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• W2016367251 abstract "Acid-sensing ion channel (ASIC) 1a and ASIC2a are acid-sensing ion channels in central and peripheral neurons. ASIC1a has been implicated in long-term potentiation of synaptic transmission and ischemic brain injury, whereas ASIC2a is involved in mechanosensation. Although the biological role and distribution of ASIC1a and ASIC2a subunits in brain have been well characterized, little is known about the intracellular regulation of these ion channels that modulates their function. Using pulldown assays and mass spectrometry, we have identified A kinase-anchoring protein (AKAP)150 and the protein phosphatase calcineurin as binding proteins to ASIC2a. Extended pulldown and co-immunoprecipitation assays showed that these regulatory proteins also interact with ASIC1a. Transfection of rat cortical neurons with constructs encoding green fluorescent protein- or hemagglutinin-tagged channels showed expression of ASIC1a and ASIC2a in punctate and clustering patterns in dendrites that co-localized with AKAP150. Inhibition of protein kinase A binding to AKAPs by Ht-31 peptide reduces ASIC currents in cortical neurons and Chinese hamster ovary cells, suggesting a role of AKAP150 in association with protein kinase A in ASIC function. We also demonstrated a regulatory function of calcineurin in ASIC1a and ASIC2a activity. Cyclosporin A, an inhibitor of calcineurin, increased ASIC currents in Chinese hamster ovary cells and in cortical neurons, suggesting that activity of ASICs is inhibited by calcineurin-dependent dephosphorylation. These data imply that ASIC down-regulation by calcineurin could play an important role under pathological conditions accompanying intracellular Ca2+ overload and tissue acidosis to circumvent harmful activities mediated by these channels. Acid-sensing ion channel (ASIC) 1a and ASIC2a are acid-sensing ion channels in central and peripheral neurons. ASIC1a has been implicated in long-term potentiation of synaptic transmission and ischemic brain injury, whereas ASIC2a is involved in mechanosensation. Although the biological role and distribution of ASIC1a and ASIC2a subunits in brain have been well characterized, little is known about the intracellular regulation of these ion channels that modulates their function. Using pulldown assays and mass spectrometry, we have identified A kinase-anchoring protein (AKAP)150 and the protein phosphatase calcineurin as binding proteins to ASIC2a. Extended pulldown and co-immunoprecipitation assays showed that these regulatory proteins also interact with ASIC1a. Transfection of rat cortical neurons with constructs encoding green fluorescent protein- or hemagglutinin-tagged channels showed expression of ASIC1a and ASIC2a in punctate and clustering patterns in dendrites that co-localized with AKAP150. Inhibition of protein kinase A binding to AKAPs by Ht-31 peptide reduces ASIC currents in cortical neurons and Chinese hamster ovary cells, suggesting a role of AKAP150 in association with protein kinase A in ASIC function. We also demonstrated a regulatory function of calcineurin in ASIC1a and ASIC2a activity. Cyclosporin A, an inhibitor of calcineurin, increased ASIC currents in Chinese hamster ovary cells and in cortical neurons, suggesting that activity of ASICs is inhibited by calcineurin-dependent dephosphorylation. These data imply that ASIC down-regulation by calcineurin could play an important role under pathological conditions accompanying intracellular Ca2+ overload and tissue acidosis to circumvent harmful activities mediated by these channels. Acid-sensing ion channels (ASIC) 3The abbreviations used are: ASIC, acid-sensing ion channel; AKAP, A kinase-anchoring protein; PKA, cAMP-dependent protein kinase (protein kinase A); PKC, protein kinase C; PICK1, protein interacting with C kinase 1; CHO cells, Chinese hamster ovarian cancer cells; GST, glutathione S-transferase; YFP, yellow fluorescent protein; HA, hemagglutinin; HEK cells, human embryonic kidney cells; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; ANOVA, analysis of variance; pF, picofarad; NMDA, N-methyl-d-aspartate; GFP, green fluorescent protein. are amiloride-sensitive Na+ channels and constitute one member of the epithelial Na+ channel/degenerin superfamily (1Garciía-Añoveros J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (296) Google Scholar, 2Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (835) Google Scholar, 3Lingueglia E. Voilley N. Waldmann R. Lazdunski M. Barbry P. FEBS. 1993; 318: 95-99Crossref PubMed Scopus (321) Google Scholar, 4Voilley N. Lingueglia E. Champigny G. Mattei M.G. Waldmann R. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 247-251Crossref PubMed Scopus (213) Google Scholar). Although epithelial Na+ channels are constitutively active in kidney epithelia, ASICs are proton-gated ion channels that are activated in response to reduction in extracellular pH. In mammalian brain and sensory neurons, isoforms of ASICs and their splice variants (ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3) form various functional ASICs that are composed of homo- or heteromultimeric subunits. Homomultimeric ASICs display different sensitivities in response to external acidification, and assemblies of heteromeric subunits have acid-responsive properties that are distinct from their parental homomultimeric ASICs (5Bassilana F. Champigny G. Waldman R. de Weille J.R. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 28819-28822Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 6Baron A. Waldmann R. Lazdunski M. J. Physiol. (Lond.). 2002; 539: 485-494Crossref Scopus (185) Google Scholar, 7Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldman R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 8Deval 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). Homomultimeric ASIC1a channels are permeable to Na+ as well as to Ca2+ (9Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1155) Google Scholar, 10Chu X-P. Miesch J. Johnson M. Root L. Zhu X.M. Chen D. Simon R.P. Xiong Z.-G. J. Neurophysiol. 2002; 87: 2555-2561Crossref PubMed Scopus (65) Google Scholar, 11Yermolaieva O. Leonard A.S. Schnizler M.K. Abboud F.M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6752-6757Crossref PubMed Scopus (336) Google Scholar). ASIC1a generates depolarizing currents that are implicated in hippocampal long term potentiation, an important physiological function of synaptic plasticity in brain and amygdala-dependent behavior in the context of fear conditioning (12Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.N. Hoshi T. Freeman Jr., J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 13Wemmie J.A. Askwith C.C. Lamani E. Cassell M.D. Freeman Jr., J.H. Welsh M.J. J. Neurosci. 2003; 23: 5496-5502Crossref PubMed Google Scholar, 14Wemmie J.A. Coryell M.W. Askwith C.C. Lamani E. Leonard A.S. Sigmun C.D. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3621-3626Crossref PubMed Scopus (168) Google Scholar). However, the acid-evoked currents associated with tissue acidosis and the Ca2+-influx/intracellular Ca2+ accumulation could result in detrimental consequences as occur after seizures and cerebral ischemia (11Yermolaieva O. Leonard A.S. Schnizler M.K. Abboud F.M. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6752-6757Crossref PubMed Scopus (336) Google Scholar, 15Xiong 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. Simon R.P. Cell. 2004; 118: 687-698Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). A mechanotransduction role of ASIC2 has been found in sensory neurons (16Price M.P. Lewin G.R. Mcllwrath S.L. Cheng C. Xie J. Heppenstall P.A. Stucky C.L. Mannsfeldt A.G. Brennan T.J. Drummond H.A. Qiao J. Benson C.J. Tarr D.E. Hrstka R.F. Yang B. Williamson R.A. Welsh M.J. Nature. 2000; 407: 1007-1011Crossref PubMed Scopus (431) Google Scholar, 17McIlwrath S.L. Hu J. Anirudhan G. Shin J.B. Lewin G.R. Neuroscience. 2005; 131: 499-511Crossref PubMed Scopus (43) Google Scholar, 18Peng B.G. Ahmad S. Chen S. Chen P. Price M.P. Lin X. J. Neurosci. 2004; 24: 10167-10175Crossref PubMed Scopus (58) Google Scholar, 19Ettaiche M. Guy N. Hofman P. Lazdunski M. Waldmann R. J. Neurosci. 2004; 24: 1005-1012Crossref PubMed Scopus (99) Google Scholar). The central nervous system expresses ASIC1a, ASIC2a, and ASIC2b. ASIC1a is activated below pH 7.0, and ASIC2a is activated below pH 5.5; ASIC2b generates no currents in response to low pH (7Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldman R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Proton-activated currents in hippocampus are largely contributed by homo- and heteromultimers composed of ASIC1a and ASIC2a (20Askwith C.C. Wemmie J.A. Price M.P. Rokhlina T. Welsh M.J. J. Biol. Chem. 2004; 279: 18296-18305Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). ASIC2 knock-out mice have shown enhanced pH sensitivity and slow desensitization of proton-induced currents with almost no changes in current amplitude in hippocampal neurons, implying a modulatory role of ASIC2a in ASIC1a/ASIC2a heteromultimers in pH sensitivity and desensitization (20Askwith C.C. Wemmie J.A. Price M.P. Rokhlina T. Welsh M.J. J. Biol. Chem. 2004; 279: 18296-18305Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). ASICs share a common structural architecture with members of the epithelial Na+ channel/degenerin super family, which have two transmembrane domains with a large extracellular loop and two short intracellular N and C termini (for review, see Ref. 21Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (447) Google Scholar). A number of regulatory proteins for ASICs have been identified. Protein interacting with C kinase 1 (PICK1) is a synaptic protein with a PDZ domain and interacts with the C termini of both ASIC1a and ASIC2a, which mediates the regulation of these channels by protein kinase C (PKC) (22Duggan A. Garciía-Añveros J. Corey D.P. J. Biol. Chem. 2002; 277: 5203-5208Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 23Hruska-Hageman A. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Crossref PubMed Scopus (103) Google Scholar, 24Baron 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 (98) Google Scholar). Similar to the involvement of MEC-2 in mechanosensation in Caenorhabditis elegans, stomatin, the mammalian homolog of MEC-2, has been shown to interact with and modulate the activity of ASIC1a, ASIC2a, and ASIC3 (25Price M. Thomson 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 (109) Google Scholar). These interacting proteins have been shown to influence the function of ASICs. Although PICK1 binding to ASIC1a is associated with phosphorylation and changes in the cellular localization of the channel (26Leonard 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 (71) Google Scholar), the interaction between stomatin and ASICs has been suggested to modulate channel gating (25Price M. Thomson 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 (109) Google Scholar). Accordingly, we sought proteins that participate in regulation of brain-expressed ASICs by protein-protein interaction. Pulldown assays in combination with mass spectrometric analyses identified A kinase-anchoring protein 150 (AKAP150) and the Ca2+/calmodulin-dependent protein phosphatase 2B, also called calcineurin, as proteins interacting with ASIC2a. These regulatory proteins also interacted with ASIC1a. AKAP150, the neuron specific rat ortholog of human AKAP79, is present in the postsynaptic density in association with cAMP-dependent protein kinase (PKA), PKC, and calcineurin (27Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Hunganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 28Coghlan V.M. Perrino B.A. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar, 29Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar, 30Faux M.C. Rollins E.N. Edwards A.S. Langeberg L.K. Newton A.C. Scott J.D. Biochem. J. 1999; 343: 443-452Crossref PubMed Scopus (76) Google Scholar). These enzymes are associated with AKAP79/150 in inactive states, and the interaction with the anchoring protein has different consequences for the activity of these enzymes. In the case of PKA, only the regulatory subunit (RII) is anchored to AKAP and the catalytic (C) subunit, a serine/threonine kinase, interacts with RII forming active enzyme (29Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar, 31Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 32Glantz S.B. Amat J.A. Rubin C.S. Mol. Biol. Chem. 1992; 3: 1215-1228Google Scholar). PKC and calcineurin, on the other hand, are inactive when associated with AKAP79/150 until they are released from the anchoring protein (28Coghlan V.M. Perrino B.A. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar, 33Faux M.C. Scott J.D. J. Biol. Chem. 1997; 272: 17038-17044Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 34Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In the present work we have shown interaction of ASICs with AKAP150 and calcineurin. Disruption of the RII binding to AKAP150 by synthetic peptide Ht-31 decreased acid-evoked currents in mouse cortical neurons and Chinese hamster ovarian cancer (CHO) cells expressing homomeric ASIC1a or ASIC2a function, implying involvement of AKAP-anchored PKA in the function of ASIC1a and ASIC2a. Furthermore, inhibition of calcineurin by cyclosporin A significantly increased acid-evoked current in neuronal cells as well as in CHO cells. Taken together, our results suggest that both AKAP150 and calcineurin are involved in regulation of ASIC1a and ASIC2a, possibly by changing the phosphorylation status of these channels. Plasmid Constructs and Purification of Fusion Proteins—The rat cDNA clone of ASIC1a (GI: 13162348) was a gift from Dr. E. W. McCleskey (Vollum Institute, Oregon Health and Science University) and Dr. M. J. Welsh (Department of Internal Medicine and Physiology and Biophysics and Howard Hughes Medical Institute, University of Iowa), and ASIC2a (GI: 1280440) was from Dr. M. Lazdunski (Institut de Pharmacologie Molecularire et Celluaire, CNRS-UNSA, Institut Paul Hamel). The GST fusion to cDNA clone of AKAP150 (pGEX::GST-AKAP150) was a gift from Dr. J. D. Scott (Vollum Institute and Howard Hughes Medical Institute, Oregon Health and Science University). From this clone the cDNA encoding AKAP150 was amplified by the PCR method and inserted into the mammalian expression vector pCDNA3.1 (Invitrogen), resulting in pCDNA-AKAP150. For GST fusion proteins of ASIC1a and ASIC2a, the coding regions of the intracellular N and C terminus were PCR-amplified from the original cDNA clones and subcloned into the plasmid pGEX-6P-3 (Amersham Biosciences). The resulting fusion proteins were designated as GST-ASIC1aN (44 amino acids from the N terminus), GST-ASIC1aC (74 amino acids from the C terminus) GST-ASIC1aE (68 amino acids from Asn-119 to Val-186), GST-ASIC2aN (41 amino acids from the N terminus), and GST-ASIC2aC (59 amino acids from the C terminus). The N-terminal fusion of YFP to ASIC1a and ASIC2a was constructed using the plasmid pCDNA6.2-N-YFP (Invitrogen), resulting pCDNA6.2-YFP-ASIC1a and pCDNA6.2-YFP-ASIC2a. For the hemagglutinin (HA) epitope tagging in the extracellular domain of ASIC1a, the nucleotide sequence encoding HA (YPYDVPDYA) was incorporated into the oligonucleotide primer sequence that was used to amplify the first 690-base pair fragment from the 5′-end, allowing the HA insertion at position 230 amino acid. The 891-base pair fragment of the ASIC1a from the 3′-end was also PCR-amplified and subcloned together with the 5′ fragment into the plasmid pCDNA3.1, resulting in full-length ASIC1a (pCDNA-ASIC1a-eHA). The HA epitope was inserted at the C terminus of the ASIC2a using PCR methods, which was then subcloned into the plasmid pCDNA3.1 (pCDNA-ASIC2a-cHA). The GFP fusion constructs of ASIC1a and ASIC2a were carried out using the plasmid pCDNAJM1-EGFP (a gift from Dr. A. Merz, Department of Biochemistry, University of Washington), allowing GFP fusion at the C terminus of ASIC1a (pCDNA-ASIC1a-EGFP) and ASIC2a (pCDNA-ASIC2a-EGFP). The overexpression and purification of the GST fusion proteins were carried out as recommended by the manufacturer (Amersham Biosciences). GST fusion proteins were affinity-purified from the bacterial lysates using GS-4B beads (supplemental Fig. S1). Cell Culture and Transfection—CHO cells and HEK293 were cultured in F-12K medium (ATCC) supplemented with 10% FBS in humidified 5% CO2 incubator. Cortical neurons from neonatal Sprague-Dawley rat and neonatal E16 Swiss mice were cultured in neurobasal medium supplemented with B-27 and GlutaMax (Invitrogen). Neurons were cultured for 5 days before immunocytochemistry and 12 days before electrophysiological recordings. Transient transfection of neurons, CHO cells, and HEK293 cells was carried out using FuGENE 6 lipid-based transfection reagent (Roche Applied Science). For electrophysiological recordings, CHO cells were transfected with pCDNA-ASIC1a-GFP or pCDNA-ASIC2a-GFP for 48 h and then re-plated in 35-mm dishes at a lower cell density. After incubation overnight, GFP-positive cells were selected for the measurement of ASIC currents. Immunocytochemistry—After transfection with pCDNA-ASIC1a-GFP and pCDNA-ASIC2a-HA, neuronal cells were washed 3 times in PBS, fixed in 10% formalin, PBS, washed three times in PBS, and permeabilized with 3% Triton X-100/PBS. For immunochemical staining with anti-AKAP150 antibody alone, cells were incubated first in blocking buffer containing 2% donkey serum and 1% bovine serum albumin in PBS. For double detection with anti-AKAP150 and anti-HA antibody, blocking buffer containing 2% donkey serum, 2% goat serum, and 1% bovine serum albumin in PBS was used. Cells were then incubated with goat anti-AKAP 150 C-20 IgG (Santa Cruz Biotechnology) with or without rabbit anti-HA probe (Y-11) IgG (Santa Cruz Biotechnology). After extensive wash, cells were incubated for 2 h with donkey anti-goat IgG-Cy3 (Jackson Immuno Research) and/or goat anti-rabbit IgG-fluorescein isothiocyanate (Jackson Immuno Research). Both primary and secondary antibodies were diluted in the appropriate blocking buffer. After extensive washing, coverslips were mounted on glass slides. Images were collected with an epifluorescence microscope (Leica DM LB) in 40× objective/10× ocular. HEK293 cells expressing AKAP150 together with GFP, YFP-ASIC1a, or YFP-ASIC2a were immunolabeled with anti-AKAP150 antibody as described above. YFP-ASICs were visualized using a primary antibody that detects both GFP wild type and its variants (BD Living colors A.v. monoclonal antibody JL-8, Clontech) and goat anti-mouse IgG-fluorescein isothiocyanate. Pulldown Assays—For mass spectrometric analyses, ∼0.5 mg of purified GST fusion proteins bound to GS-4B beads were used to incubate with lysate from one rat brain. Rat brain lysate was prepared by Dounce homogenization in 10 ml of HEN buffer (50 mm HEPES, 10 mm EDTA, 25 mm NaCl, pH 7.4) and subsequently diluted with 10 ml of HEN buffer containing 2% Triton X-100. After incubation at 4 °C with rotation, insoluble material was separated from the lysate by centrifugation for 15 min at 14,000 × g. To preclear, supernatant was incubated with 0.5 ml of GS-4B beads at 4 °C for 30 min. The beads were removed by centrifugation for 15 min at 14,000 × g. The GST fusion proteins bound to GS-4B were added to the supernatant and incubated for 2 h at 4°C with shaking. The complexes of bead-protein(s) were washed 6 times with PBS buffer containing 1% Triton X-100 and finally resuspended in SDS-PAGE loading buffer and stored at -20 °C. In smaller scale pulldown experiments, the same procedure was applied as described above, except only half-amount of all the reagents were used. Co-immunoprecipitation Assay—HEK293 cells were co-transfected with pCDNA-AKAP150 and pCDNA-GFP, pCDNA-AKAP150 and pCDNA6.2-YFP-ASIC1a, or pCDNA-AKAP150 and pCDNA6.2-YFP-ASIC2a in 6-well plates. After 24 h of transfection, cells were harvested and lysed in 300 μl of ice-cold HEN buffer supplemented with 1% Triton X-100 and complete protease inhibitor mixture (Roche Applied Science). The insoluble material including nuclei (pellet) was removed by centrifugation at 760 × g for 10 min, and supernatant was precleared with 5 mg of protein A-Sepharose (Amersham Biosciences) and 1 μg of anti-cascade blue antibody (Molecular Probes) at 4 °C. Beads and antibody were removed from the lysate by centrifugation at 10,000 rpm for 15 min. For co-immunoprecipitation, lysates were first incubated overnight at 4 °C with 2 μg of affinity-purified anti-GFP antibody and then with 5 mg of protein A-Sepharose for an additional 3 h. After centrifugation at 760 × g for 5 min, the pellet containing beads bound to antibody-protein(s) complex was washed 5 times with HEN buffer containing 0.5% Triton X-100 and resuspended in SDS-PAGE sample loading buffer for Western blot analysis. Sample Preparation for Mass Spectrometry—Bead-bound protein samples from pulldown assays were denatured at 95 °C for 5 min and separated by one-dimensional SDS-PAGE. One whole lane was cut in 5 gel slices. As described previously (35Farr C. Grafken P.R. Norbeck A.D. Doneanu C.E. Stapels M.D. Barofsky D.F. Minami M. Saugstad J.A. J. Neurochem. 2004; 91: 438-450Crossref PubMed Scopus (69) Google Scholar), the trypsin digestion of the protein samples in the gel slices and liquid chromatography/electrospray ionization/tandem mass spectroscopy were carried out by Proteomics facility, Fred Hutchinson Cancer Research Center (Seattle, WA). Western Blot Analysis—Protein samples from pulldown assays and co-immunoprecipitation assays were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in PBS containing 5% milk (blocking buffer) and probed with primary antibody diluted in blocking buffer for 2 h. After an extensive wash in PBS, membranes were probed with appropriate secondary antibody-horseradish peroxidase conjugates, donkey anti-goat IgG (Santa Cruz Biotechnology), goat anti-rabbit IgG (Sigma), and goat anti-mouse IgG (Sigma). Horseradish peroxidase bound to immunoblot was visualized with enhanced chemiluminescence (ECL, Amersham Biosciences) and Eastman Kodak BioMax Chemiluminescence film. Electrophysiology—ASIC currents were recorded with the conventional whole-cell patch clamp technique at room temperature (20-22 °C). In general, cells were voltage-clamped at holding potentials of -60 mV. Data were acquired using an AXOPATCH 200B amplifier with pCLAMP 8.2 software (Axon Instruments, CA). Data were filtered at 2 KHz and digitized at 5 Hz using Digidata 1322A (Axon Instruments). For rapid changes of extracellular solutions, a multibarrel perfusion system (SF-77, Warner Instruments, Hamden, CT) was used. Low pH extracellular solutions were applied at 2-min intervals to allow for complete recovery of ASICs from desensitization. The neutral extracellular solutions contained 140 mm NaCl, 5.4 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 20 mm HEPES, and 10 mm glucose, 320-335 mOsm. In the extracellular solutions with pH ≤ 6.0, 20 mm HEPES was replaced by 10 mm MES for more reliable pH buffering. The pH of the extracellular solution was adjusted with NaOH/HCl. Patch pipettes were pulled from borosilicate glass (1.5-mm diameter; WPI, Sarasota, FL) on a two-stage puller (PP83, Narishige, Tokyo, Japan). Pipettes had a resistance of 2-4 megaohms when filled with the intracellular solution, which contained 140 mm CsF, 2 mm triethylammonium chloride, 11 mm EGTA, 10 mm HEPES, 1 mm CaCl2, 4 mm MgCl2 in pH 7.3 adjusted with CsOH/HCl, 290-300 mOsm. Cyclosporin A (Fluka) and FK-506 (LC laboratories) were first dissolved in 100% ethanol to make a 10 mm stock solution and diluted to the final concentration of 30 μm and 10 μm in the pipette solution, respectively. The AKAP inhibitor peptide Ht-31 and Ht-31P (Promega) were dissolved in water to make a 10 mm stock solution and then diluted to the final concentration of 20 μm in the pipette solution. To ensure high quality voltage-clamp, only the recordings with an access resistance of less than 10 megaohms and a leak current less than 100 pA at -60 mV were included for analysis. Data are presented as the mean ± S.E. Statistical significance was determined using two-way ANOVA and Student t test where appropriate. Differences were considered significant when p < 0.05. Identification of AKAP150 and Calcineurin B as Binding Partners with ASIC2a N Terminus—Like the other members of the degenerin/epithelial Na+ channel superfamily, N and C termini of ASIC2a are the only cytoplasmic domains of this channel (36Renard S. Lingueglia E. Voilley N. Lazdunsky M. Barbry P. J. Biol. Chem. 1994; 269: 12981-12986Abstract Full Text PDF PubMed Google Scholar, 37Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 38Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267 (-C1690): C1682Crossref PubMed Google Scholar, 39Lai C-C. Hong K. Chalfie M. Driscoll M. J. Cell Biol. 1996; 133: 1071-1081Crossref PubMed Scopus (97) Google Scholar). Homomeric ASIC1a and ASIC2a channels or ASIC2b in heteromeric ASIC3/ASIC2b have been shown to interact with the PICK1 and, thereby with PKC via their C terminus (8Deval 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, 23Hruska-Hageman A. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Crossref PubMed Scopus (103) Google Scholar, 24Baron 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 (98) Google Scholar, 26Leonard 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 (71) Google Scholar). We questioned whether the N terminus of these channels is also involved in the regulation of function or surface expression. To identify potential regulatory proteins in brain, we first used in vitro protein-protein binding assays using the N terminus (41 amino acids) of ASIC2a fused to GST protein (GST-ASIC2aN). The fusion protein was affinity-purified using GS-4B beads from bacterial lysate and mixed with rat brain lysate to pull down any interacting proteins. The bead-protein complexes were purified and separated by one-dimensional SDS-gel electrophoresis (Fig. 1A). In parallel, a control experiment was performed with GST protein bound to GS-4B to identify nonspecific interaction. Both samples were analyzed by tandem mass spectrometry, and identified proteins were searched in the rat protein data base of international protein index. Of particular interest were a peptide fragment from AKAP150 and one peptide fragment from A subunit isoform α of protein phosphatase 2B (also called calcineurin). The identified peptides of AKAP150 and calcineurin A subunit were nine amino acids long (TPGSEKEAK) and 33 amino acids long (VTEMLVNVLNICSDDELGSEEDGFDGATAAARK), respectively. AKAP150/79 is a neuronal protein enriched in postsynaptic fractions (31Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar). A number of reports indicated the involvement of AKAP150/79 in trafficking and function of channels and receptors in neuronal cells (for review, see Ref. 41Wong W. Scott J.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (862) Google Scholar). Moreover, AKAP150/79 associates with PKA, PKC, and calcineurin (28Coghlan V.M. Perrino B.A. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar, 29Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar, 31Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar). To verify the mass spectrometry data, we repeated the pulldown assay with GST and GST-ASIC2aN protein from rat brain lysates. Western blot analyses with anti-AKAP150" @default.
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• W2016367251 title "A Kinase-anchoring Protein 150 and Calcineurin Are Involved in Regulation of Acid-sensing Ion Channels ASIC1a and ASIC2a" @default.
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