Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics

BOUZAT, C.; ROCCAMO, A.M.; GARBUS, I.; BARRANTES, F.J.

MOLECULAR PHARMACOLOGY

AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS

Año: 1998 vol. 54 p. 146 - 153

0026-895X

The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of aM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in aM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing aC418A and aT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type a/aT422A AChR show that the closing rate depends on the number of a subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the  closing process by ;0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in aM4 in AChRchannel gating mechanism.