MUTATIONS IN THE M4 DOMAIN OF TORPEDO-CALIFORNICA ACETYLCHOLINE-RECEPTOR DRAMATICALLY ALTER ION-CHANNEL FUNCTION

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys 447 in the M4 domain of Torpedo californica acetylcholine receptor exp ressed in Xenopus laevis oocytes. The M4 region is a transmembrane dom ain thought to be located at the lipid-protein interface. By whole-cel l voltage clamp analysis, mutation of both a subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC(50) for acetylcholine from 32 mu M to 13 alpha M. Patch measur ements of single-channel currents revealed that the alpha Trp418 incre ased channel open times similar to 28-fold at 13 degrees C with no eff ect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is a ltered by the mutation. Our results show that changes in protein struc ture at the putative lipid-protein interface can dramatically affect r eceptor function.