Title: PSD-95 and Lin-7b Interact with Acid-sensing Ion Channel-3 and Have Opposite Effects on H+-gated Current
Abstract: The acid-sensing ion channel-3 (ASIC3) is a degenerin/epithelial sodium channel expressed in the peripheral nervous system. Previous studies indicate that it participates in the response to mechanical and painful stimuli, perhaps contributing to mechanoreceptor and/or H+-gated nociceptor function. ASIC3 subunits contain intracellular N and C termini that may control channel localization and function. We found that a PDZ-binding motif at the ASIC3 C terminus interacts with four different proteins that contain PDZ domains: PSD-95, Lin-7b, MAGI-1b, and PIST. ASIC3 and these interacting proteins were expressed in dorsal root ganglia and spinal cord, and PSD-95 co-precipitated ASIC3 from spinal cord. When expressed in heterologous cells, PSD-95 reduced the amplitude of ASIC3 acid-evoked currents, whereas Lin-7b increased current amplitude. PSD-95 and Lin-7b altered current density by decreasing or increasing, respectively, the amount of ASIC3 on the cell surface. The finding that multiple PDZ-containing proteins bind ASIC3 and can influence its presence in the plasma membrane suggests that they may play an important role in the contribution of ASIC3 to nociception and mechanosensation. The acid-sensing ion channel-3 (ASIC3) is a degenerin/epithelial sodium channel expressed in the peripheral nervous system. Previous studies indicate that it participates in the response to mechanical and painful stimuli, perhaps contributing to mechanoreceptor and/or H+-gated nociceptor function. ASIC3 subunits contain intracellular N and C termini that may control channel localization and function. We found that a PDZ-binding motif at the ASIC3 C terminus interacts with four different proteins that contain PDZ domains: PSD-95, Lin-7b, MAGI-1b, and PIST. ASIC3 and these interacting proteins were expressed in dorsal root ganglia and spinal cord, and PSD-95 co-precipitated ASIC3 from spinal cord. When expressed in heterologous cells, PSD-95 reduced the amplitude of ASIC3 acid-evoked currents, whereas Lin-7b increased current amplitude. PSD-95 and Lin-7b altered current density by decreasing or increasing, respectively, the amount of ASIC3 on the cell surface. The finding that multiple PDZ-containing proteins bind ASIC3 and can influence its presence in the plasma membrane suggests that they may play an important role in the contribution of ASIC3 to nociception and mechanosensation. ASIC3 1The abbreviations used are: ASIC, acid-sensing ion channel; DEG/ENaC, degenerin/epithelial sodium channel; PDZ, PSD-95, Drosophila discs-large protein, zonula occludens protein-1; PSD-95, postsynaptic density-95 protein; PICK1, protein interacting with C kinase-1; CIPP, channel-interacting PDZ domain protein; MAGI, membrane-associated guanylate kinase with inverted orientation; PIST, PDZ protein interacting specifically with TC10; NR2, N-methyl-d-aspartate receptor subunit 2; RT, reverse transcription; DRG, dorsal root ganglion; GFP, green fluorescent protein; HA, hemagglutinin; CHO, Chinese hamster ovary; MES, 4-morpholineethanesulfonic acid; CAL, CFTR-associated ligand; CFTR, cystic fibrosis transmembrane conductance regulator.1The abbreviations used are: ASIC, acid-sensing ion channel; DEG/ENaC, degenerin/epithelial sodium channel; PDZ, PSD-95, Drosophila discs-large protein, zonula occludens protein-1; PSD-95, postsynaptic density-95 protein; PICK1, protein interacting with C kinase-1; CIPP, channel-interacting PDZ domain protein; MAGI, membrane-associated guanylate kinase with inverted orientation; PIST, PDZ protein interacting specifically with TC10; NR2, N-methyl-d-aspartate receptor subunit 2; RT, reverse transcription; DRG, dorsal root ganglion; GFP, green fluorescent protein; HA, hemagglutinin; CHO, Chinese hamster ovary; MES, 4-morpholineethanesulfonic acid; CAL, CFTR-associated ligand; CFTR, cystic fibrosis transmembrane conductance regulator. is a non-voltage-gated Na+ channel activated by acidic extracellular solutions (1Waldmann 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, 2Babinski K. Le K.T. Séguéla P. J. Neurochem. 1999; 72: 51-57Crossref PubMed Scopus (158) Google Scholar). It is expressed in the peripheral nervous system, including the dorsal root ganglia and trigeminal ganglia. Immunocytochemical studies have localized it to several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and nociception (3Price M.P. McIllwrath 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). The importance of ASIC3 for sensory function was revealed by studies of mice bearing targeted disruptions of the ASIC3 gene (3Price M.P. McIllwrath 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, 4Chen C.C. Zimmer A. Sun W.H. Hall J. Brownstein M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8992-8997Crossref PubMed Scopus (260) Google Scholar). In single fiber recordings from cutaneous nerves, loss of ASIC3 increased the sensitivity of mechanoreceptors detecting light touch but reduced the sensitivity of a mechanoreceptor responding to noxious pinch. In behavioral studies, ASIC3 modulated the response to noxious acid, heat, and mechanical stimuli. Moreover chronic mechanical hyperalgesia produced by intramuscular acid injections was prevented in ASIC3 null animals (5Sluka 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). Functional studies suggest that ASIC3 may also mediate the pain associated with myocardial ischemia (6Benson C.J. Eckert S.P. McCleskey E.W. Circ. Res. 1999; 84: 921-928Crossref PubMed Scopus (196) Google Scholar, 7Sutherland 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). Thus, in different cellular contexts, ASIC3 may participate in the responses to both mechanical and acidotic stimuli to mediate normal touch and pain sensation. ASIC3, a member of the DEG/ENaC family, forms heteromultimers in neurons with other DEG/ENaC subunits, ASIC1 and ASIC2, to generate H+-gated cation channels (8Xie J. Price M.P. Berger A.L. Welsh M.J. J. Neurophysiol. 2002; 87: 2835-2843Crossref PubMed Scopus (82) Google Scholar, 9Alvarez de la Rosa D. Zhang P. Shao D. White F. Canessa C.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2326-2331Crossref PubMed Scopus (208) Google Scholar, 10Benson C.J. Xie J. Wemmie J.A. Price M.P. Henss J.M. Welsh M.J. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2338-2343Crossref PubMed Scopus (308) Google Scholar). ASIC3 shares the overall structure of DEG/ENaC proteins, including two transmembrane domains, a large extracellular loop containing 14 conserved cysteines, and intracellular N and C termini (11Mano I. Driscoll M. Bioessays. 1999; 21: 568-578Crossref PubMed Scopus (135) Google Scholar, 12Benos D.J. Stanton B.A. J. Physiol. (Lond). 1999; 520: 631-644Crossref Scopus (153) Google Scholar). The intracellular domains of ASIC subunits might have several functions, including localizing the channels, controlling the number of channels on the cell surface, regulating channel function, and forming part of a scaffold important for the response to mechanical stimuli (11Mano I. Driscoll M. Bioessays. 1999; 21: 568-578Crossref PubMed Scopus (135) Google Scholar, 12Benos D.J. Stanton B.A. J. Physiol. (Lond). 1999; 520: 631-644Crossref Scopus (153) Google Scholar, 13Welsh M.J. Price M.P. Xie J. J. Biol. Chem. 2002; 277: 2369-2372Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Two proteins have been shown to interact with the intracellular domains of ASIC channels. The C termini of ASIC1 and ASIC2 share homology with type II PDZ (PSD-95, Drosophila discslarge protein, zonula occludens protein-1)-binding domains and bind PICK1 (protein interacting with C kinase-1) (14Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Crossref PubMed Scopus (102) Google Scholar, 15Duggan A. Garcia-Anoveros J. Corey D.P. J. Biol. Chem. 2002; 277: 5203-5208Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). PICK1 may facilitate the interaction of ASIC2a with protein kinase C, and the interaction between ASIC1 and PICK1, regulated by phosphorylation, may provide a mechanism to control the cellular localization of ASIC1 (16Baron A. Deval E. Salinas M. Lingueglia E. Voilley N. Lazdunski M. J. Biol. Chem. 2002; 277: 50463-50468Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Leonard A.S. Yermolaieva O. Hruska-Hageman A. Askwith C.C. Price M.P. Wemmie J.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2029-2034Crossref PubMed Scopus (70) Google Scholar). The ASIC3 C terminus shares homology with type I PDZ-binding motifs. CIPP (channel-interacting PDZ domain protein), which contains four PDZ domains, is reported to interact with the ASIC3 C terminus and increase H+-gated current (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 potential importance of protein-protein interactions for ASIC3 function led us to ask what proteins bind its intracellular domains. To identify interacting proteins, we used the yeast two-hybrid system and a candidate gene approach. Yeast Two-hybrid Assay—Construction of the bait plasmid, hASIC3 (residues 476–531), in the GAL4(DB) vector pAS2-1 (Clontech) was described previously (14Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Crossref PubMed Scopus (102) Google Scholar). This bait construct was transformed into the PJ692A yeast strain using the lithium acetate procedure and mated with yeast strain Y187 pretransformed with the human brain Match-maker cDNA library (Clontech). The mRNA source was a normal, whole brain from a 37-year old Caucasian male as reported on the Clontech product analysis certificate. Mated cells were plated on Leu-/Trp-/His- plates supplemented with 5 mm 3-amino-1,2,4-triazole and grown for 10 days at 30 °C before positive clones were picked and streaked on plates lacking adenine, His, Leu, and Trp. Library plasmids from clones that grew in the absence of adenine, His, Leu, and Trp and that tested positive for β-galactosidase expression were isolated. They were cotransformed with either the bait vector or the original pAS2-1 vector into PJ69-2A to confirm the interaction. Those that were specific for the bait were sequenced. DNA Constructs—hASIC3 (residues 476–531), hASIC1 (residues 459–528), hASIC2 (residues 470–512), hαENaC (residues 589–669), and hβENaC (residues 559–640) in the GAL4(DB) vector pAS2-1 and the full-length mASIC3 construct in pMT3 were described previously (14Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Crossref PubMed Scopus (102) Google Scholar). The hASIC3 deletion constructs used for the yeast two-hybrid experiments were made by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) for residues 476–527 and subcloning using PCR to amplify and insert the C-terminal deletions of hASIC3 residues 476–523, 476–513, and 513–531 into the unique EcoRI and BamHI sites of the GAL(DB) vector. Point mutations in the C terminus of the hASIC3 (residues 476–531) in pAS2-1 (see Fig. 1C) were made using the QuikChange site-directed mutagenesis kit. The mASIC3Δ4 (mASIC3 minus the C-terminal 4 amino acids) construct in pMT3 was also made using the QuikChange mutagenesis kit. The GFP-tagged rat PSD-95 construct was a gift from David Bredt, the Myc-tagged mouse Lin-7b construct was a gift from Ben Margolis, the FLAG-tagged mouse MAGI-1b used in the coimmunoprecipitation experiments was a gift from Irina Dobrosotskaya, the Myc-tagged PSD-95 construct was a gift from Johannes Hell, and the HA-tagged mouse PIST (PDZ protein interacting specifically with TC10) was a gift from Ian Macara. The rat ASIC3 cDNA (used in the electrophysiology studies) was cloned by reverse transcription (RT)-PCR of RNA isolated from Sprague-Dawley rat dorsal root ganglia using the 5′ primer 5′-CCA TCG ATG GAG CCA TGA AAC CTC GCT CCG GAC TGG AGG AGG CCC AG-3′ and the 3′ primer 5′-TCC CAC CGT ACC TGT TAC CTC GTC ACA AGG CTC TAG GGG GTA CCC C-3′. Mouse ASIC1a was cloned as described previously (10Benson C.J. Xie J. Wemmie J.A. Price M.P. Henss J.M. Welsh M.J. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2338-2343Crossref PubMed Scopus (308) Google Scholar). The rat ASIC3Δ4 construct (rASIC3 minus the C-terminal 4 amino acids) and the mouse ASIC1vtrl construct in which the residues VTRL were substituted for the last 4 amino acids of ASIC1a were generated using the QuikChange site-directed mutagenesis kit (Stratagene) and were cloned into pMT3 for expression in CHO cells. All plasmid constructs were confirmed by DNA sequencing. Antibodies—Anti-mASIC3 (anti-DRASIC) and anti-hASIC1 (anti-hASIC) were described previously (3Price M.P. McIllwrath 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, 14Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Crossref PubMed Scopus (102) Google Scholar, 19Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.C. Yoder P.G. Lamani E. Hoshi T. Freeman Jr., J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar), guinea pig anti-ASIC3 was purchased from Chemicon (Temecula, CA), anti-PSD-95 and anti-Lin-7 were purchased from Sigma, anti-HA was purchased from Roche Applied Science, anti-MAGI-1 (sc-11523) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and mouse monoclonal anti-Myc (clone 9E10) was from the Developmental Studies Hybridoma Bank (Iowa City, IA). COS-7 and CHO Cell Culture and Transfection—COS-7 cells were maintained in culture with Dulbecco's modified Eagle's medium plus 10% fetal calf serum in a humidified atmosphere of 5% CO2 in air at 37 °C. COS-7 cells were transfected by electroporation using 107 cells with 20–30 μg of plasmid DNA at a 1:1 ratio (ASIC construct or empty vector:interacting protein construct or empty vector). CHO cells were cultured in F12 medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C. CHO cells were transfected with cDNAs using cationic lipid (TransFast, Promega) following the manufacturer's recommendations. ASIC subunits (0.18 μg/1.5 ml) and PDZ proteins (or dsRed as a control cDNA) (1.82 μg/1.5 ml) were cotransfected at a 1:10 ratio. cDNA for green fluorescent protein (0.33 μg/1.5 ml) was also expressed to facilitate detection of transfected cells by epifluorescence. We have found that >90% of green cells generated ASIC-like current and <5% of non-green cells have acid-activated current. CHO cells were studied 48–72 h after transfection. Immunoprecipitation from COS-7 Cells—Transfected cells were lysed 48 h post-transfection at 4 °C in lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.4 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstatin A) for PSD-95-GFP and (20 mm Tris, pH 7.4, 100 mm NaCl, 1 mm EDTA, pH 8, 1% Triton X-100, 0.4 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstatin A) for FLAG-MAGI-1b, Myc-Lin-7b, and HA-PIST as described previously (20Adams C.M. Snyder P.M. Welsh M.J. J. Biol. Chem. 1997; 272: 27295-27300Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We used 5% of the lysate for Western blot, and the remainder was incubated with the indicated antibody overnight at 4 °C. Proteins were separated on 8% (10% for Myc-Lin-7b) SDS-polyacrylamide gels. Western blots were blocked with 5% bovine serum albumin and incubated first with primary antibody (anti-mASIC3 serum (1:5000), anti-hASIC serum (1: 5000), anti-PSD-95 (1:15,000), anti-MAGI-1 (1:250), anti-Lin-7 (1:2500), or anti-HA (1:7500)) and then with either a horseradish peroxidase-coupled secondary antibody (1:5000) (Amersham Biosciences) or horseradish peroxidase-linked Protein A (1:10,000) (Amersham Biosciences). Proteins were detected by enhanced chemiluminescence (Pierce). Immunoflourescence—COS-7 cells were grown on chamber slides coated with collagen. Cells were fixed with 4% formaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline, blocked with SuperBlock (Pierce), and incubated with primary antibodies anti-mASIC3 serum (1:750) and anti-PSD-95 (1:8000), anti-Myc (1:1000), or anti-HA (1:250); guinea pig anti-ASIC3 (1:600) and anti-Myc (1:1000) followed by the secondary antibodies goat anti-rabbit Alexa 568 (1:1250) and goat anti-mouse Alexa 488 (1:1250); or goat anti-guinea pig Alexa 568 (1:1250) and goat anti-mouse Alexa 488 (1:1250) (Molecular Probes, Eugene, OR). Staining was visualized using a confocal microscope (Bio-Rad 1024). RT-PCR—For RT-PCR analysis, first strand cDNA was synthesized with Superscript II (Invitrogen) with random hexamer primers using mouse brain, spinal cord, and dorsal root ganglion RNA. The following primers were used for PCR amplification: MAGI-1b, 5′-ATGATCCCTCCTAAAATCGCT-3′ and 5′-CTTCCGGAACTCCTTGTGCAC-3′ (138-bp band); PIST, 5′-GGACAGCCTGCGGATAGATGT-3′ and 5′-ACGATAGCGGTGTCCACTCTC-3′ (215-bp band); Lin-7b, 5′-AGCGGAGAGCTGCCCCCGCAG-3′ and 5′-ATGAGCCCGCACCTCGGCGCT-3′ (139-bp band); ASIC3, 5′-GGGGAGTTCCACCATAAGACCACC-3′ and 5′-CTTCCAGATGGGCAGATACTCCTC-3′ (450-bp band); PSD-95, 5′-TCAGGTCTGGGCTTCAGCATC-3′ and 5′-CCGGCGCATGACGTAGAGGCG-3′ (113-bp band). The cycling parameters using the RoboCycler thermocycler (Stratagene) consisted of 40 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min. Immunoprecipitation from Tissue—Rat tissues (brain, spinal cord, and dorsal root ganglia) were isolated and frozen at -80 °C. Lysates were made from the frozen tissue as described previously (21Leonard A.S. Hell J.W. J. Biol. Chem. 1997; 272: 12107-12115Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). PSD-95, ASIC3, or ASIC1 was precipitated by adding anti-PSD-95, anti-mA-SIC3 (3Price M.P. McIllwrath 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), or anti-hASIC 6.4 affinity-purified antibodies (19Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.C. Yoder P.G. Lamani E. Hoshi T. Freeman Jr., J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar) to 500 μg of tissue extracts in a final buffer of 25 mm HEPES, pH 7.4, 100 mm NaCl, 5 mm EGTA, 5 mm EDTA, 1% deoxycholate, 1 mm β-mercaptoethanol, 0.4 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstatin A and rocking at 4 °C. Protein A-Sepharose (Pierce) (3–5 mg, preswollen and washed three times with TBS (10 mm Tris-Cl, pH 7.4, 150 mm NaCl) was added to the samples and mixed for 2.5 h. The immunocomplexes were sedimented by centrifugation and washed three times with 1% Triton X-100 in TBS and once with 10 mm Tris-Cl, pH 7.4, before being extracted with 20 μl of SDS sample buffer (2% SDS, 20 mm dithiothreitol, 10% sucrose, 125 mm Tris-Cl, pH 6.8) for 20 min at 60 °C. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 3% bovine serum albumin in TBS (TBS-bovine serum albumin) and incubated with anti-PSD-95 (1:1000), anti-mASIC3 (1: 250), or anti-hASIC 6.4 (1:1000) affinity-purified antibody for 2 h. The blots were washed five times with TBS-bovine serum albumin, incubated with horseradish-peroxidase-labeled protein A (1:10,000), washed with 0.05% Tween 20 in TBS, and developed with the enhanced chemiluminescence reagent (Pierce). Electrophysiology—Whole-cell patch clamp recordings (at -70 mV) from CHO cells were performed with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and acquired and analyzed with Pulse/Pulsefit 8.30 (HEKA Electronics, Lambrecht, Germany) and Igor Pro 3.16 (WaveMetrics, Lake Oswego, OR) software. Experiments were performed at room temperature. Currents were filtered at 5 kHz and sampled at 2 or 0.2 kHz. Series resistance was compensated by at least 50%. Micropipettes (2–5 megaohms) were filled with internal solution: 100 mm KCl, 10 mm EGTA, 40 mm HEPES, and 5 mm MgCl2, pH 7.4 with KOH. External solution contained: 120 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, 10 mm MES, pH adjusted with tetramethylammonium hydroxide, and osmolarity adjusted with tetramethylammonium chloride. Extracellular solutions were changed within 20 ms using a computer-driven solenoid valve system (10Benson C.J. Xie J. Wemmie J.A. Price M.P. Henss J.M. Welsh M.J. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2338-2343Crossref PubMed Scopus (308) Google Scholar). Data are means ± S.E. Kinetics of desensitization were fit with single exponential equations, and time constants (τ) are reported. Statistical differences were assessed by two-tailed Student's t test. Surface Biotinylation—Surface proteins on 48-h post-transfected COS-7 cells were labeled with cell-impermeable EZ-Link sulfo-N-hydroxysuccinimidobiotin (Pierce), 0.25 mg/ml in phosphate-buffered saline, at 4 °C for 20 min following the manufacturer's recommendations. After labeling, cells were washed two times with TBS and lysed in lysis buffer (25 mm HEPES, pH 7.4, 100 mm NaCl, 5 mm EGTA, 5 mm EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 0.4 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstatin A). Cell surface proteins were isolated by incubating 500 μg of the lysate with immobilized neutravidin beads (Pierce) at 4 °C for 2 h. Total ASIC3 protein was isolated by incubating 500 μg of the same lysate as above with anti-mASIC3 antibodies at 4 °C for 2 h followed by incubation with protein A-Sepharose (prewashed as above). The bound proteins were eluted with sample buffer (2% SDS, 20 mm dithiothreitol, 10% sucrose, 125 mm Tris-Cl, pH 6.8) for 20 min at 65 °C, subjected to SDS-PAGE, Western blotted with anti-mASIC3 antibodies, and developed with the enhanced chemiluminescence reagent (Pierce). We used 10 μg of the total lysate for Western blotting. Identification of MAGI-1b, Lin-7b, and PIST with a Yeast Two-hybrid Screen—To find proteins that interact with ASIC3, we used its intracellular C terminus as the bait to screen a human brain cDNA library in the yeast two-hybrid system. We identified three clones specific for the bait: MAGI-1b (amino acids 642–1287), Lin-7b (amino acids 40–207), and PIST (amino acids 46–462). Each clone was missing sequence that encoded the N-terminal portion of the protein but included the C-terminal end. All three clones encoded at least one PDZ domain. MAGI-1b is a membrane-associated guanylate kinase that contains two WW domains, a guanylate kinase domain, and five PDZ domains (22Dobrosotskaya I. Guy R.K. James G.L. J. Biol. Chem. 1997; 272: 31589-31597Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The clone we identified contained PDZ domains 2–5. MAGI-1b is a member of a family of MAGI proteins, and several different splice variants of MAGI-1 have been identified including MAGI-1a, -1b, and -1c (22Dobrosotskaya I. Guy R.K. James G.L. J. Biol. Chem. 1997; 272: 31589-31597Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 23Laura R.P. Ross S. Koeppen H. Lasky L.A. Exp. Cell Res. 2002; 275: 155-170Crossref PubMed Scopus (76) Google Scholar). Lin-7b, also named MALS (mammalian Lin-7 protein) or Veli (vertebrate LIN-7 homologs) contains an N-terminal domain that binds CASK and a PDZ domain at its C terminus (24Borg J.P. Straight S.W. Kaech S.M. de Taddeo-Borg M. Kroon D.E. Karnak D. Turner R.S. Kim S.K. Margolis B. J. Biol. Chem. 1998; 273: 31633-31636Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 25Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 26Jo K. Derin R. Li M. Bredt D.S. J. Neurosci. 1999; 19: 4189-4199Crossref PubMed Google Scholar). Both Lin-7b and MAGI isoforms are thought to provide a scaffold linking receptors and channels with cytoskeletal proteins and enzymes at sites of cell-cell contact such as tight junctions in epithelial cells and synapses in neurons. PIST is an intracellular protein first identified by its interaction with TC10, a Rho GTPase (27Neudauer C.L. Joberty G. Macara I.G. Biochem. Biophys. Res. Commun. 2001; 280: 541-547Crossref PubMed Scopus (72) Google Scholar). PIST contains two coiled-coil domains, a leucine zipper domain embedded in the second coiled-coil domain, and a PDZ domain at its C terminus. Splice variants of PIST, such as CAL (CFTR-associated ligand) and FIG (fused in glioblastoma), are thought to participate in the trafficking of proteins out of the trans-Golgi network and retention of membrane proteins inside the cell (28Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 29Charest A. Lane K. McMahon K. Housman D.E. J. Biol. Chem. 2001; 276: 29456-29465Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The interaction of MAGI-1b, Lin-7b, and PIST was specific for the ASIC3 C terminus; the C termini of ASIC1, ASIC2, αENaC, and βENaC failed to interact with these proteins (Fig. 1A). Deleting the distal half of the ASIC3 C terminus eliminated the interaction with all three proteins, whereas eliminating the N-terminal half had no effect (Fig. 1B). Moreover deleting the last 4 or 8 C-terminal residues also abolished the interaction. These results suggested that ASIC3 interacts with the three PDZ domain proteins via a PDZ-binding motif at its C terminus. The C-terminal 4 residues of hASIC3 (VTQL) correspond to a type I PDZ-binding motif (X(S/T)X(V/L/I) where X is any amino acid) (30Songyang 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). Mutating the Leu at the 0 position to Ala abolished the interaction with MAGI-1b, Lin-7b, and PIST (Fig. 1C). In addition, mutating the Thr at the -2 position abolished the interaction with MAGI-1b and Lin-7b (PIST was not tested). In contrast, mutating the -3 Val had no effect on the interactions. Mutation of the -1 Gln did not alter the interaction with MAGI-1b or Lin-7b but disrupted the interaction with PIST. Thus, binding conforms to an interaction between the ASIC3 C terminus and PDZ domains in MAGI-1b, Lin-7b, and PIST most likely through their PDZ domains. ASIC3 Interacts with MAGI-1b, Lin-7b, and PIST in COS-7 Cells—To test the interaction between ASIC3 and these proteins, we transfected COS-7 cells with mouse ASIC3 and epitope-tagged MAGI-1b, Lin-7b, and PIST. Immunoprecipitating ASIC3 co-precipitated each of these PDZ proteins (Fig. 2). We used PIST to test the importance of the C-terminal 4 residues in the interaction with PDZ domain-containing proteins. Deleting the last 4 residues of ASIC3 (ASIC3Δ4) prevented co-precipitation of PIST. PSD-95 Interacts with ASIC3 and Requires Its PDZ-binding Domain—In addition to their interaction with ASIC3, previous studies showed that the PDZ domain-containing proteins MAGI-2 (the major neuronal MAGI protein) and Lin-7 interact with the N-methyl-d-aspartate receptor subunits (26Jo K. Derin R. Li M. Bredt D.S. J. Neurosci. 1999; 19: 4189-4199Crossref PubMed Google Scholar, 31Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Previous studies showed that the PDZ domain scaffolding protein PSD-95 also interacts with NR2 subunits (32Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1622) Google Scholar). PSD-95 contains a Src homology 3 domain, a guanylate kinase domain, and three N-terminal PDZ domains (33Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). In addition, both MAGI-2 and PSD-95 interacted with shaker type K+ channels and the neuronal cell surface molecule neuroligin (31Hirao K. Hata Y. Ide N. Takeuchi M. Irie M. Yao I. Deguchi M. Toyoda A. Sudhof T.C. Takai Y. J. Biol. Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 34Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (893) Google Scholar, 35Irie M. Hata Y. Takeuchi M. Ichtchenko K. Toyoda A. Hirao K. Takai Y. Rosahl T.W. Sudhof T.C. Science. 1997; 277: 1511-1515Crossref PubMed Scopus (602) Google Scholar). Therefore, we hypothesized that ASIC3 may bind PSD-95 via its PDZ-binding domain. Supporting this hypothesis, we found that ASIC3 co-immunoprecipitated PSD-95, and PSD-95 co-precipitated ASIC3 in COS-7 cells (Fig. 3, A and B). As controls, neither ASIC1a nor ASIC3 missing its C-terminal 4 residues co-precipitated PSD-95 (Fig. 3C). These data suggest that ASIC3 interacts with PSD-95 through its PDZ-bind