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

G SPITZMAUL,; J CORRADI,; CECILIA BEATRIZ BOUZAT

MOLECULAR MEMBRANE BIOLOGY

Taylor & Francis Healthsciences

Año: 2004 vol. 21 p. 39 - 50

0968-7688

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an a2bed composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15’ of the M1 domain is phenylalanine in a subunits while it is isoleucine in non-a 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 a subunit (aF15’I) as well as those containing the reverse mutations in the non-a subunits (bI15’F, dI15’F, and eI15’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 b subunit being the one that produces the most significant stabilization of the open state. Kinetic analysis of bI15’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 bI15’ 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 b15’ 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.