title: Functional prokaryotic-eukaryotic chimera from the pentameric ligand-gated ion channel family.
abstract: Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.
authors: Institut Pasteur, Groupe Récepteurs-Canaux, F-75015 Paris, France.
authors: Guillaume
authors: G
authors: Duret
authors: Catherine
authors: C
authors: Van Renterghem
authors: Yun
authors: Y
authors: Weng
authors: Marie
authors: M
authors: Prevost
authors: Gustavo
authors: G
authors: Moraga-Cid
authors: Christèle
authors: C
authors: Huon
authors: James M
authors: JM
authors: Sonner
authors: Pierre-Jean
authors: PJ
authors: Corringer
mesh_terms: D000438:Alcohols; D000595:Amino Acid Sequence; D018681:Anesthetics, General; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D007559:Ivermectin; D058446:Ligand-Gated Ion Channels / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015742:Propofol; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology*; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014981:Xenopus
chemical_list: D000438:Alcohols; D018681:Anesthetics, General; D001426:Bacterial Proteins; D018076:DNA, Complementary; D058446:Ligand-Gated Ion Channels; D018009:Receptors, Glycine; D011993:Recombinant Fusion Proteins; D007559:Ivermectin; D015742:Propofol