A Gating Mutation in the Internal Pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore. Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore. The epithelial sodium channel (ENaC) 2The abbreviations used are: ENaC, epithelial Na+ channel; ASIC, acid-sensing ion channel; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; IASIC, current induced by acidification to pH 6.0; MES, 4-morpholineethanesulfonic acid; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate; MTS-PTrEA, [3-(triethylammonium)propyl] methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; TM1/TM2, transmembrane segment 1/2; wt, wild type. 2The abbreviations used are: ENaC, epithelial Na+ channel; ASIC, acid-sensing ion channel; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; IASIC, current induced by acidification to pH 6.0; MES, 4-morpholineethanesulfonic acid; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate; MTS-PTrEA, [3-(triethylammonium)propyl] methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; TM1/TM2, transmembrane segment 1/2; wt, wild type. and the acid-sensing ion channels (ASICs) in mammals are members of the recently identified ENaC/degenerin family of voltage-insensitive channels (1Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (852) Google Scholar). The epithelial sodium channel mediates Na+ transport in renal and airway-tight epithelia (2Rossier B.C. Pradervand S. Schild L. Hummler E. Annu. Rev. Physiol. 2002; 64: 877-897Crossref PubMed Scopus (318) Google Scholar). The ASICs are expressed in the central and peripheral nervous system and are possibly involved in nociception, learning, or mechanosensation (3Krishtal O. Trends Neurosci. 2003; 26: 477-483Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). ENaC and ASICs are likely formed by four homologous subunits. The membrane topology of the channel subunits predicts an amino and a carboxyl terminus facing the inside of the cell, the presence of two transmembrane segments, and a large extracellular loop (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.-D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1766) Google Scholar, 5Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (368) Google Scholar, 6Canessa C.M. Merillat A.-M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, 7Saugstad J.A. Roberts J.A. Dong J. Zeitouni S. Evans R.J. J. Biol. Chem. 2004; 279: 55514-55519Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The structural basis of ENaC/ASIC function remains poorly understood. Amino acid residues in the extracellular loop of ENaC preceding the second transmembrane domain (TM2) bind the pore blocker amiloride (8Kellenberger S. Gautschi I. Schild L. Mol. Pharmacol. 2003; 64: 848-856Crossref PubMed Scopus (70) Google Scholar, 9Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (238) Google Scholar). Based on previous studies on the interaction between permeant Na+ ions and pore blockers of different sizes, it has been proposed that the external pore vestibule of ENaC, where amiloride binds, narrows down to the selectivity filter allowing a Na+ or Li+ ion to selectively permeate the channel (10Palmer L.G. Ren. Physiol. Biochem. 1990; 13: 51-58PubMed Google Scholar). This model is supported by the identification of residues at the external end of ENaC TM2 near the amiloride binding site, which are important for maintaining its high selectivity for Na+ (11Kellenberger S. Hoffmann-Pochon N. Gautschi I. Schneeberger E. Schild L. J. Gen. Physiol. 1999; 114: 13-30Crossref PubMed Scopus (109) Google Scholar, 12Kellenberger S. Auberson M. Gautschi I. Schneeberger E. Schild L. J. Gen. Physiol. 2001; 118: 679-692Crossref PubMed Scopus (69) Google Scholar, 13Kellenberger S. Gautschi I. Schild L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4170-4175Crossref PubMed Scopus (133) Google Scholar, 14Sheng S. Li J. Mcnulty K.A. Avery D. Kleyman T.R. J. Biol. Chem. 2000; 275: 8572-8581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Sheng S.H. McNulty K.A. Harvey J.M. Kleyman T.R. J. Biol. Chem. 2001; 276: 44091-44098Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Beyond the external selectivity filter in TM2, the channel structures lining the ion permeation pathway that are accessible from the cytosol have not yet been identified. In this study, we have used the homomultimeric ASIC1a as a model to investigate the structure of the internal ASIC/ENaC pore. Recently, we have observed that multiple cysteine residues in the amino terminus of ENaC subunits are at least, in part, responsible for the sensitivity of ENaCs to inhibition by intracellular sulfhydryl-modifying agents (16Kellenberger S. Gautschi I. Pfister Y. Schild L. J. Biol. Chem. 2005; 280: 7739-7747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This suggests that the amino terminus participates in ENaC gating and/or constitutes the internal pore of the channel. In the present study, we have investigated the role of the amino terminus and the start of the first transmembrane segment in controlling the ion flux through ASIC1a. We have found that cysteine substitutions in the amino terminus and in TM1 are modified by intracellularly applied sulfhydryl reagents, thereby inhibiting ASIC1a currents. We provide evidence that the cytosolic part of the TM1 is located at the internal channel pore and undergoes conformational changes during channel gating. ASIC1a Expression—Complementary cDNA of the human ASIC1a (17Garcia-Anoveros J. Derfler B. Nevillegolden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (292) Google Scholar) was subcloned in the pSDEasy cloning vector for in vitro transcription and expression in Xenopus oocytes. Stage V and VI Xenopus oocytes were injected with 5 ng of cRNA encoding hASIC1a. Electrophysiology—Electrophysiological measurements were performed 24-36 h after oocyte injection with ASIC cRNA. Macroscopic ASIC currents (IASIC) were elicited every 30 s by rapid changes in extracellular pH from 7.4 to 6.0 and were measured using either the two-electrode voltage clamp for whole-cell currents or the cut-open oocyte technique when intracellular perfusion was needed. The two-electrode voltage clamp measurements were performed as described previously (9Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (238) Google Scholar). The bathing solution contained (in mm) NaCl 120, MgCl2 2, HEPES·H+ 10 mm, adjusted to pH 7.5 with NaOH. Changes in extracellular pH were achieved using the same bathing solution with MES·H+ 10 mm buffered at pH 6.0. The cut-open configuration of the Xenopus oocyte allows the recording of macroscopic ASIC currents while continuously perfusing the inside (2 μl·min-1) and the outside (>8 μl·min-1) of the oocytes (16Kellenberger S. Gautschi I. Pfister Y. Schild L. J. Biol. Chem. 2005; 280: 7739-7747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). A microperfusion pipette, in which two thin capillaries (Microfil, World Precision instruments) had been inserted, was used for the intracellular perfusion and served as an intracellular electrode potential measurement. The intracellular solution contained (in mm) potassium gluconate 90, KCl 10, sodium gluconate 2, MgCl2 1, BAPTA 0.2, HEPES-N-methyl-d-glucamine 10, adjusted to pH 7.35. Methanethiosulfonates or Cd2+ (1 mm) were added to the solution. The holding potential was -100 mV. The extracellular solution corresponded to the bathing solution in the two-electrode voltage clamp experiments. Site-directed Mutagenesis—Introduction of cysteine residues in the ASIC1a sequence was performed as described previously for ENaC mutagenesis (9Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (238) Google Scholar). The presence of the mutation in the ASIC1a cDNA was verified by sequencing. cRNA was synthesized in vitro for injection into the oocytes. Chemicals—The methanethiosulfonate (MTS) reagents purchased from Toronto Research Chemicals (Toronto, Canada) were MTSET, MTSEA, and MTS-PtrEA. Stock solutions with MTS reagents were prepared in Me2SO and diluted at least 100-fold, immediately before use, into the intracellular solution. Typical recordings of ASIC1a wild type (Fig. 1) in internally perfused oocytes (cut-open configuration) show that ASIC currents (IASIC) elicited by short external pH changes (2 s duration) from 7.4 to 6.0, were stable for several minutes. The perfusion of intracellular methanethio-sulfonates (MTSEA, MTSET, MTS-PTrEA at 1 mm) did not affect the magnitude of IASIC. Similar recordings were obtained in the presence of MTSET in the external medium (data not shown). The insensitivity of ASIC1a to inhibition by intracellular and extracellular MTSET reagents indicates that the cysteine residues Cys-49, Cys-59, and Cys-61 in the first transmembrane segment (TM1) of ASIC1a wt (see Fig. 2A) are not modified by MTSET or, if modified, do not inhibit ASIC currents. Data reported on an ortholog of ENaC, the peptide-gated Na+ channel (FaNaCh), using a systematic cysteine substitution in TM1 showed 50-60% inhibition of the FaNaCh current by external MTS reagents (18Poet M. Tauc M. Lingueglia E. Cance P. Poujeol P. Lazdunski M. Counillon L. EMBO J. 2001; 20: 5595-5602Crossref PubMed Scopus (26) Google Scholar). It was concluded that the TM1 of FaNaCh is lining the external pore of the channel. We could not confirm these findings on ASIC1a.FIGURE 2Sensitivity of ASIC1a mutants to internal MTSET. A, sequence alignment of the amino terminus and the first transmembrane helix (TM1) of ASIC1a and ENaC α, β, and γ subunits. Dark lines represent putative α helices predicted from the amino acid sequence. • are residues mutated into cysteine, and ▾ are cysteine residues Cys-49, Cys-59, and Cys-61 that were mutated in Ala, Val, and Ser, respectively. B, ASIC current (IASIC) recorded from A22C and R43C mutants before and after the internal perfusion of MTSET at 1 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test the solvent accessibility of the amino terminus of ASIC1a, cysteine residues were introduced by site-directed mutagenesis from position Ile-18 in the amino terminus to Trp-46 in TM1, and the mutants were expressed in Xenopus oocytes (Fig. 2A). Fig. 2B illustrates typical IASIC expressed by mutants of ASIC1a with cysteine substituted at positions A22C and R43C. For these mutants, IASIC decreased after the application of internal MTSET (1 mm), indicating that cysteine residues at position Ala-22 and Arg-43 are modified. R43C was similarly inhibited by the larger methanethiosulfonates MTS-PTrEA 1 mm (73.9 ± 4.4% inhibition of IASIC after 2 min), indicating that R43C is accessible to large molecules. Fig. 3 summarizes the results obtained from the systematic cysteine substitution and analysis of modification by intracellular MTSET of residues Ile-18 to Trp-46. Substitutions at positions His-28, Gly-29, and His-32 were not investigated, because the corresponding mutations in ENaC result in channel loss of function (19Grunder S. Jaeger N.F. Gautschi I. Schild L. Rossier B.C. Pfluegers Arch. Eur. J. Physiol. 1999; 438: 709-715Crossref PubMed Scopus (69) Google Scholar). A few other cysteine substitutions, T26C, S35C, E37C, and W46C resulted in a complete loss of function. The substituted cysteine residues at positions Ala-22, Ile-33, Phe-34, or Arg-43 are modified by intracellular MTSET resulting in ASIC1a inhibition. The A22C, I33C, F34C, or R43C mutants were unresponsive to external MTSET at 2.5 mm (data not shown). The R38C mutant showed a weak but significant inhibition by internal MTSET. The cysteine residue at position Ala-22 in the ASIC1a sequence corresponds to the conserved cysteine residues αCys-88, βCys-30, γCys-33 in rat ENaC subunit sequences (see Fig. 2A) that have been shown to be involved in the block of ENaC by intracellular MTS reagents (16Kellenberger S. Gautschi I. Pfister Y. Schild L. J. Biol. Chem. 2005; 280: 7739-7747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Thus, ENaC inhibition by an internal MTS reagent can be reproduced in ASIC1a with the A22C substitution. Additional cysteine residues in ENaC corresponding to Ser-35 and Leu-30 in ASIC1a also participate in ENaC inhibition by MTS reagents. The ASIC S35C mutant was not functional, but the adjacent cysteine substitutions I33C and F34C resulted in IASIC inhibition by internal MTSET. By contrast to ENaC, exposure of L30C to MTSET did not result in current inhibition. Taken together, these results indicate that cysteine residues flanking the HG motif in both ENaC and ASIC1a are accessible from the cytosol by MTS reagents and result in channel inhibition upon modification by sulfhydryl reagents. State-dependent Accessibility—We next determined whether the substituted cysteine residues at position Ala-22, Ile-33, or Arg-43 were preferentially modified by MTS reagents in the open or closed conformation of the channel. We have compared the time course of ASIC1a inhibition by MTSET during repeated pH pulses applied every 0.5 min or after maintaining the channel in the resting state for 2 min. It is clear from the recordings (Fig. 4A) and from the time course of IASIC inhibition (Fig. 4B) that maintaining ASIC1aR43C in the resting state in the presence of internal MTSET (1 mm) ASIC block and the time course of channel inhibition (Fig. are with a accessibility of R43C to internal MTSET when the channel is closed and indicate that the TM1 undergoes conformational changes with channel gating. of the current inhibition of the A22C, I33C, and R43C ASIC1a mutants using the same that the channel in the resting state (Fig. did not affect the magnitude of inhibition of the A22C and I33C This indicates that the of A22C and I33C by contrast to R43C are that Ala-22 conformational changes during channel gating that affect of Cd2+ to sulfhydryl and is of a as Na+ Cd2+ (1 mm) ASIC1a wt, and this inhibition is by the mutation in the TM1 (Fig. that is accessible to intracellular Cd2+ By contrast to MTS reagents, internal Cd2+ did not inhibit IASIC of R43C The effect of Cd2+ on R43C was a of the IASIC by of open channel inactivation. are in Fig. and Cd2+ the IASIC of ASIC1a wt and of the and By the substitution ASIC1a inhibition by Cd2+ and IASIC by in the presence of Fig. that internal Cd2+ the time for the of the R43C mutant by compared with ASIC wt (Fig. We that Cd2+ by IASIC of R43C to the of the channel in the presence of Cd2+ did affect the current magnitude the time course of current of the A22C and I33C mutants for wt ASIC1a (data not shown). Taken together, indicate first that MTSET not the A22C, I33C, F34C, and R43C mutants, that the of the sulfhydryl-modifying is important for channel the block by MTSET and the effect of Cd2+ on the R43C mutant evidence that TM1 undergoes conformational changes during channel inactivation. from the is to as S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). It is likely because of its Cd2+ also the channel. We if the substituted cysteine R43C or Cys-61 is located the ion permeation it also bind Cd2+ from the extracellular We whether the application of extracellular Cd2+ the of internal Cd2+ on the R43C and Cd2+ and 10 mm) the IASIC current expressed by ASIC wt ± and ± (Fig. A and The for IASIC inhibition by external Cd2+ as determined from was for ASIC1a wt ± mm), for the R43C ± mm), and for the mutant in the TM1 ± Thus, both extracellular and intracellular Cd2+ ASIC1a. mutation the ASIC1a inhibition by internal Cd2+ but has effect on ASIC1a inhibition by external This indicates that external Cd2+ either to Cys-61 the ion flux through the pore or the Cys-61 residue from the external This is with the of ASIC1a inhibition by external MTS reagents and do not a of Cys-61 residue in the TM1 the ion permeation In contrast to ASIC1a wt, external Cd2+ the current of the R43C mutant (Fig. B, and resulting in a in Thus, intracellular and extracellular Cd2+ have on the time of IASIC of the R43C indicating that R43C Cd2+ applied from either of the This evidence that Arg-43 is located in the ion permeation show that TM1 not during channel gating but that part of it also lines the internal pore. results have identified amino acid residues in the amino terminus and the TM1 of ASIC1a when substituted with cysteine, are modified by intracellular MTS reagents, resulting in channel inhibition. The sequence of ASIC1a to a high of between the members of the ENaC/degenerin family with a conserved HG motif (see Fig. 2A). to the current of the membrane topology of the amino terminus to the first transmembrane segment TM1 of ASIC is facing the cytosolic of the The of this domain has been first by a mutation in the HG motif of ENaC to a form of a S. Firsov D. Jaeger N.F. Gautschi I. Schild L. Rossier B.C. EMBO J. 1997; PubMed Scopus Google Scholar). The ENaCs with mutations of the HG motif show gating by and short channel recently it has been shown that the high sensitivity of ENaC to intracellular sulfhydryl reagents and agents can be to conserved cysteine residues in the amino terminus of ENaC subunits (16Kellenberger S. Gautschi I. Pfister Y. Schild L. J. Biol. Chem. 2005; 280: 7739-7747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The mutants A22C, I33C, and F34C in the amino terminus of ASIC1a the ENaC sensitivity to inhibition by internal MTS reagents, indicating that the in the amino terminus are conserved ENaCs and do not to that the inhibition of the A22C, I33C, or F34C mutants by internal MTS reagents results from a block of the channel pore. The amino terminus of ENaC has been shown to a role in channel gating S. Firsov D. Jaeger N.F. Gautschi I. Schild L. Rossier B.C. EMBO J. 1997; PubMed Scopus Google Scholar). We have been to show that the permeant Cd2+ to A22C or I33C when applied from either of the as if A22C or I33C be located the ion permeation Thus, we are evidence that A22C and I33C are lining the internal pore of results also show that A22C and I33C do not conformational changes that accessibility to intracellular MTS reagents. Thus, we have evidence that A22C and I33C are part of the gating of the channel. It be that mutations in the at positions corresponding to and in the ASIC1a sequence (Fig. 2A) the ion selectivity of the that this of the amino terminus to the channel pore structure S. Lingueglia E. Lazdunski M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the the inhibition of the A22C and I33C mutants by MTS reagents remains to be to the of these residues in the channel gating and/or in the permeation of the channel. It is that the A22C or I33C ASIC1a mutants are to by of intracellular ENaC with cysteine residues at positions corresponding to Ala-22 and Ser-35 is to by internal Cd2+ (see Fig. 2A) (16Kellenberger S. Gautschi I. Pfister Y. Schild L. J. Biol. Chem. 2005; 280: 7739-7747Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). It is that channel inhibition by internal Cd2+ requires the binding interaction of Cd2+ with several cysteine as Ala-22 and Ser-35 or other present in ENaC but in ASIC1a The of R43C in the ion permeation pathway is supported by the that R43C is accessible to Cd2+ from both the extracellular and intracellular the modification of R43C by internal MTS reagents indicates that this residue lines the internal channel pore. with the accessibility of R43C to MTS reagents, this suggests the presence of an internal that the of ions to the internal pore of the channel. of Cd2+ to R43C as a in the channel inactivation. the Cys-49, Cys-59, and Cys-61 located in the TM1, we do not have evidence that these residues the channel Cys-61 to a role in the ASIC1a inhibition and to be accessible by internal The a model of the transmembrane of ASIC and ENaC that summarizes the structures to be involved in the ion permeation The external ion channel pore is of a short putative α helix that a when modified by external MTS in the open conformation of the the channel in the open state J. Gen. Physiol. 2000; PubMed Scopus Google Scholar, S. Gautschi I. Schild L. J. Physiol. 2002; Scopus Google Scholar). the binding for the external pore blocker amiloride has been identified in ENaC (8Kellenberger S. Gautschi I. Schild L. Mol. Pharmacol. 2003; 64: 848-856Crossref PubMed Scopus (70) Google Scholar, 9Schild L. Schneeberger E. Gautschi I. Firsov D. J. Gen. Physiol. 1997; 109: 15-26Crossref PubMed Scopus (238) Google Scholar). The first residues in TM2 of ENaC are accessible to external Cd2+ and are important for maintaining the ion selectivity and the of the channel (11Kellenberger S. Hoffmann-Pochon N. Gautschi I. Schneeberger E. Schild L. J. Gen. Physiol. 1999; 114: 13-30Crossref PubMed Scopus (109) Google Scholar, 12Kellenberger S. Auberson M. Gautschi I. Schneeberger E. Schild L. J. Gen. Physiol. 2001; 118: 679-692Crossref PubMed Scopus (69) Google Scholar, 13Kellenberger S. Gautschi I. Schild L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4170-4175Crossref PubMed Scopus (133) Google Scholar, 14Sheng S. Li J. Mcnulty K.A. Avery D. Kleyman T.R. J. Biol. Chem. 2000; 275: 8572-8581Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Sheng S.H. McNulty K.A. Harvey J.M. Kleyman T.R. J. Biol. Chem. 2001; 276: 44091-44098Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). is evidence a of the part of TM2 in the internal pore We have and the substitution mutants and in the cytosolic end of to 1 mm internal MTSET did not result in current inhibition in of these mutants, that TM2 not participate in the internal of the channel. represent the first that TM1 is involved in the pore lining and participates in channel this the amino terminus of the as an important domain accessible from the cytosol and as controlling the ion flux through the channel.

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