The Extracellular Domain Determines the Kinetics of Desensitization in Acid-sensitive Ion Channel 1

Abstract

The acid-sensitive ion channel 1 (ASIC1α or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that has the broadest distribution in the nervous system. Hallmarks of ASIC1α are gating by external protons and rapid desensitization. In sensory neurons ASIC1 may constitute a nociceptor for pain induced by local acidification, whereas in central neurons it may modulate synaptic activity. To gain insight into the functional roles of ASIC1, we cloned and examined the properties of the evolutionarily distant species toadfish (Opsanus tau), ∼420-million year divergent from mammals. Analysis of the protein sequence from fish ASIC1 revealed 76% amino acid identity with the rat orthologue. The regions of highest conservation are the second transmembrane domain and the ectodomain, whereas the amino and carboxyl termini and first transmembrane domain are poorly conserved. At the functional level, fish ASIC1 is gated by external protons with a half-maximal activation at pH o 5.6 and a half-maximal inactivation at pH o 7.30. The fish differs from the rat channel on having a 25-fold faster rate of desensitization. Functional studies of chimeras made from rat and fish ASIC1 indicate that the extracellular domain specifically, a cluster of three residues, confers the faster desensitization rate to the fish ASIC1. The acid-sensitive ion channel 1 (ASIC1α or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that has the broadest distribution in the nervous system. Hallmarks of ASIC1α are gating by external protons and rapid desensitization. In sensory neurons ASIC1 may constitute a nociceptor for pain induced by local acidification, whereas in central neurons it may modulate synaptic activity. To gain insight into the functional roles of ASIC1, we cloned and examined the properties of the evolutionarily distant species toadfish (Opsanus tau), ∼420-million year divergent from mammals. Analysis of the protein sequence from fish ASIC1 revealed 76% amino acid identity with the rat orthologue. The regions of highest conservation are the second transmembrane domain and the ectodomain, whereas the amino and carboxyl termini and first transmembrane domain are poorly conserved. At the functional level, fish ASIC1 is gated by external protons with a half-maximal activation at pH o 5.6 and a half-maximal inactivation at pH o 7.30. The fish differs from the rat channel on having a 25-fold faster rate of desensitization. Functional studies of chimeras made from rat and fish ASIC1 indicate that the extracellular domain specifically, a cluster of three residues, confers the faster desensitization rate to the fish ASIC1. The acid-sensitive ion channels (ASICs) 1The abbreviations used are: ASIC, acid-sensitive ion channel; TEVC, two-microelectrode voltage clamp; ENaC, epithelial sodium channel; MES, 4-morpholineethanesulfonic acid.1The abbreviations used are: ASIC, acid-sensitive ion channel; TEVC, two-microelectrode voltage clamp; ENaC, epithelial sodium channel; MES, 4-morpholineethanesulfonic acid. constitute a subfamily of the large epithelial sodium channel (ENaC)/DEG family of ion channels (17Kellenberger S. Hoffmann-Pochon N. Gatschi I. Scheeberger E. Schild L. J. Gen. Physiol. 1999; 114: 13-30Crossref PubMed Scopus (105) Google Scholar, 27Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (175) Google Scholar). The ASIC1α protein (or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that is expressed in most neurons of the central and peripheral nervous systems (1Alvarez de la Rosa D. Zhang P. Shao D. White F. Canessa C.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 99: 2326-2331Crossref Scopus (203) Google Scholar, 2Alvarez de La Rosa D.A. Krueger S.R. Kolar A. Shao D. Fitzsimonds R.M. Canessa C.M. J. Physiol. 2003; 546: 77-87Crossref PubMed Scopus (173) Google Scholar). The mammalian ASIC1, ASIC2, and ASIC3 are all activated by protons but the degree and rate of desensitization markedly differ in each type of channel. For instance, rat ASIC1 and ASIC3 exhibit rapid and complete desensitization at pH o 5.0, whereas ASIC2 has a slow and incomplete desensitization, leaving a substantial component of persistent current in the continual presence of protons (29Zhang P. Canessa C.M. J. Gen. Physiol. 2002; 120: 553-566Crossref PubMed Scopus (61) Google Scholar). Indeed, most of the functional differences in the currents generated by the ASICs can be attributed to differences in desensitization rate, suggesting that this property may be important in conferring specificity to the response of the various ASICs in different regions of the nervous system. Numerous functions have been proposed for ASIC1 including a role in sensory transduction, specifically in nociception (pain induced by ischemia and inflammation) (7Benson C.J. Sutherland S.P. Ann. N. Y. Acad. Sci. 2001; 940: 96-109Crossref PubMed Scopus (25) Google Scholar, 9Chen C. England S. Akopian A.N. Wood J.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10240-10245Crossref PubMed Scopus (396) Google Scholar, 15Immke D.C. McCleskey E.W. Nat. Neurosci. 2001; 4: 869-870Crossref PubMed Scopus (317) Google Scholar, 20Mamet J. Baron A. Lazdunski M. Voilley N. J. Neurosci. 2002; 22: 10662-10670Crossref PubMed Google Scholar, 21Price M.P. McIIwrath 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 (501) Google Scholar, 24Xie J. Price M.P. Wemmie J.A. Askwith C.C. Welsh M.J. J. Neurophysiol. 2003; 89: 2459-2465Crossref PubMed Scopus (71) Google Scholar), and as modulators of synaptic transmission and long term plasticity (2Alvarez de La Rosa D.A. Krueger S.R. Kolar A. Shao D. Fitzsimonds R.M. Canessa C.M. J. Physiol. 2003; 546: 77-87Crossref PubMed Scopus (173) Google Scholar, 28Wemmie 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. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). For many of these functions, in particular nociception, proton sensitivity plays a fundamental role in the physiology of these channels. In order to gain more insight in the structure-function of these channels, we cloned and examined the functional properties of ASIC1 from an evolutionarily distant species, the fish Opsanus tau. Comparison of the properties of the fish and mammalian ASIC1 revealed significant differences, primarily Ca2+ dependence for activation and faster desensitization rate of the fish ASIC1. To identify the structural determinants of the desensitization process, we constructed a series of chimeras with the cDNAs of fish and rat and expressed them in Xenopus laevis oocytes for functional analysis with the two-electrode voltage clamp and patch clamp techniques. Cloning of Fish ASIC1 cDNA—Poly(A)+ was extracted from brain and spinal cord of toadfish using oligo(dT)-cellulose and proteinase K (Roche Applied Science) as described (8Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar). First strand cDNA synthesis was performed using oligo(dT) primers and SuperDNA reverse transcriptase (Roche Applied Science). We designed degenerated primers from highly conserved sequences among all the mammalian ASIC proteins. We selected the protein sequence NCNCRMVHMPG to make the sense primer: AA(C/T)TG(C/T)AG(A/G)ATGGTICA(C/T)ATGCCIGG. The antisense primer was designed from the protein sequence GDIGGQMG: CCCAT(C/T)TGICCICCIAT(A/G)TCICC, where I indicates inosine. The position of the primers is indicated in Fig. 1. PCR was performed on first strand cDNA with Taq polymerase (Roche Applied Science) with the following parameters: denaturing for 20 s at 94 °C, annealing for 30 s at 55 °C, and extension for 30 s at 72 °C, repeated for 30 cycles. The PCR product was sequenced and used to design specific primers for 5′- and 3′-rapid amplification of cDNA ends performed with 5′- and 3′-rapid amplification of cDNA end system kits (Roche Applied Science) according to the protocols provided by the supplier. Once the 5′- and 3′-untranslated sequences were obtained, we designed a new set of oligonucleotides to amplify the whole DNA coding sequence using high fidelity Taq plus Tgo polymerases (Roche Applied Science). To the antisense 3′ primer was added the sequence of the HA epitope, such that translation of the final cDNA produced a protein with the HA epitope in the carboxyl terminus. The PCR product was subcloned in pCR.2 vector (Invitrogen). Several clones were sequenced with an automatic DNA sequencer at the Keck Facility at Yale University. Construction of Rat and Fish ASIC Chimeras and Site-directed Mutagenesis—Fish and rat ASIC1 chimeras were made by PCR using hybrid primers and a combination of Taq and Tgo DNA polymerases (Roche Applied Science) as described (14Fyfe G.F. Zhang P. Canessa C.M. J. Biol. Chem. 1999; 274: 36415-36421Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Constructs were subcloned in pCDNA-3.1 vector (Invitrogen). Site-directed mutageneses of cDNAs were performed with the QuickExchange kit according to the manufacturer's instructions (Stratagene). All constructs underwent DNA sequencing at the Keck Facility of Yale University prior to further use. Synthesis of cRNA and Injection of X. laevis Oocytes—cRNAs were synthesized with T7 RNA polymerase from linearized plasmids containing the entire coding sequence of the genes using mMESSAGEmMA-CHINE (Ambion). Stage V and VI oocytes were injected with 2 ng of cRNA from rASIC1, fASIC1 or each of the chimeras. Oocytes were incubated in frog Ringer: 96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES, adjusted to pH 7.4 for 2–3 days at 19 °C before making recordings. Prior to patching, oocytes were placed in an hyperosmotic solution: 220 mm N-methyl-d-glucamine, 220 mm aspartic acid, 2 mm MgCl2, 10 mm EGTA, 10 mm HEPES, adjusted to pH 7.4 with KOH) to remove the vitelline membrane with fine forceps. Single-channel Recordings—Unitary currents were recorded using the outside-out configuration of the patch clamp technique. Channels were activated by rapidly moving squared glass tubes (inner diameter 0.7 mm) delivering solutions of desired pH o values in front of the tip of the patch pipette. The delivery device achieves complete solution changes within 20 ms (SF-77B; Perfusion Fast-Step, Warner were from glass using a and to a final tip diameter of 1 were with the was channel currents were recorded with an using a series and from were recorded at 10 at 5 and on a for For were with a to solution was as mm NaCl, 5 mm 5 mm HEPES, adjusted to pH 7.4 with in the was as mm NaCl, mm and 10 mm adjusted to pH The tip of the patch was with solutions of to the extracellular but at pH 5.0, or as indicated in the To the of this ion was added to the external solution in indicated in the particular of Oocytes with a and voltage were from glass and with The of was in the of were recorded with an and with a rate of and the were in the of a The of for rapid of was at a rate of The was with a to the of the solutions in the To the Ca2+ the external solution the mm NaCl, 1 mm KCl, 10 mm HEPES, 10 mm MES, at pH o or and various of Ca2+ as indicated in the were at Analysis and current values from currents were used to the half-maximal activation and for and according to the I where the external of or is the half-maximal activation for the and is the are the of at is in the for each were by the of currents of to a where 1 the component of the current that with the rate and is a that for the of the Cloning of ASIC1 cDNAs from the fish ASIC1, we highly conserved sequences among all of the mammalian ASIC cloned to We designed degenerated primers to the amino acid sequences NCNCRMVHMPG and that are highly conserved. primers were used to amplify a from strand cDNA synthesized from extracted from toadfish brain and spinal The DNA sequence of the PCR product indicated that it to the fish ASIC1 The complete coding sequence of the cDNA was using and 3′-rapid amplification of cDNA ends as described and Fig. 1 an of rat R. Champigny G. F. Heurteaux C. Lazdunski M. Nature. PubMed Scopus Google and fish ASIC proteins. The exhibit 76% amino acid The of highest are and the whereas the amino carboxyl and are more Functional of Fish ASIC1 in Xenopus Fish ASIC1 cRNA was injected in Xenopus and currents were 2 days with the two-microelectrode voltage Fig. currents by a in pH o from 7.4 to The current has a rapid of activation and rapidly in the presence of the oocytes were and for The fASIC1 protein was revealed with protein of was in injected oocytes but in of Fish examined the properties of currents of fish ASIC1 expressed in X. oocytes with the in the outside-out Channels were activated by a rapid of external pH o from 7.4 to Fig. of fASIC1 recorded in mm For Fig. from The in the of the by the system the solutions are a fASIC1 channels by rapid and in the continual presence of external The of the fish currents in to the rASIC1, in to the channel. are at the end of the of in the and from Fig. M.P. McIIwrath 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 (501) Google Scholar). are significant differences in channel the of fASIC1 is by with and the of that a to the significant these channels is the of the of of the fish the rat channels. the in the in Fig. and B. indicates that the desensitization rate of fish is faster rate were from the of patch clamp from fASIC1 or The in Fig. the of the with a The desensitization rate were and for the fish and of for and and the of fASIC1 for external we the half-maximal activation from currents by the pH o using The of with pH o of 7.4 before the external solution was to each of the currents were to the with a pH o of The in Fig. the of the to the with half-maximal proton activation of 5.6 The of pH o was by currents induced by changes in solutions from a series of pH o values to a solution of pH o of The dependence of desensitization on proton was to be The of the indicates that desensitization by a The of fASIC1 currents were from recorded in the presence of mm or mm and with 1 mm Ca2+ in the external Fig. as by the of the ASIC family of ion channels. were of in the presence of in The cord in the voltage of to was and 20 for in In the presence of the currents were in and Ca2+ was the external the were to be as of the channels is the by or The half-maximal for of fASIC1 was by currents induced by a in pH o from 7.4 to in the presence of of external The was and the was The for external was by currents induced by pH o in the presence of of external from 30 to The of the solutions was by the of The were to the with a of mm indicated that the rate of desensitization of the mammalian ASIC1 (2Alvarez de La Rosa D.A. Krueger S.R. Kolar A. Shao D. Fitzsimonds R.M. Canessa C.M. J. Physiol. 2003; 546: 77-87Crossref PubMed Scopus (173) Google Scholar). was that this property modulate the of the ASICs in peripheral where the is a in the central nervous system. In to the of is and it a in making of the of fASIC1 in the but in the central nervous system. We the of fASIC1 is to changes in Fig. of currents activated by pH o at or °C, is the of by most currents and the rate of desensitization differ at or for of Ca2+ has been in the of the rat ASIC1 in Scholar, S. Schild L. Physiol. 2002; PubMed Scopus Google Scholar, 21Price M.P. McIIwrath 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 (501) Google and of ASIC3 E. Baron A. Lingueglia E. J. Lazdunski M. 2003; PubMed Scopus Google Scholar). We examined the of external Ca2+ on the of fASIC1 by first whole currents with 1 mm external Ca2+ or Ca2+ Fig. of fASIC1 currents activated by a in pH o from 7.4 to with 1 mm or The of external Ca2+ and the pH o are indicated the current In the of the the was with 1 mm Ca2+ in the o and o response was was to be the The second a but Ca2+ in the pH o to the solution 1 mm Ca2+ and the solution mm a current of of the response was the solution Ca2+ and the solution 1 mm the current was of the The were of the sequence on the different of solutions were indicate that Ca2+ be to the channel prior to the of protons in order to The for external Ca2+ was by currents by pH o solutions with of For each Ca2+ the and the solutions the of Fig. a of with the the of the with half-maximal activation of mm and of We that the of Ca2+ was 1 the of fASIC1 but it was external Ca2+ was to To the of we examined the of Ca2+ on the were with pH o solutions containing mm and of in Fig. a in the of the of the of currents to mm external Ca2+ is in Fig. where the the of the to the with values for of mm and of of the of of of the functional differences fish and rat ASIC1 is the rate of desensitization. these channels exhibit a high degree of amino acid we that it be to identify the sequences conferring this property by the fish and rat ASIC1. First we examined the of the structural of amino and carboxyl and and the Fig. a of the first set of where in are sequences from fish ASIC1, in are sequences from rat ASIC1, and in are sequences from The the chimeras indicate the amino in the protein sequence to the rat or fish The cRNA of each was injected in and and currents were The chimeras were functional expressed in The rate of desensitization was from currents by pH o and 1 mm and were designed to the of the transmembrane chimeras at the to fish and rat ASIC1, that the transmembrane the and were designed to the of the amino and carboxyl chimeras desensitization of the fish and rat ASIC1, that the of the channel to desensitization. the extracellular and the desensitization of fish and rat ASIC1, We the of the extracellular domain by making and amino and carboxyl termini from the rapid fish ASIC1 and the from and J. Baron A. Lazdunski M. Voilley N. J. Neurosci. 2002; 22: 10662-10670Crossref PubMed Google Scholar). a slow and incomplete desensitization rate, from the role of the in Fig. of patch of the three where the whole has been To further the regions in the extracellular domain important for desensitization, we made of the ectodomain, to the carboxyl of the from a of whereas the amino of the from a of indicates that the first of the of fish confers the faster of 20 and of the properties as the the amino and is important to the to this Fig. of the most chimeras. sequence of the by amino from rat and fish is in Fig. We the in the rat ASIC1 to and The channel a desensitization rate of faster the type rASIC1, whereas the a desensitization rate different from of the from rat into in a significant of of three in the extracellular domain of the rat channel for the in fish is to a desensitization. amino acid changes in that the desensitization rate on the of the three and Functional Comparison of Fish and Rat of the sequences of fish and rat ASIC1 76% identity at the amino acid of the differences are to and the whereas and the are highly conserved R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (175) Google Scholar). We that the properties of the channels from the species are ion channel and 30 for fish and and of and 10 for fish and (29Zhang P. Canessa C.M. J. Gen. Physiol. 2002; 120: 553-566Crossref PubMed Scopus (61) Google Scholar). functional with the highly conserved sequence of the that the ion (17Kellenberger S. Hoffmann-Pochon N. Gatschi I. Scheeberger E. Schild L. J. Gen. Physiol. 1999; 114: 13-30Crossref PubMed Scopus (105) Google Scholar, S. M. Gatschi I. Scheeberger E. Schild L. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar). the has been in ion S. De Weille J.R. Lingueglia E. Lazdunski M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), we of the poorly conserved amino termini with the The that a high degree of identity is the extracellular domain conservation of extracellular is a of all of the For instance, the of from rat and frog the functional properties of from these species are A. A. Canessa C.M. Horisberger J.D. Schild L. Rossier B.C. J. Physiol. PubMed Google Scholar). the high degree of conservation of the that it is important in the ASIC Indeed, this domain has been in the gating by external protons and Ca2+ Weille J. F. 2001; PubMed Scopus Google Scholar, D.C. McCleskey E.W. Neuron. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Canessa C.M. J. Gen. Physiol. 2002; 120: 553-566Crossref PubMed Scopus (61) Google Scholar). that fASIC1 is activated by protons with an of to the mammalian ASIC1 (29Zhang P. Canessa C.M. J. Gen. Physiol. 2002; 120: 553-566Crossref PubMed Scopus (61) Google Scholar), but in to the rASIC1, the fish channel more external Ca2+ for activation of important the fish and mammalian channels is the desensitization is a that in most channels. The rate and of desensitization are important properties that the and of the channel 25-fold faster desensitization rate that for the in pH o the of into fish neurons is in mammalian The of this on the physiology of ASIC1 in the nervous system are with current of of these channels. in the it has been that a of different and modulate the rate and of desensitization of the mammalian external protons (29Zhang P. Canessa C.M. J. Gen. Physiol. 2002; 120: 553-566Crossref PubMed Scopus (61) Google Scholar), Ca2+ Weille J. F. 2001; PubMed Scopus Google Scholar, D.C. McCleskey E.W. Neuron. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Canessa C.M. J. Gen. Physiol. 2002; 120: 553-566Crossref PubMed Scopus (61) Google Scholar), A. L. Lingueglia E. Champigny G. Lazdunski M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), C.C. Cheng C. M. C. Welsh M.J. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Baron A. Lingueglia E. J. Lazdunski M. 2003; PubMed Scopus Google Scholar, R. Champigny G. Voilley N. I. Lazdunski M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), of various F. Champigny G. R. de Weille J.R. Heurteaux C. Lazdunski M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), to the in the R. Champigny G. F. Heurteaux C. Lazdunski M. Nature. PubMed Scopus Google Scholar), and C.C. C.J. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). these is the the desensitization of ASIC1 or the structural that in the To gain insight into these we of the differences in desensitization fish and mammalian ASIC1 to identify structural determinants that from functional analysis of many chimeras indicate that the extracellular domain the rate of desensitization of ASIC1. of this domain to identify a sequence by three in rat and in that is for most of the in in rat and fish ASIC1. The of the three was by in of the three but a The was of amino in the the for or the desensitization We all of the different amino in the by the of the the from to 20 to the degree as the of the whole and of 20 and properties of the channels were such as the of activation or rate of from desensitization The the changes in be by we can that these amino may a that the from the to the in the these may in with of the protein such that the the and the desensitization the the the the desensitization rate faster the of amino we have is are in the that the rate of desensitization, of these the to the of the channels. important from these is that activation and desensitization are in the of activation in type fish and rat ASIC1 or in the chimeras. In functional analysis of evolutionarily distant ASIC1 has provided the first that the ectodomain, in particular a sequence of three residues, the desensitization of ASIC1.