Photoaffinity Labeling the Propofol Binding Site in GLIC

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• 作者：David C. Chiara ; Jonathan F. Gill ; Qiang Chen ; Tommy Tillman ; William P. Dailey ; Roderic G. Eckenhoff ; Yan Xu ; Pei Tang ; Jonathan B. Cohen
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：January 14, 2014
• 年：2014
• 卷：53
• 期：1
• 页码：135-142
• 全文大小：416K
• ISSN：1520-4995

文摘

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 伪-helices (Nury, H, et al. (2011) Nature 469, 428鈥?31). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABA_AR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC₂₀) with IC₅₀ values of 20 and 50 渭M, respectively. When [³H]AziPm (7 渭M) 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 AziPm 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.