is Expressed in Native DRG Neurons, Where Its Distribution Partly Overlaps with That of ASIC3—We next examined the expression of NHERF-1 and NHERF-2 in native DRG neurons. NHERF-1 transcripts were detected in rat DRG by reverse transcription-PCR (Fig. 4A). The levels seemed lower than in epithelial tissues like lung and colon. However, the NHERF-1 protein was easily detected by Western blot in lysates from both rat and mouse DRG (Fig. 4B). These data confirmed the previous observation of high levels of the NHERF-1 protein in rat DRG neurons (27Melendez-Vasquez C.V. Rios J.C. Zanazzi G. Lambert S. Bretscher A. Salzer J.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1235-1240Crossref PubMed Scopus (113) Google Scholar). On the other hand, NHERF-2 transcripts are barely detected in rat DRG (Fig. 4A), and the protein was not detected in DRG neurons by Western blot (Fig. 4C) and immunohistochemistry (data not shown). The overlap in expression between ASIC3 and NHERF-1 was next analyzed in rat DRG by double in situ hybridization and immunohistochemistry. We used in situ hybridization to detect ASIC3 mRNA, and the NHERF-1 protein was subsequently identified on the same samples by immunohistochemistry (Fig. 4E). ASIC3, which has been previously shown to be expressed in nociceptive sensory neurons (9Waldmann R. Bassilana F. De Weille J.R. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 10Voilley N. de Weille J. Mamet J. Lazdunski M. J. Neurosci. 2001; 21: 8026-8033Crossref PubMed Google Scholar), exhibited significant
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