Nicotinic receptor M3 transmembrane domain: Position 8′ contributes to channel gating

Abstract

The nicotinic acetylcholine receptor (nAChR) is a pentamer of homologous subunits with composition α2βεδ in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 8′ of the M3 domain is phenylalanine in all heteromeric α subunits, whereas it is a hydrophobic nonaromatic residue in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle nAChR activation by combining mutagenesis with single-channel kinetic analysis. Construction of nAChRs carrying different numbers of phenylalanine residues at 8′ reveals that the mean open time decreases as a function of the number of phenylalanine residues. Thus, all subunits contribute through this position independently and additively to the channel closing rate. The impairment of channel opening increases when the number of phenylalanine residues at 8′ increases from two (wild-type nAChR) to five. The gating equilibrium constant of the latter mutant nAChR is 13-fold lower than that of the wild-type nAChR. The replacement of αF8′, βL8′, δV8′ and εV8′ by a series of hydrophobic amino acids reveals that the structural bases of the observed kinetic effects are nonequivalent among subunits. In the α subunit, hydrophobic amino acids at 8′ lead to prolonged channel lifetimes, whereas they lead either to normal kinetics (δ and ε subunits) or impaired channel gating (β subunit) in the non-α subunits. The overall results indicate that 8′ positions of the M3 domains of all subunits contribute to channel gating.