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Original Paper

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

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Pages 39-50 | Received 25 Apr 2003, Published online: 09 Jul 2009
 

Abstract

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1–M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.

AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; M1, first transmembrane domain; HEK cells, human embryonic kidney cells; α-BTX, α-bungarotoxin; Popen, channel open probability

AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; M1, first transmembrane domain; HEK cells, human embryonic kidney cells; α-BTX, α-bungarotoxin; Popen, channel open probability

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