MUTATIONS AT LIPID-EXPOSED RESIDUES OF THE ACETYLCHOLINE-RECEPTOR AFFECT ITS GATING KINETICS

The firmest candidate among the transmembrane portions of the nicotini c acetylcholine receptor (AChR) to be in contact with the lipid bilaye r is the fourth segment, M4. To explore the contribution of alpha M4 a mino acid residues of mouse AChR to channel gating, we combined site-d irected mutagenesis with single-channel recordings. Two residues in al pha M4, Cys418 and Thr422, were found to significantly affect gating k inetics when replaced by alanine. AChRs containing alpha C418A and alp ha T422A subunits form channels characterized by a 3- and 5-fold reduc tion in the mean open time, respectively, suggesting an increase in th e closing rate due to the mutations. The calculated changes in the ene rgy barrier for the channel closing process show unequal and coupled c ontributions of both positions to channel gating. Single-channel recor dings of hybrid wild-type alpha/alpha T422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substituti on of threonine to alanine changes the energy barrier of the closing p rocess by similar to 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair c hannel opening. Both Cys418 and Thr422 have been postulated to be in c ontact with the lipid milieu and are highly conserved among species an d subunits. Our results support the involvement of lipid-exposed resid ues in alpha M4 in AChR channel gating mechanism.