Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label
- Washington Univ. School of Medicine, St. Louis, MO (United States)
The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.
- OSTI ID:
- 5028959
- Journal Information:
- Biochemistry; (United States), Vol. 30:28; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NICOTINE
RADIORECEPTOR ASSAY
RECEPTORS
MOLECULAR STRUCTURE
ACETYLCHOLINE
LIGANDS
TRITIUM COMPOUNDS
ALKALOIDS
AMINES
AMMONIUM COMPOUNDS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZINES
AZOLES
DRUGS
ESTERS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
MEMBRANE PROTEINS
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASYMPATHOLYTICS
PARASYMPATHOMIMETICS
PROTEINS
PYRIDINES
PYRROLES
PYRROLIDINES
QUATERNARY COMPOUNDS
TRACER TECHNIQUES
550601* - Medicine- Unsealed Radionuclides in Diagnostics