Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels

Abstract

AbstractAbstract Using ELIC, a prokaryotic pentameric ligand-gated ion channel (pLGIC) Erwinia chrysanthemi , as a model, propofol is shown to bind in a transmembrane intrasubunit pocket that overlaps anesthetic-binding sites previously identified in other pLGICs. The functional relevance of this binding site is demonstrated by analysis of chimeric receptors, which suggests that the transmembrane intrasubunit site is a common binding site for anesthetic inhibition of cationic pLGICs. Supplemental Digital Content is available in the text. Background Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. Methods We photolabeled ELIC with a light-activated derivative of propofol (AziP m ) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABA A R) chimera that replaced the ELIC-TMD with the α1β3GABA A R-TMD and compared functional responses of ELIC-GABA A R and ELIC with propofol modulations. Results Photolabeling showed multiple AziP m -binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABA A R showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. Conclusions The distinctly different responses of ELIC and ELIC-GABA A R to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.