Photoaffinity Labeling the Propofol Binding Site in GLIC

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABA_AR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus. In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) <i>Nature 469</i>, 428–431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3<i>H</i>-diazirin-3-yl]­phenol or AziP<i>m</i>) that acts as an anesthetic in vivo and potentiates GABA_AR in vitro. For GLIC expressed in <i>Xenopus</i> oocytes, propofol and AziP<i>m</i> inhibited current responses at pH 5.5 (EC₂₀) with IC₅₀ values of 20 and 50 μM, respectively. When [³H]­AziP<i>m</i> (7 μM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziP<i>m</i> bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket