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• W2073163983 abstract "The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity. The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity. Acid-sensing ion channels (ASICs) 2The abbreviations used are: ASIC, acid-sensing ion channel; NMDA, N-methyl-d-aspartate; CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein (GFP); HA, hemagglutinin; PBS, phosphate-buffered saline; siRNA, small interfering RNA; MES, 4-morpholineethanesulfonic acid; RT, reverse transcription; TMAOH, tetramethylammonium hydroxide. are H+-gated members of the DEG/ENaC family (1Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci... 1999; 868: 67-76Google Scholar, 2Kellenberger S. Schild L. Physiol. Rev... 2002; 82: 735-767Google Scholar, 3Wemmie J.A. Price M.P. Welsh M.J. Trends Neurosci... 2006; 29: 578-586Google Scholar). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci... 1999; 868: 67-76Google Scholar, 2Kellenberger S. Schild L. Physiol. Rev... 2002; 82: 735-767Google Scholar, 3Wemmie J.A. Price M.P. Welsh M.J. Trends Neurosci... 2006; 29: 578-586Google Scholar, 4Jasti J. Furukawa H. Gonzales E.B. Gouaux E. Nature.. 2007; 449: 316-323Google Scholar). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5Wemmie 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 J.H.J. Welsh M.J. Neuron.. 2002; 34: 463-477Google Scholar, 6Baron A. Waldmann R. Lazdunski M. J. Physiol. (Lond.).. 2002; 539: 485-494Google Scholar, 7Askwith C.C. Wemmie J.A. Price M.P. Rokhlina T. Welsh M.J. J. Biol. Chem... 2004; 279: 18296-18305Google Scholar). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8Benson 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: 2338-2343Google Scholar, 9Xiong Z.G. Zhu X.M. Chu X.P. Minami M. Hey J. Wei W.L. MacDonald J.F. Wemmie J.A. Price M.P. Welsh M.J. Simon R.P. Cell.. 2004; 118: 687-698Google Scholar, 10Babini E. Paukert M. Geisler H.S. Gründer S. J. Biol. Chem... 2002; 277: 41597-41603Google Scholar). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca2+ (9Xiong Z.G. Zhu X.M. Chu X.P. Minami M. Hey J. Wei W.L. MacDonald J.F. Wemmie J.A. Price M.P. Welsh M.J. Simon R.P. Cell.. 2004; 118: 687-698Google Scholar, 11Bassler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P. Grunder S. J. Biol. Chem... 2001; 276: 33782-33787Google Scholar, 12Yermolaieva O. Leonard A.S. Schnizler M.K. Abboud F.M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 6752-6757Google Scholar, 13Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature.. 1997; 386: 173-177Google Scholar). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H+-gated currents unless extracellular pH is reduced below pH 5.0 (5Wemmie 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 J.H.J. Welsh M.J. Neuron.. 2002; 34: 463-477Google Scholar, 7Askwith C.C. Wemmie J.A. Price M.P. Rokhlina T. Welsh M.J. J. Biol. Chem... 2004; 279: 18296-18305Google Scholar). Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5Wemmie 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 J.H.J. Welsh M.J. Neuron.. 2002; 34: 463-477Google Scholar, 14Zha X.-M. Wemmie J.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 16556-16561Google Scholar, 15Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J... 2002; 361: 443-450Google Scholar). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5Wemmie 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 J.H.J. Welsh M.J. Neuron.. 2002; 34: 463-477Google Scholar, 16Wemmie J.A. Askwith C.C. Lamani E. Cassell M.D. Freeman J.H.J. Welsh M.J. J. Neurosci... 2003; 23: 5496-5502Google Scholar). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17Miesenbock G. De Angelis D.A. Rothman J.E. Nature.. 1998; 394: 192-195Google Scholar, 18Krishtal O.A. Osipchuk Y.V. Shelest T.N. Smirnoff S.V. Brain Res... 1987; 436: 352-356Google Scholar, 19Chesler M. Kaila K. Trends Neurosci... 1992; 15: 396-402Google Scholar). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20Levitan I.B. Nat. Neurosci... 2006; 9: 305-310Google Scholar, 21Kim E. Sheng M. Nat. Rev. Neurosci... 2004; 5: 771-781Google Scholar). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J... 2002; 361: 443-450Google Scholar, 22Duggan A. Garcia-Anoveros J. Corey D.P. J. Biol. Chem... 2002; 277: 5203-5208Google Scholar, 23Leonard 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: 2029-2034Google Scholar). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24Donier E. Rugiero F. Okuse K. Wood J.N. J. Biol. Chem... 2005; 280: 38666-38672Google Scholar) and with Ca2+/calmodulin-dependent protein kinase II to phosphorylate the channel (25Gao J. Duan B. Wang D.G. Deng X.H. Zhang G.Y. Xu L. Xu T.L. Neuron.. 2005; 48: 635-646Google Scholar). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function. In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26Otey C.A. Carpen O. Cell Motil. Cytoskeleton.. 2004; 58: 104-111Google Scholar). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26Otey C.A. Carpen O. Cell Motil. Cytoskeleton.. 2004; 58: 104-111Google Scholar, 27Flood G. Kahana E. Gilmore A.P. Rowe A.J. Gratzer W.B. Critchley D.R. J. Mol. Biol... 1995; 252: 227-234Google Scholar, 28Djinovic-Carugo K. Gautel M. Ylanne J. Young P. FEBS Lett... 2002; 513: 119-123Google Scholar). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29Wyszynski M. Kharazia V. Shanghvi R. Rao A. Beggs A.H. Craig A.M. Weinberg R. Sheng M. J. Neurosci... 1998; 18: 1383-1392Google Scholar, 30Rao A. Kim E. Sheng M. Craig A.M. J. Neurosci... 1998; 18: 1217-1229Google Scholar, 31Allison D.W. Gelfand V.I. Spector I. Craig A.M. J. Neurosci... 1998; 18: 2423-2436Google Scholar, 32Asanuma K. Kim K. Oh J. Giardino L. Chabanis S. Faul C. Reiser J. Mundel P. J. Clin. Investig... 2005; 115: 1188-1198Google Scholar, 33Walikonis R.S. Oguni A. Khorosheva E.M. Jeng C.J. Asuncion F.J. Kennedy M.B. J. Neurosci... 2001; 21: 423-433Google Scholar, 34Nuriya M. Oh S. Huganir R.L. J. Neurochem... 2005; 95: 544-552Google Scholar, 35Nakagawa T. Engler J.A. Sheng M. Neuropharmacology.. 2004; 47: 734-745Google Scholar). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca2+-dependent inhibition of NMDA receptors (36Rosenmund C. Westbrook G.L. Neuron.. 1993; 10: 805-814Google Scholar, 37Wyszynski M. Lin J. Rao A. Nigh E. Beggs A.H. Craig A.M. Sheng M. Nature.. 1997; 385: 439-442Google Scholar, 38Krupp J.J. Vissel B. Thomas C.G. Heinemann S.F. Westbrook G.L. J. Neurosci... 1999; 19: 1165-1178Google Scholar). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca2+ channels, K+ channels, and TRP channels (39Sadeghi A. Doyle A.D. Johnson B.D. Am. J. Physiol. Cell Physiol... 2002; 282: 1502-1511Google Scholar, 40Maruoka N.D. Steele D.F. Au B.P. Dan P. Zhang X. Moore E.D. Fedida D. FEBS Lett... 2000; 473: 188-194Google Scholar, 41Li Q. Dai X.Q. Shen P.Y. Wu Y. Long W. Chen C.X. Hussain Z. Wang S. Chen X.Z. J. Neurochem... 2007; 103: 2391-2400Google Scholar). To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na+ channel (ENaC) interacts with the cytoskeleton (42Mazzochi C. Benos D.J. Smith P.R. Am. J. Physiol. Renal Physiol... 2006; 291: 1113-1122Google Scholar, 43Zuckerman J.B. Chen X. Jacobs J.D. Hu B. Kleyman T.R. Smith P.R. J. Biol. Chem... 1999; 274: 23286-23295Google Scholar). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a. Expression Constructs—Mouse ASIC1a was cloned into pMT3 (44Swick A.G. Janicot M. Cheneval-Kastelic T. McLenithan J.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A... 1992; 89: 1812-1816Google Scholar) for heterologous expression. Human α-actinin-1 was a gift of C. Otey, University of North Carolina, and α-actinin-2, -3, and -4 were gifts of M. Sheng, Massachusetts Institute of Technology. Full-length human α-actinin-1–4 were cloned into pEGFP vectors (Clontech Laboratories) to generate enhanced green fluorescent protein (EGFP)-α-actinin fusion proteins; previous studies showed that the localization and function of α-actinins were unaffected by GFP tags (45Fraley T.S. Pereira C.B. Tran T.C. Singleton C. Greenwood J.A. J. Biol. Chem... 2005; 280: 15479-15482Google Scholar, 46Zhang W. Gunst S.J. J Physiol... 2006; 572: 659-676Google Scholar, 47Rajfur Z. Roy P. Otey C. Romer L. Jacobson K. Nat. Cell Biol... 2002; 4: 286-293Google Scholar). A dominant-negative N-terminal-truncated (missing amino acid 3–249) human α-actinin-1 (rod-actinin) (48Bhatt A. Kaverina I. Otey C. Huttenlocher A. J. Cell Sci... 2002; 115: 3415-3425Google Scholar) was a gift of A. Huttenlocher, University of Wisconsin. Rod-actinin contains the central α-actinin spectrin domain including the C terminus but lacks the N-terminal head domain. Dimers containing rodactinin can bind other proteins, but the absence of the head domain interrupts cross-linking to the filamentous actin cytoskeleton (48Bhatt A. Kaverina I. Otey C. Huttenlocher A. J. Cell Sci... 2002; 115: 3415-3425Google Scholar, 49Wang J. Shaner N. Mittal B. Zhou Q. Chen J. Sanger J.M. Sanger J.W. Cell Motil. Cytoskeleton.. 2005; 61: 34-48Google Scholar). Rod-actinin would be expected to interfere with all α-actinins, and consistent with that conclusion, it disrupted the effect of α-actinins in human embryonic kidney cells, which express both α-actinin-1 and -4. 3J. W. Hell, unpublished observation. Chinese hamster ovary (CHO) cells also express α-actinin-1 and α-actinin-4 (50Celli L. Ryckewaert J.J. Delachanal E. Duperray A. J. Immunol... 2006; 177: 4113-4121Google Scholar). HA-tagged ASIC1a was made by adding the influenza hemagglutinin epitope (YPYDVPDYAGV) to the N terminus of ASIC1a. Cell Culture and Transfection—For patch clamp studies CHO cells were transfected with 1–6 μg of DNA using the TransFast Lipid™ reagent (Promega Madison, WI) and cultured on glass coverslips in 35-mm Petri dishes. To identify transfected cells using epifluorescence, we used EGFP at an ASIC1a:EGFP ratio of 6:1. Channel and α-actinin constructs were transfected at 1:1 ratio. Empty vector DNA was used to maintain a constant final DNA concentration for all transfections. Experiments were performed at room temperature 2–3 days after transfection. Hippocampal Neuronal Culture and Transfection—Rat E18 hippocampal neurons (Brain Bits, Springfield, IL) were transfected after 7–8 days in culture using calcium phosphate transfection. Conditioned medium was collected and replaced by freshly prepared neurobasal medium with B-27 supplement (Invitrogen) 30 min before transfection. A total of 5 μg of vector DNA was added in a transfection mix consisting of 50 ml of water, 5 ml of CaCl2 (2.5 m), and 60 ml of 2× PBS per dish. After 3 h of incubation at 37 °C and after an 8-min Hanks' balanced salt solution washing step, conditioned media was reapplied to cultures. At day 10, neurons were used for whole-cell, patch clamp experiments. Immunoprecipitation and Immunoblotting—Whole mouse brains from adult animals were homogenized in ice-cold buffer (5 ml/brain) containing 300 mm sucrose, 10 mm Tris (pH 7.5), 20 mm NaCl plus protease inhibitors (Complete Mini, EDTA-free, Roche Applied Science) using a glass Teflon homogenizer. Samples were spun for 2 min in a tabletop centrifuge at 2000 × g to remove large debris. Supernatants were centrifuged for 30 min at 135,000 × g in a Beckman TLA100.3 rotor at 4 °C. The membrane pellet was washed once on ice and then solubilized in 6 ml of 50 mm Tris (pH 7.5), 150 mm NaCl, protease inhibitors (Complete Mini) with 1% Triton X-100. For immunoprecipitation, samples were centrifuged (10 min, 700 × g) to remove particulate debris and precleared by the addition of protein G-agarose (Roche Applied Science, 50 μl, 15 mg/ml). Either undiluted affinity-purified ASIC1 antibody (raised in rabbits against the 22-amino acid peptide from the C terminus of ASIC1 and purified as described previously (16Wemmie J.A. Askwith C.C. Lamani E. Cassell M.D. Freeman J.H.J. Welsh M.J. J. Neurosci... 2003; 23: 5496-5502Google Scholar)) or a mouse monoclonal α-actinin antibody BM-75.2 that binds to all four actinin isoforms (1 μl, Sigma-Aldrich) was added to 750 μl of protein extract and incubated for 3 h while rotating at 4 °C. For precipitation, protein G-agarose beads (Roche Applied Science, 50 μl 15 mg/ml) were added, and samples were rotated overnight at 4 °C. After centrifugation, 3 wash steps were performed to clear samples (buffer 1: 50 mm Tris (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycolate, protease inhibitors (Complete Mini); buffer 2: 50 mm Tris (pH 7.5), 500 mm NaCl, 0.1% Nonidet P-40, 0.05% sodium deoxycolate; buffer 3: 10 mm Tris (pH 7.5), 0.1% Nonidet P-40, 0.05% sodium deoxycolate). Bound proteins were extracted at 95 °C in SDS sample buffer (0.125 mm Tris (pH 7.5), 3.4% SDS, 17% glycerol, 67 mm dithiothreitol, 0.008% bromphenol blue) for 10 min and were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Co-immunoprecipitation from COS-7 cells was performed 48 h after electroporation (106 cells, 10 or 15 μg of DNA at 1:1 ASIC1a:α-actinin ratio per sample). Electroporation was performed with 400 μl of prechilled cell suspension (120 mm KCl, 25 mm HEPES, 0.15 mm NaCl, 10 mm KPO4, 2 mm EGTA, 5 mm MgCl2 with 2 mm ATP and 5 mm glutathione using a single 25-s pulse of 0.320 V and 975 microfarads. Cells in 100-mm dishes were washed twice with ice-cold PBS and harvested in lysis buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, and protease inhibitors (Complete Mini). Supernatants from cell extracts were pre-cleared with protein A-Sepharose (Pierce) gently agitated for 1 h at 4 °C. Either ASIC1 antibody (16Wemmie J.A. Askwith C.C. Lamani E. Cassell M.D. Freeman J.H.J. Welsh M.J. J. Neurosci... 2003; 23: 5496-5502Google Scholar) or rabbit polyclonal anti-GFP antibody (Clontech, Living Colors® Full-length A.v. polyclonal antibody) were added to 250–500 μl of protein extract after 1 h of rotation at 4 °C. Protein A-Sepharose was added followed by incubation overnight with rotation at 4 °C. Immunoprecipitates absorbed to protein A-Sepharose were washed three times as described above. The immunoprecipitated proteins were resuspended in SDS sample buffer and electrophoresed. For immunoblotting, primary antibodies were detected with horseradish peroxidase-conjugated goat-anti rabbit or anti-mouse IgG from ECL (GE Healthcare). SuperSignal West Pico Chemiluminescent Substrate (Pierce) was used for detection. CHO cells were transfected with HA-ASIC1a:α-actinin:GFP at 1:1:1 ratio. For control, α-actinin was replaced with GFP so the ratio was 1:2 for HA-ASIC1a:GFP. Biotinylation of CHO cells was performed 2 days after Lipofectamine-mediated transfection. Cells were washed 3× with ice-cold PBS+/+, and 3 ml of 0.5 mg/ml NHS-biotin in PBS+/+ was added to each 10-cm dish followed by incubation at 4 °C for 30 min with gentle rocking. Cells were washed once with PBS+/+, and 0.1 m glycine in PBS+/+ was added to quench the reaction followed by 2 washes with PBS+/+. Cells were lysed in PBS with 1% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, and freshly added proteinase inhibitors (Roche Applied Science). Cell lysate was sonicated briefly and cleared by centrifugation. Protein concentration was quantified using a modified Bradford assay kit (Bio-Rad). For Neutravidin pulldown, 60 μl of a 50% slurry of Neutravidin beads were added to 200 μl of cell lysate and incubated at 4 °C overnight with gentle rotation. Beads were washed 3× with wash buffer (Tris 50 mm (pH 7.4), 1% Triton X-100). Beads were then boiled in 80 μl of SDS sample buffer with or without reducing agent. RT-PCR—Rat hippocampal neurons and rat muscle tissue were lysed in cell lysis buffer, and first-strand cDNA was synthesized using the Cells-to-cDNA II Kit (Ambion). For PCR, the primer pairs for detection of α-actinin-1 were 5′-gatgcagacaaggagcgcct-3′ (bp 1887–1906) and 5′-gggacccaacgtgccggag-3′ (bp 2495–2513); for α-actinin-2 they were 5′-agaatgaggtggagaaggtga-3′ (bp 1787–1807) and 5′-tggaaggtgaccgtgccttg-3′ (bp 2467–2486); for α-actinin-3 they were 5′-ctgcagctggagtttgctcg-3′ (bp 1607–1625) and 5′-tgtgctccatgctgtagacc-3′ (bp 2196–2215), and for α-actinin-4 they were 5′-agcaatcacatcaagctgtcg-3′ (bp 1906–1927) and 5′-ccacactcatgatccggttg-3′ (bp 2542–2561). RT-PCR products were sequenced to confirm identity. RNA Interference—We generated small interfering RNAs (siRNAs) against each of the four rat α-actinin isoforms using OligoEngine™ to locate 19 nucleotides within exons of the target and immediately downstream of AA dinucleotides and to exclude identity to other sequences in the NCBI data base. Sequences with potential polymerase III termination sites and mRNA splice sites were avoided. The 19-nucleotide sense sequence and the inverted antisense sequence were connected by a 9-nucleotide spacer to allow stem-loop formation. At the 3′ end, a penta-thymidine motif provided a polymerase III termination site. siRNAs were cloned into pSilencer™ vector (Ambion) for expression of the respective sequences under the U6 promotor. DsRed and its cytomegalovirus promoter from pDsRed2-N1 were cloned into the Kpn site upstream of the U6 promoter within the pSilencer™ vector; this vector was used for identification of transfected neurons. To test for efficacy and specificity of siRNA, CHO cells were co-transfected with either one of the siRNAs and EGFP-tagged α-actinin at a 10:1 cDNA ratio (total 11 μg DNA) using Lipofectamine 2000 in Opti-MEM I media according to the manufacturer's recommendation (Invitrogen). RNAi1 targeted at 476–494 from the start sequence of rat α-actinin-1 (the sense chain was 5′-gatctccaacgtcaacaagttcaagagacttgttgacgttggagatctttttt-3′. RNAi2 targeted at 224–242 from the start sequence of rat α-actinin-2; the sense chain was 5′-tggcctgatggatcatgagttcaagagactcatgatccatcaggccatttttt-3′. RNAi3 targeted at 455–473 from the start sequence of rat α-actinin-3; the sense chain was 5′-aacgagaagctgatggaggttcaagagacctccatcagcttctcgtttttttt-3′. RNAi4 targeted at 493–511 from the start sequence of rat α-actinin-4; the sense chain was 5′-gactatccaggagatgcagttcaagagactgcatctcctggatagtctttttt-3′. Electrophysiology—Whole-cell, patch clamp recordings were performed on CHO cells on 10-mm glass coverslips continuously superfused with a bath solution containing: 128 mm NaCl, 5.4 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm MES, 5.55 mm glucose, adjusted to pH 7.4 with TMAOH. The standard pipette solution contained 10 mm NaCl, 121 mm KCl, 5 mm EGTA, 2 mm MgCl2, 2 mm Na2ATP, 10 mm HEPES adjusted to pH 7.25 with TMAOH. For whole-cell, patch clamp experiments with hippocampal neurons the bath solution contained 100 mm NaCl, 5.4 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm MES, 10 mm glucose adjusted to pH 7.4 with TMAOH. The pipette solution contained 10 mm NaCl, 70 mm potassium gluconate, 10 mm KCl, 10 mm EGTA, 1 mm MgCl2, 3 mm Na2ATP, 25 mm HEPES adjusted to pH 7.25 with TMAOH. Bath solutions with different pH values were adjusted with TMAOH and applied with a rapid solution exchanger (RSC-200 & EVH-9, Biologic Science Instruments). Recording pipettes were pulled from capillary glass with a micropipette puller (Sutter instruments) and polished (MF830, Narishige, Japan). Pipette resistances ranged from 2 to 5.5 megaohms. Recordings were made at room temperature using an AXO-PATCH 200B amplifier with pCLAMPex 8.1 software (Axon Instruments). Data were analyzed using Clampfit (Axon Instruments). Amplitude was determined by subtracting the base-line current at pH 7.4 from the peak current amplitude. The τ of desensitization (τdes) was calculated by fitting the data to a single exponential equation with Clampfit (Axon Instruments). Membrane potential was held at –70 mV. Data are shown as the mean ± S.E. Statistical differences were determined by two-tailed Student's t test. ASIC1a Contains a Putative Binding Site for α-Actinin—In the C terminus of ASIC1a, we identified an amino acid motif that resembled sequences in the cytosolic portions of the NMDA receptor subunit 1 and the P2X7 purinoceptor, two ion channels known to physically interact with α-actinin (Fig. 1) (38Krupp J.J. Vissel B. Thomas C.G. Heinemann S.F. Westbrook G.L. J. Neurosci... 1999; 19: 1165-1178Google Scholar, 51Leonard A.S. Bayer K.U. Merrill M.A. Lim I.A. Shea M.A. Schulman H. Hell J.W. J. Biol. Chem... 2002; 277: 48441-48448Google Scholar, 52Kim M. Jiang L.H. Wilson H.L. North R.A. Surprenant A. EMBO J.. 2001; 20: 6347-6358Google Scholar). The potential α-actinin-binding site in ASIC1a lies in the cytosolic portion of the subunit and would, therefore, be accessible for interaction with other cytoskeletal proteins. The sequence was not present in ASIC2 or ASIC3, suggesting that α-actinin might specifically interact with ASIC1. Because ASIC1a and -1b splice variants share the same C terminus, interactions would be predicted to occur with both subunits. ASIC1a Associates with α-Actinin-1 and α-Actinin-4—To test the hypothesis that ASIC1a and α-actinin interact, we expressed ASIC1a with each of the four α-actinin isoforms in COS-7 cells. Immunoprecipitating ASIC1a co-precipitated α-actinin-1 and α-actinin-4 (Fig. 2). Conversely, precipitating α-actinin-1 or α-actinin-4 co-precipitated ASIC1a. ASIC1a weakly co-precipitated α-actinin-3, but not the converse, and we did not detect an interaction with α-actinin-2. These data suggest that ASIC1a resides in close proximity with at least two of the actinins, α-actinin-1 and α-actinin-4. To learn whether the potential α-actinin binding motif in the C terminus of ASIC1a was involved in the interaction between ASIC1a and α-actinin, we expressed α-actinin-4 with an ASIC1a variant that contained mutations in the potential α-actinin binding motif (ASIC1amut residues 484LSLDDVK490 mutated to 484LSADAVA490). Mutating the C-terminal motif prevented ASIC1a from co-precipitating α-actinin-4 (Fig. 3). Likewise, α-actinin-4 failed to co-precipitate ASIC1amut. These results suggest a direct association between α-actinin-4 and ASIC1a that depends on the α-actinin binding motif. For most of the remainder of the studies, we focused on α-actinin-4. α-Actinin Did Not Affect Cell Surface Expression of ASIC1a—To determine whether α-actinin might regulate surface expression of ASIC1a, we biotinylated cell surface proteins in CHO cells transfected with ASIC1a. Co-expressing ASIC1a with α-actinin-4, α-actinin-1, or rod-actinin did not alter the fraction of ASIC1a on the cell surface (Fig. 4, A and B). α-Actinin Influences ASIC1a Current Density—To learn whether α-actinin also regulates the function of ASIC1a, we co-expressed them and measured currents. α-Actinin-4 reduced ASIC1a current density, whereas α-actinin-1 had no effect (Fig. 5, A and B). These results suggest that even though both α-actinins interact with ASIC1a, they have selective functional effects. As an additional test of the effect of α-actinin, we co-expressed ASIC1a with rod-actinin, a dominant-negative construct (48Bhatt A. Kaverina I. Otey C. Huttenlocher A. J. Cell Sci... 2002; 115: 3415-3425Google Scholar, 49Wang J. Shaner N. Mittal B. Zhou Q. Chen J. Sanger J.M. Sanger J.W. Cell Motil. Cytoskeleton.. 2005; 61: 34-48Google Scholar). We found that the dominant-negative rod-actinin fragment had the opposite effect of α-actinin-4 and increased current density (Fig. 5, A and B). Finding that rod-actinin increased current amplitude suggested that it disrupted an interaction between ASIC1a and an endogenous α-actinin. Such an interaction predicts that ASIC1amut, which did not interact with α-actinin, would have a greater current density than ASIC1a. Consistent with this idea, ASIC1amut generated more current than ASIC1a (Fig. 5, A and B). In addition, α-actinin-1 and α-actinin-4 failed to alter current produced by ASIC1amut. These data also suggest that the effect of α-actinin-4 on current results from a direct interaction with ASIC1a. α-Actinin Alters the Properties of ASIC1a Currents—The findings that α-actinin-4 did not change the amount of ASIC1a on the cell surface whereas it decreased current density suggested that α-actinin-4 must have also altered the properties of the current generated by ASIC1a or increased the proportion of silent channels. To test this possibility, we examined several characteristics of ASIC1a current, including the pH sensitivity, the rate of desensitization, and the time course of recovery from desensitization. Co-expressing α-actinin-4 increased the pH sensitivity of ASIC1a (Fig. 6A, Table 1). Conversely, the dominant-negative rod-actinin had the opposite effect, reducing pH sensitivity. Consistent with these data, when we disrupted the ASIC1a α-actinin-binding site (ASIC1amut), pH sensitivity fell, and α-actinin-4 and rod-actinin failed to alter the pH sensitivity of this variant (Fig. 6B, Table 1). These results indicate that the interaction with α-actinin influenced the sensitivity of ASIC1a to extracellular protons.TABLE 1α-Actinin and pH sensitivity of ASIC1a in CHO c" @default.
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• W2073163983 title "The Cytoskeletal Protein α-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction" @default.
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