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 Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect 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 altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.
Lee
Y H
YH
Section of Molecular and Cellular Biology, University of California, Davis 95616.
Li
L
L
Lasalde
J
J
Rojas
L
L
McNamee
M
M
Ortiz-Miranda
S I
SI
Pappone
P
P
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005260:Female; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D014101:Torpedo; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D007473:Ion Channels; D011950:Receptors, Cholinergic; D014364:Tryptophan; D003545:Cysteine; D000109:Acetylcholine