A transmembrane site determines sensitivity of neuronal nicotinic acetylcholine receptors to general anesthetics

Neuronal nicotinic acetylcholine receptors (nAChRs) are potential targets f or a wide variety of general anesthetics. We recently showed that alpha (4) beta (2) nAChRS are more sensitive than alpha (4)beta (4) receptors to the gaseous anesthetics nitrous oxide and xenon. The present study examines chi meric and point mutant rat nAChRs expressed in Xenopus oocytes and identifi es a single amino acid residue (beta (2)-Val(253) or beta (4)-Phe(255)) nea r the middle of the second transmembrane segment (TM2) that determines gase ous anesthetic sensitivity. Mutations of this residue in beta subunits and the homologous residue of alpha (4) subunits (alpha (4)-Val(254)) showed th at this position also determines sensitivities of nAChRs to acetylcholine, isoflurane, pentobarbital, and hexanol, In contrast, these mutations did no t affect actions of ketamine, The positively charged sulfhydryl-specific re agent methanethiosulfonate ethylammonium reacted with a cysteine introduced at alpha (4)-Val(254) or beta (2)-Val(258), and irreversibly reduced anest hetic sensitivities of nAChRs, Propyl methanethiosulfonate is an anesthetic analog that covalently binds to a TM2 site of gamma -aminobutyric acid, an d glycine receptors and irreversibly enhances receptor function. However, p ropyl methanethiosulfonate reversibly inhibited cysteine-substitution mutan ts at alpha (4)-Val(254) or beta (2)-Val(253) of nAChRs, and did not affect anesthetic sensitivity. Thus, residues alpha (4)-Val(254) and beta (2)-Val (253) alter channel gating and determine anesthetic sensitivity of nAChRs, but are not likely to be anesthetic-binding sites.