Subunit-selective contribution of the fourth transmembrane domain of the nicotinic receptor to channel gating

F. GUMILAR; M.C. ESANDI; C. BOUZAT

Buenos Aires, Argentina.

Congreso; ISN/ASN Joint Meeting.; 2001

ISN, ASN, SAN

The muscle nicotinic receptor (AChR) is a pentamer of four different subunits, each one containing four transmembrane domains. Among the transmembrane domains, the fourth segment (M4) is the outermost and the firmest candidate to be in contact with the lipid bilayer. We have recently shown that efficient gating of the AChR depends on a hydrogen bond involving the side chain at position 422 of the alpha subunit (alphaT422). To determine if the equivalent positions in epsilon, delta and beta subunits similarly contribute to channel gating, we mutated deltaT422, betaT422 and epsilonS422, expressed the resulting AchRs in HEK cells and evaluated changes in activation kinetics by single-channels recordings. The mutant epsilonS422A subunit form channels characterized by a significant increase in the duration of the open state when co-expressed with wild-type alpha, beta and delta subunits. The mean open time of these mutant channels is about 8-9 times longer than that of wild-type AchR. In contrast, opening events of AchRs carrying the mutation alphaT422A are about 8-fold briefer than those of the control. Double-mutant AchR channels resulting from the combination of alphaT422A and eS422A subunits are similar to wild-type AchRs. Mutation to alanine of the conserved threonine in either beta or delta subunit does not affect significantly the channel properties. Mutation of each subunit to serine (or threonine in epsilon) which preserves the hydrogen bonding ability of the original amino acid supports nearly normal gating kinetics. Thus, we here delineate subunit-specific contributions of the M4 domain to channel gating.