Functional characterization of two prokaryotic pentameric ligand-gated ion channel chimeras – role of the GLIC transmembrane domain in proton sensing

• Published in: Biochimica et biophysica acta. Biomembranes 2017-02, Vol.1859 (2), p.218-227
• Main Authors: Hénault, Camille M, Baenziger, John E
• Format: Article
• Language: English
• Subjects: Animals; Channel gating; Chimera; Chimera - metabolism; Crystallography, X-Ray - methods; Cysteamine - metabolism; ELIC; GLIC; Histidine - metabolism; Inter-domain complementarity; Ion Channel Gating - physiology; Ligand-Gated Ion Channels - metabolism; Ligands; Lipid-protein interactions; M4 lipid-sensor; Models, Molecular; Mutation - genetics; nAChR; Oocytes - metabolism; Pentameric ligand-gated ion channels; Prokaryotic Cells - metabolism; Protein Domains - physiology; Protons; Xenopus laevis - metabolism
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2016.11.006

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. [Display omitted] •GLIC-extracellular/ELIC transmembrane chimera exhibits impaired function•ELIC-extracellular/GLIC transmembrane chimera (EG) gates open to an ELIC agonist•EG gates open to protons at pH values below 4.5•Protons inhibit the EG agonist response 7≤pH≤4.5, but potentiate below pH4.5•Thermal stability of chimeras is similar to that of the parent transmembrane domain