title: Functional characterization of two prokaryotic pentameric ligand-gated ion channel chimeras - role of the GLIC transmembrane domain in proton sensing.
abstract: With the long-term goal of using a chimeric approach to dissect the distinct lipid sensitivities and thermal stabilities of the pentameric ligand-gated ion channels (pLGIC), GLIC and ELIC, we constructed chimeras by cross-combining their extracellular (ECD) and transmembrane (TMD) domains. As expected, the chimera formed between GLIC-ECD and ELIC-TMD (GE) responded to protons, the agonist for GLIC, but not cysteamine, the agonist for ELIC, although GE exhibited a 25-fold decrease in proton-sensitivity relative to wild type. The chimera formed between ELIC-ECD and the GLIC-TMD (EG) was usually toxic, unless it contained a pore-lining Ile9'Ala gain-of-function mutation. No significant improvements in expression/toxicity were observed with extensive loop substitutions at the ECD/TMD interface. Surprisingly, oocytes expressing EG-I9'A responded to both the ELIC agonist, cysteamine and the GLIC agonist, protons - the latter at pH values ≤4.0. The cysteamine- and proton-induced currents in EG-I9'A were inhibited by the GLIC TMD pore blocker, amantadine. The cysteamine-induced response of EG-I9'A was also inhibited by protons at pH values down to 4.5, but potentiated at lower pH values. Proton-induced gating at low pH was not abolished by mutation of an intramembrane histidine residue previously implicated in GLIC TMD function. We show that the TMD plays a major role governing the thermal stability of a pLGIC, and identify three distinct mechanisms by which agonists and protons influence the gating of the EG chimera. A structural basis for the impaired function of GE is suggested.
authors.affiliation: Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd, Ottawa, ON K1H 8M5, Canada.
authors.forename: Camille M
authors.initials: CM
authors.lastname: Hénault
authors.affiliation: Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Rd, Ottawa, ON K1H 8M5, Canada. Electronic address: John.Baenziger@uottawa.ca.
authors.forename: John E
authors.initials: JE
authors.lastname: Baenziger
mesh_terms: D000818:Animals; D002678:Chimera / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D003543:Cysteamine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D058446:Ligand-Gated Ion Channels / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D011387:Prokaryotic Cells / Q000378:metabolism*; D000072417:Protein Domains / Q000502:physiology; D011522:Protons; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D058446:Ligand-Gated Ion Channels; D008024:Ligands; D011522:Protons; D006639:Histidine; D003543:Cysteamine
keywords: Channel gating; Chimera; ELIC; GLIC; Inter-domain complementarity; Lipid-protein interactions; M4 lipid-sensor; Pentameric ligand-gated ion channels; nAChR