Abstract

We performed macroscopic and single-channel current measurements on wild-type (WT) and two mutant muscle-type nicotinic acetylcholine (ACh) receptor channels transiently expressed in HEK-293 cells. The mutants contained polar-to-nonpolar substitutions at the 10′ (α₂S10′AβT10′Aγδ) and 6′ positions (α₂S6′AβγδS6′A) in the M2 pore region of the channel. We studied the behavior of these channels in the absence and presence of the volatile general anesthetic isoflurane. Both mutations changed the gating behavior of the channel. A comparison of the α₂S10′AβT10′Aγδ mutant to WT receptors revealed faster desensitization kinetics, increased sensitivity to ACh, a higher efficacy for activation by the partial nicotinic agonist decamethonium, and a greater number of openings per burst. A comparison of the α₂S6′AβγδS6′A mutant to WT receptors also revealed increased sensitivity to ACh and an increased burst duration at the single-channel level with ACh as agonist. The α₂S10′AβT10′Aγδ mutation increased the sensitivity of the ACh receptor to isoflurane, whereas the α₂S6′AβγδS6′A mutation did not. These changes were probably not caused by the differential effects of the mutation on channel gating and desensitization. The increased sensitivity of the α₂S10′AβT10′Aγδ receptor to isoflurane is state-dependent; the mutation changes the affinity of the closed state but not that of the open state of the channel.