Use of chemical modifications and site-directed mutagenesis to probe the functional role of thiol groups on the gamma subunit of Torpedo californica acetylcholine receptor.
Alkylation of Torpedo californica purified nicotinic acetylcholine receptor (AChR) with N-phenylmaleimide (NPM) under nonreducing conditions led to ion flux inhibition without affecting ligand binding properties [Yee, A. S., Corey, D. E. & McNamee, M. G. (1986) Biochemistry 25, 2110-2119]. The gamma subunit was shown to be preferentially labeled by [3H]NPM with partial labeling of the alpha subunit at higher NPM concentrations. Alkylation occurs at cysteine residues as confirmed by amino acid analysis. Cyanogen bromide peptide mapping of the gamma subunit indicates that at least two residues corresponding to Cys-416, -420, or -451 are labeled. Residues 416 and 420 are part of the proposed amphipathic helix, and the functional role of these two cysteines is further investigated by site-directed mutagenesis of T. californica AChR cDNAs and expression of the mutants in Xenopus laevis oocytes following injection of SP6 transcripts. Several features of SP6 transcripts are shown to be important for efficient translation in vivo. Mutations Cys----Ser gamma 416,420 and Cys----Phe gamma 416 did not perturb either the receptor functional properties or its expression levels. The double mutant Cys----Phe gamma 416,420 displayed a 30% decrease of normalized AChR activity. The relatively small effect of large steric mutations in the amphipathic helix argues against its presence in the tightly packed transmembrane domain of the protein.
Pradier
L
L
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Yee
A S
AS
McNamee
M G
MG
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine; D066298:In Vitro Techniques; D009154:Mutation; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D014101:Torpedo; D014982:Xenopus laevis
D011950:Receptors, Cholinergic; D013438:Sulfhydryl Compounds; D003545:Cysteine