期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2011
卷号：108
期号：29
页码：12143-12148
DOI：10.1073/pnas.1104494108
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 1 glycine receptor (1GlyR). 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 1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on 1GlyR. 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 1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and 1GlyR 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.
关键词：Gloeobacter violaceus ; allosteric effector ; evolution ; fusion protein ; membrane protein
