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

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are potential targets for a wide variety of general anesthetics. We recently showed that α4β2 nAChRs are more sensitive than α4β4 receptors to the gaseous anesthetics nitrous oxide and xenon. The present study examines chimeric and point mutant rat nAChRs expressed in Xenopus oocytes and identifies a single amino acid residue (β2-Val253 or β4-Phe255) near the middle of the second transmembrane segment (TM2) that determines gaseous anesthetic sensitivity. Mutations of this residue in β subunits and the homologous residue of α4 subunits (α4-Val254) showed that this position also determines sensitivities of nAChRs to acetylcholine, isoflurane, pentobarbital, and hexanol. In contrast, these mutations did not affect actions of ketamine. The positively charged sulfhydryl-specific reagent methanethiosulfonate ethylammonium reacted with a cysteine introduced at α4-Val254 or β2-Val253, and irreversibly reduced anesthetic sensitivities of nAChRs. Propyl methanethiosulfonate is an anesthetic analog that covalently binds to a TM2 site of γ-aminobutyric acidA and glycine receptors and irreversibly enhances receptor function. However, propyl methanethiosulfonate reversibly inhibited cysteine-substitution mutants at α4-Val254 or β2-Val253 of nAChRs, and did not affect anesthetic sensitivity. Thus, residues α4-Val254 and β2-Val253 alter channel gating and determine anesthetic sensitivity of nAChRs, but are not likely to be anesthetic-binding sites.