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

Journal Article · Tue Aug 08 00:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00442a006· OSTI ID:5443344

Pradier, L; Yee, A S; McNamee, M G ^[1]

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1. Univ. of California, Davis (USA)

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. The {gamma} subunit was shown to be preferentially labeled by ({sup 3}H)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 {yields} Ser{gamma}416,420 and Cys {yields} Phe{gamma}416 did not perturb either the receptor functional properties or its expression levels. The double mutant Cys {yields} 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.

• OSTI ID: 5443344
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:16; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
ALKYLATION
AMINES
AMINO ACIDS
AMMONIUM COMPOUNDS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOLOGICAL EFFECTS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CYSTEINE
DRUGS
ESTERS
GENETIC EFFECTS
HYDROGEN COMPOUNDS
HYDROXY ACIDS
IMIDES
LABELLING
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
MUTAGENESIS
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASYMPATHOMIMETICS
PHENYLALANINE
PROTEINS
QUATERNARY COMPOUNDS
RECEPTORS
SERINE
STEREOCHEMISTRY
THIOLS
TRITIUM COMPOUNDS