Nmr studies of recombinant active site peptides of the nicotinic acetylcholine receptor

Interactions of ligands with recombinant cholinergic binding sites have been monitored by NMR. Monitoring the selective T₁relaxation of the protons of acetylcholine, nicotine, d-tubocurarine, and gallamine reveals specific binding to peptide constructs containing the a183-204 or shorter sequences of the nicotinic acetylcholine receptor of Torpedo, Human, Chicken, Xenopus, Mouse, Calf, and Drosophila. The trend of the kdvalues of the different ligands shows that the binding of the low molecular weight agonists and antagonists is very weak to the Drosophila sequence which is different from the vertebrate sequences in the N and C terminals. Within the vertebrates, the antagonists d-tubocurarine and gallamine display a kd trend different from that of acetylcholine and a-bungarotoxin. Specificity of binding is proven by the fact that atropine, a muscarinic inhibitor, binds non-specifically. Temperature dependence indicates a fast exchange limit (Ti bound τbound) for gallamine bound to the Torpedo α184-200 sequence. This limit should apply also for the other ligands which have weaker binding constants.