A Family of Acetylcholine-gated Chloride Channel Subunits in Caenorhabditis elegans

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by α-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by α-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. Fast (ionotropic) cholinergic neurotransmission is generally mediated by nicotinic acetylcholine (ACh) 1The abbreviations used are: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; LGIC, ligand-gated ion channel; C6, hexamethonium; dβe, dihydro-β-erythroidine; α-BT, α-bungarotoxin; d-TC, d-tubocurarine. receptors (nAChRs). These are cation-selective channels and hence mediate excitatory neurotransmission. However, electrophysiological evidence of ionotropic, ACh-gated chloride channels in molluscs suggests the existence of fast inhibitory cholinergic neurotransmission as well (1Kehoe J. J. Physiol. 1972; 225: 85-114Crossref PubMed Scopus (136) Google Scholar, 2Kehoe J. J. Physiol. 1972; 225: 115-146Crossref PubMed Scopus (238) Google Scholar, 3Kehoe J. McIntosh J.M. J. Neurosci. 1998; 18: 8198-8213Crossref PubMed Google Scholar). The ACh-gated chloride channels in Aplysia neurons respond to several agonists and antagonists of nAChRs, indicating that, like the nAChRs, they may belong to the superfamily of Cys loop ligand-gated ion channel (LGIC) subunits. Otherwise, little is known about the molecular nature of the receptors that mediate fast inhibitory cholinergic neurotransmission, whether this type of neurotransmission is widespread in the animal kingdom, or how it evolved. The Cys loop LGICs are encoded by a large and diverse gene superfamily. These channels are pentameric and can be homomers or heteromers consisting of as many as four different subunits, each encoded by a different gene (4Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (914) Google Scholar). Subunits of the Cys loop LGIC superfamily share a topology that consists of a large extracellular ligand-binding domain and four transmembrane domains that form the ion-selective pore (4Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (914) Google Scholar, 5Hille B. Ionic Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA1992: 405-422Google Scholar, 6Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1088) Google Scholar). In vertebrates, the LGIC superfamily consists of two families of cation-selective channels, the nicotinic ACh receptors and the 5-hydroxytryptamine type 3 receptors, and two families of anion channels, the GABAA receptors and the glycine receptors (7Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18: 121-127Abstract Full Text PDF PubMed Scopus (471) Google Scholar). The repertoire of invertebrate LGICs is larger, including, in addition to homologues of vertebrate channels, histaminegated chloride channels (8Gisselmann G. Pusch H. Hovemann B.T. Hatt H. Nat. Neurosci. 2002; 5: 11-12Crossref PubMed Scopus (118) Google Scholar, 9Zheng Y. Hirschberg B. Yuan J. Wang A.P. Hunt D.C. Ludmerer S.W. Schmatz D.M. Cully D.F. J. Biol. Chem. 2002; 277: 2000-2005Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), a GABA-gated cation channel (10Beg A.A. Jorgensen E.M. Nat. Neurosci. 2003; 6: 1145-1152Crossref PubMed Scopus (127) Google Scholar), a serotonin-gated anion channel (11Ranganathan R. Cannon S.C. Horvitz H.R. Nature. 2000; 408: 470-475Crossref PubMed Scopus (184) Google Scholar), several glutamategated anion channels (12Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H. Schaeffer J.M. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (583) Google Scholar, 13Dent J.A. Davis M.W. Avery L. EMBO J. 1997; 16: 5867-5879Crossref PubMed Scopus (281) Google Scholar, 14Vassilatis D.K. Arena J.P. Plasterk R.H. Wilkinson H.A. Schaeffer J.M. Cully D.F. Van der Ploeg L.H. J. Biol. Chem. 1997; 272: 33167-33174Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 15Dent J.A. Smith M. Vassilatis D.K. Avery L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2674-2679Crossref PubMed Scopus (321) Google Scholar, 16Horoszok L. Raymond V. Sattelle D.B. Wolstenholme A.J. Br. J. Pharmacol. 2001; 132: 1247-1254Crossref PubMed Scopus (89) Google Scholar), and a divergent choline-gated nAChR (17Yassin L. Boaz G. Kahan T. Halevi S. Eshel M. Treinin M. Mol. Cell. Neurosci. 2001; 17: 589-599Crossref PubMed Scopus (64) Google Scholar). Thus, the LGIC channel structure appears flexible enough to accommodate diverse ligands and ligand/ion selectivity pairings. Although no genes encoding ACh-gated chloride channels have been previously identified, it is likely that many invertebrate receptors with unusual properties remain to be characterized. The genomes of Caenorhabditis elegans and Drosophila melanogaster reveal numerous predicted Cys loop LGICs that do not obviously belong to any family of known ion or ligand specificity (18The C. elegans Genome Consortium Science. 1998; 282: 2012-2018Crossref PubMed Scopus (3634) Google Scholar, 19Bargmann C.I. Science. 1998; 282: 2028-2033Crossref PubMed Scopus (734) Google Scholar, 20Gelbart W.M. Crosby M. Matthews B. Rindone W.P. Chillemi J. Russo Twombly S. Emmert D. Ashburner M. Drysdale R.A. Whitfield E. Millburn G.H. de Grey A. Kaufman T. Matthews K. Gilbert D. Strelets V. Tolstoshev C. Nucleic Acids Res. 1997; 25: 63-66Crossref PubMed Scopus (107) Google Scholar, 21Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. Sutton G.G. Wortman J.R. Yandell M.D. Zhang Q. Chen L.X. Brandon R.C. Rogers Y.H. Blazej R.G. Champe M. Pfeiffer B.D. Wan K.H. Doyle C. Baxter E.G. Helt G. Nelson C.R. Gabor G.L. Abril J.F. Agbayani A. An H.J. Andrews-Pfannkoch C. Baldwin D. Ballew R.M. Basu A. Baxendale J. Bayraktaroglu L. Beasley E.M. Beeson K.Y. Benos P.V. Berman B.P. Bhandari D. Bolshakov S. Borkova D. Botchan M.R. Bouck J. Brokstein P. Brottier P. Burtis K.C. Busam D.A. Butler H. Cadieu E. Center A. Chandra I. Cherry J.M. Cawley S. Dahlke C. Davenport L.B. Davies P. de Pablos B. Delcher A. Deng Z. Mays A.D. Dew I. Dietz S.M. Dodson K. Doup L.E. Downes M. Dugan-Rocha S. Dunkov B.C. Dunn P. Durbin K.J. Evangelista C.C. Ferraz C. Ferriera S. Fleischmann W. Fosler C. Gabrielian A.E. Garg N.S. Gelbart W.M. Glasser K. Glodek A. Gong F. Gorrell J.H. Gu Z. Guan P. Harris M. Harris N.L. Harvey D. Heiman T.J. Hernandez J.R. Houck J. Hostin D. Houston K.A. Howland T.J. Wei M.H. Ibegwam C. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4854) Google Scholar). C. elegans in particular encodes ∼70 LGIC subunit genes, of which fewer than 20 have been characterized pharmacologically (19Bargmann C.I. Science. 1998; 282: 2028-2033Crossref PubMed Scopus (734) Google Scholar). The function of such a large and diverse LGIC superfamily in a single species is unclear. To better understand the constraints on Cys loop LGIC structure and evolution and to identify new modes of neurotransmission, we have characterized several members of a novel family of channel subunits from C. elegans. These form ACh-gated chloride channels exhibiting an unusual pharmacology that appears to reflect a unique ACh-binding site. Cloning ACC cDNAs—Poly(A+) RNA was purified from adult worms (Bristol N2 strain). First strand cDNA was synthesized with oligo(dT) primer using the avian myeloblastosis virus reverse transcriptase system (Invitrogen Canada Inc., Burlington, Ontario, Canada). The open reading frame of ACC-1, -2, and -4, as predicted in Wormbase (available on the World Wide Web at www.wormbase.org/), was amplified by PCR using the following primers: ACC-1, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCATATGAGTCATCCGGGTTGGATTAT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGATTAAGGTTGATCAATATTCACA-3′; ACC-2, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCATATGATATTTACTCTTTTATCAACACTGCCT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGATTATCCGTCAACTCGATT-3′; ACC-4, 5′-GGGGTACCATATGCGACTAATCATATTAGTAATCT-3′ and 5′-GCTCTAGATTAGATAGTTCTAACCAATAGTTTTCC-3′. PCR products were subcloned either into pDONR201 (ACC-1 and ACC-2) via recombination reaction using the Gateway Cloning Technology kit (Invitrogen) or into pBluescript (Stratagene Inc., La Jolla, CA) using the KpnI and XbaI sites (ACC-4). ACC-3 was first amplified from cDNA using a primer to the SL-1 transpliced leader sequence 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATCCTTTAATTACCCAAGTTTGAG-3′ and a 3′ primer corresponding to the end of the putative open reading frame, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGCAGTCATGTGTTAACAGTAAGGTAATAT-3′. The resulting PCR product was cloned into pDONR and sequenced. A new 5′-primer corresponding to the 5′-end of the open reading frame was used with the previous 3′-primer to amplify the open reading frame from the cloned ACC-3 cDNA. Three cDNA clones of each gene were sequenced to determine the true open reading frames and to find possible mutations resulting from reverse transcription-PCR. Nonsilent mutations were fixed either by overlap extension PCR (22Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (432) Google Scholar) or by splicing together mutation-free cDNA fragments using convenient restriction sites. Sequence Analysis—Amino acid sequences were aligned using the ClustalW program (available on the World Wide Web at clustalw.genome.ad.jp). Transmembrane domains were predicted using the TMHMM method based on a hidden Markov model (23Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9290) Google Scholar). SignalP 2.0, NetGlyc 1.0, and NetPhos 2.0 programs based on artificial neuronal networks were used to predict signal peptide sequences (24Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4942) Google Scholar). All of the above mentioned prediction programs are available on the World Wide Web at www.cbs.dtu.dk/services. Expression in Xenopus Oocytes and Electrophysiology—cDNAs were subcloned into the pT7N expression vector (25Cary R.B. Klymkowsky M.W. Evans R.M. Domingo A. Dent J.A. Backhus L.E. J. Cell Sci. 1994; 107: 1609-1622Crossref PubMed Google Scholar). The pT7NcDNA constructs were linearized with SalI (ACC-2) or BamHI (ACC-1, -3, and -4), and capped cRNAs were transcribed using the MEGAscript Kit (Ambion, Austin, TX). Synthesized cRNAs were recovered by LiCl precipitation and resuspended in nuclease-free H2O at a final concentration of 1 μg/ml. Oocytes were harvested from mature female Xenopus laevis according to standard procedures (26Goldin A.L. Methods Enzymol. 1992; 207: 266-279Crossref PubMed Scopus (236) Google Scholar). Oocytes were maintained at 20 °C in ND96 solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm Hepes, pH 7.5) supplemented with 100 mg/ml gentamycin and 550 mg/ml pyruvate. Oocytes were injected with 40 nl of cRNA using the Nanoject system (Drummond Scientific, Broomall, PA) and incubated for 2 days before measurements were taken. Two-electrode voltage clamp recordings were performed using the AxoClamp 2B amplifier (Axon Instruments, Foster City, CA). Oocytes were perfused in an RC-12 recording chamber (Warner Instrument Inc., Hamden, CT) or a Maltese Cross chamber (ALA Scientific Instruments, Westbury, NY). Data were acquired at 1 kHz using Clampex software (Axon Instruments, Foster City, CA). All were from for agonists were by of by To determine the and as in were using the as the is the by a concentration of is the concentration of is the and is the at Oocytes were with for 1 for to of the with either 1 or ACh for ACC-1 or ACC-2, The of to of ACh and was to that of the to ACh the ACC-1 and channels in the of ACh, were using voltage of in the of either 2 or 20 ACh for ACC-1 or ACC-2, ion or were for in the ND96 The was to the in ND96 at Cloning of of the ACC of predicted C. elegans LGIC subunit genes the of a of LGIC genes characterized was the serotonin-gated chloride channel subunit (11Ranganathan R. Cannon S.C. Horvitz H.R. Nature. 2000; 408: 470-475Crossref PubMed Scopus (184) Google Scholar). of this have no in Drosophila or vertebrate genomes not The to into that we to families of channels with ligand We and sequenced four from ACC-1 known as ACC-3 and reading frames of the ACC-1 and to in the genome The ACC-3 cDNA was transpliced with an leader sequence and from the predicted open reading frame in the first and All to LGIC subunits, such as a signal an extracellular domain with the Cys four transmembrane domains and a large loop and were in the acid sequences of these The ACC-1 and putative are to each at the acid and and to In to a C. elegans GABA-gated chloride channel subunit A.A. Jorgensen E.M. J. Neurosci. PubMed Google Scholar), and is to a C. elegans nAChR ACC-1 and ACh-gated determine the ligand specificity of the ACC subunits, we ACC cRNAs in Xenopus and the at and an with from to A and The no at ACh the ACC-1 to ACh with a concentration of and an of was with an of and a of Oocytes injected with ACC-3 cRNAs a to 1 mm ACh, and injected with no An was not in or Oocytes ACC cRNAs not respond to 1 mm or to 1 mm of the ACh and to but no to these was in ACC subunits. ACC-1, -2, and share a of the transmembrane domain to the of that been to anion selectivity in vertebrate and glycine receptors D.B. A. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, S.C. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google Scholar, A. N. S. J.P. D. Nature. 1992; PubMed Scopus Google Scholar) To determine whether ACC-1 and are chloride channels, we The in ND96 for ACC-1 or homomeric channels were and with the for chloride A and W.M. J. Biol. PubMed Scopus Google Scholar). the anion was for chloride mm final was a in the of and In was for the in was ACC-1, and ACC-2) and We of and in the for a in chloride concentration for ACC-1 and ACC-2, is in with the of predicted by the for channels. The of the ACC Channels a sequence the and in extracellular ligand-binding domains is the ligand-binding as predicted from with nAChRs, are not well and the we predicted that the have a unique pharmacological We agonists and antagonists of vertebrate on the ACC receptors. The nAChR at 1 mm was a of the ACC channels was a of ACC-1 but not ACC-2. nAChR as a of channels and as an of ACC-1 and The antagonists the d-tubocurarine for ACC-1, and for 2 and and 1 as an and an of 20 d-TC, a of and 5-hydroxytryptamine type 3 receptors, blocked ACC-1 and by and and by d-TC, and are of the vertebrate nAChR channels J. S. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, M. E. 2000; PubMed Scopus Google Scholar) and of the Aplysia ACh-gated chloride channel J. McIntosh J.M. J. Neurosci. 1998; 18: 8198-8213Crossref PubMed Google Scholar). However, these channels, ACC-1 and were not blocked by Although nicotinic agonists little on ACC-1 and ACC-2, arecoline, an of ACh receptors, a that was of the A and a in the and with an of and and A was the of in of receptors, an of open channel D. A. F. N. C. S. M. J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, J. Physiol. 1992; PubMed Scopus Google Scholar). We that the for of and for the and are not different from the for the to ACh, indicating a of of ACh and a of ACh receptors and a of nAChRs, the channel with an of with an of but of In we no to 1 of the blocked the of ACC-1 channels to 1 ACh with an of Thus, the pharmacological of ACC-1 and a ACC ligand-binding site. ACC-3 and ACC-3 and do not respond to ACh as we the that they form heteromers with ACh-gated chloride channel subunits. ACC-3 a channel with of ACC-1 ACC-3 a channel that a with the ACC-1 the of ACC-1 to ACh was than than that of homomeric ACC-1 channels. The of that the ACC-1 ACC-3 fewer ACh sites than the ACC-1 of ACC-1 with not the or to the ACC-1 Thus, is no that with Oocytes the cRNA with the ACC-3 or cRNAs either a of to 1 mm ACh or not respond to 1 mm or was for ACC-2, ACC-3 and not expression of a chloride channel or expression of ACC-1 We this as indicating that ACC-3 and are to with in a channel and with or We have a new family of Cys loop the nematode ACh-gated chloride channels. We report the first molecular characterization of an anion-selective ACh receptor and that a of Cys loop LGICs to mediate inhibitory cholinergic neurotransmission. is the first evidence of ACh-gated chloride channels in and suggests that fast inhibitory cholinergic neurotransmission is widespread in the animal than previously The ACh-gated chloride channel subunits in C. elegans belong to the superfamily of Cys loop ligand-gated ion channels. we predict that the ACC channels are We that ACC-1 and form homomeric channels in Xenopus but that ACC-1 with ACC-3 and with ACC-3 and The of ACC-1 with ACC-3 a channel that function in with a to ACh than the ACC-1 The of with ACC-3 and is these subunits to ACC-2. We that these subunits into heteromers but additional subunits to form a However, we the that, of into a these subunits are from in or that ACC-3 and ACC-2. which subunits are in this the of the ACC-3 and to with ACC-1 and is with the that the a family of ACh-gated chloride channel subunits. of the unusual of these channels is the of by ACh with anion We can to a that for the anion The transmembrane domains the pore of the channel and determine ion selectivity A. N. S. J.P. D. Nature. 1992; PubMed Scopus Google Scholar). the end of this domain is a that is in anion channels in C. elegans. are in vertebrate channels, and of this in anion selectivity in GABAA and glycine receptors D.B. A. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, S.C. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google Scholar, A. N. S. J.P. D. Nature. 1992; PubMed Scopus Google Scholar). The of the is unusual ligand site. The sequences of ACC-1 and subunits substantially from nematode and vertebrate in extracellular ligand-binding domains not and A. Nat. Neurosci. 2002; PubMed Scopus Google Scholar), by of the structure of acetylcholine-binding K. M. J. Nature. 2001; PubMed Scopus Google Scholar), have that the ACh at the of two subunits. on of the subunit in and C. on the of the and in and which form the of the However, the the ligand-binding are not the and the the of the a of the ligand-binding nAChR subunits, are from the Thus, may have the to ACh of the The unusual pharmacological of the ACC subunits a unique acetylcholine-binding site. nicotine, the of nAChRs, and a are agonists antagonists of ACC-1 and the from nicotinic receptors. The is The C. elegans receptors are to nicotine, and it been that C. elegans and cation-selective ACh receptors Jorgensen E.M. Nat. Neurosci. PubMed Scopus Google Scholar). and are antagonists of the vertebrate M. E. 2000; PubMed Scopus Google Scholar). is that is an efficient of ACC-1 and -2, with substantially than been to on cation-selective nematode and this to may be a of invertebrate ACh receptors L.R. P. P.W. Cell. 2001; 107: Full Text Full Text PDF PubMed Scopus Google Scholar, F. A. B. J. Biol. 1994; PubMed Google Scholar). α-BT, which is for vertebrate and in the system and the of the not the ACC channels. to the loop in M. R. A. J.M. M. M. K. E. S. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the of on ACC channels the of to the ACC divergent loop the Aplysia ACh-gated ion channels of the are pharmacological that they be J. McIntosh J.M. J. Neurosci. 1998; 18: 8198-8213Crossref PubMed Google Scholar). of the two Aplysia channels a of ACC-1 homomers and and were agonists of the Aplysia of the antagonists for nematode and receptors were in the However, Aplysia receptors to have an for ACh of the Aplysia channels were blocked by α-BT, indicating that they share a to the nicotinic receptors, at in the than do the ACC subunits. whether the Aplysia channels have to be by sequence Cys loop is an of a of acetylcholine receptors Sci. 16: Scopus Google Scholar), and glutamategated chloride channels (12Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H. Schaeffer J.M. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (583) Google Scholar). have properties that are unique to it possible to that are agonists of channels in the vertebrate The receptors are to the vertebrate in sequence but are not chloride channels are not in Jorgensen E.M. Nat. Neurosci. PubMed Scopus Google Scholar). are not in vertebrates, they are for the of We E. for reading of the

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