title: Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.
abstract: General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.
authors: Molecular Biophysics Program, University of Wisconsin, Madison, Wisconsin 53711, USA.
authors: Borna
authors: B
authors: Ghosh
authors: Kenneth A
authors: KA
authors: Satyshur
authors: Cynthia
authors: C
authors: Czajkowski
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D018686:Anesthetics, Intravenous / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000458:Cyanobacteria; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D058446:Ligand-Gated Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D015742:Propofol / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D040681:Structural Homology, Protein; D014982:Xenopus laevis
chemical_list: D018686:Anesthetics, Intravenous; D001426:Bacterial Proteins; D058446:Ligand-Gated Ion Channels; D021122:Protein Subunits; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D003545:Cysteine; D015742:Propofol
keywords: Allosteric Regulation; Anesthetics; Cys-loop Receptors; Electrophysiology; Ligand-binding Protein; Membrane Proteins; Protein Conformation; Protein Structure; Substituted Cysteine Accessibility Method
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