Aplysia cys-loop Glutamate-Gated Chloride Channels Reveal Convergent Evolution of Ligand Specificity
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Among the members of the superfamily of cys-loop ligand-gated ion channels (LGICs) are receptors distinguished by the presence of two cys-loops in the ligand-binding domain, for example, the glycine receptor. Such receptors have thus far been cloned only from vertebrates and from ecdysozoa (arthropods and nematodes). We have now cloned and expressed two 2-cys-loop receptors from Aplysia californica, a lophotrocozoan, and have shown that they form homomeric glutamate receptors. We have also built up a database including the two receptors cloned here, previously cloned vertebrate and ecdysozoan 2-cys-loop receptors taken from GenBank, and the same type of receptors obtained by a search of recently cloned genomes, including two non-vertebrate chordates, an echinoderm, a crustacean, an annelid, and another mollusk. We subjected these receptors to phylogenetic analysis, alone and in combination with GABA-A receptors from the same phyla and from a recently cloned cnidarian. The phylogenetic analysis revealed the presence of two independent clades of glutamate receptors: one from lophotrocozoa and other from ecdysozoa, and suggests that the ancestors of the current 2-cys-loop receptor types diverged from the GABA-A receptors and from each other before the bilateria-cnidaria split. Finally, combining the results from the phylogenetic analysis with those obtained from an analysis of the 2-cys-loop receptors in light of recently published hypotheses concerning the glycine binding pocket, we predict that glycine receptors are not exclusively a vertebrate-receptor type.
KeywordsAplysia Glutamate Glycine LGICs Chloride channels Phylogeny Glycine binding pocket motif
JacSue Kehoe would like to thank Cristina Alberini for her invaluable instruction at the beginning of the cloning of the two subunits; Philippe Djian, Eric Krejci, and Bruno della Gaspera for generously making their equipment available to her; Russell English for his help with preparation of the figures; Jean Deutsch and David Enard for their help and patience while introducing her to some of the basics of phylogenetic analysis, and Robert Zucker of Cell and Molecular Biology at U.C. Berkeley for welcoming her as a Visiting Scholar. A last but not least word of thanks to the DOE Joint Genome Institute for making their work in progress available to the scientific community. This work was supported in part by the NEUROCYPRES grant from the European Commission Seventh Framework Programme (for S.B.).
- Eguchi Y, Ihara M, Ochi E, Shibata Y, Matsuda K, Fushiki S, Sugama H, Hamasaki Y, Niwa H, Wada M, Ozoe F, Ozoe Y (2006) Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes. Insect Mol Biol 15:773–783PubMedCrossRefGoogle Scholar
- Hille B (2001) Ionic channels of excitable membranes, 3rd edn. Sinauer Associates, Sunderland, p 814Google Scholar
- Kehoe JS (1976) Electrogenic effects of neutral amino acids on neurons of Aplysia californica. Cold Spring Harb Symp of Quant Biol 40:145–155Google Scholar
- Lynch JW (2004) Molecular structure and function of the glycine receptor chloride channel. Physiol Rev 84:1061–1095Google Scholar
- Moroz LL, Edwards JR, Puthanveettil SV, Kohn AB, Ha T, Heyland A, Knudsen B, Sahni A, Yu F, Li L, Jezzini S, Lovell P, Iannucculli W, Chen M, Nguyen T, Sheng H, Shaw R, Kalachikov S, Panchin YV, Farmerie W, Russo JJ, Ju J, Kandel ER (2006) Neuronal transcriptome of Aplysia: neuronal compartments and circuitry. Cell 127:1453–1467PubMedCrossRefGoogle Scholar
- Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, Salamov A, Terry A, Shapiro H, Lindquist E, Kapitonov VV, Jurka J, Genikhovich G, Grigoriev IV, Lucas SM, Steele RE, Finnerty JR, Technau U, Martindale MQ, Rokhsar DS (2007) Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317:86–94PubMedCrossRefGoogle Scholar
- Sunesen M, de Carvalho LP, Dufresne V, Grailhe R, Savatier-Duclert N, Gibor G, Peretz A, Attali B, Changeux J-P, Pass Y (2006) Mechanism of Cl-selection by a glutamate-gated chloride (GluCl) receptor revealed through mutations in the selectivity filter. J Biol Chem 281:14875–14881PubMedCrossRefGoogle Scholar
- Wotring VE, Weiss DS (2008) Charge scan reveals an extended region at the intracellular end of the GABA receptor pore that can influence ion selectivity. J Gen Physiol 131:887–897Google Scholar