Structural differences in the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor revealed by time-resolved fluorescence spectroscopy

The nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata carries two nonequivalent agonist binding sites at the alpha delta and alpha gamma subunit interfaces. These sites have been characterized by time-resolved f luorescence with the partial nicotinic agonist dansyl-C-6-choline (Dnscho). When bound to the detergent-solubilized receptor, the fluorescence Lifetim e distribution of Dnscho displays a characteristic signature, with four sep arable components at 0.2, 1.8, 7.2, and 18.3 ns, respectively. Competition experiments with the antagonist d-tubocurarine (dTC), known to bind prefere ntially to the alpha gamma site, result in substantial changes of this sign ature, associated with a strong decrease in average fluorescence lifetime. Comparisons with two other competitive antagonists, alpha-conotoxin M1 and alpha-bungarotoxin, demonstrate that Dnscho binds with a similar affinity t o the two sites but that the microenvironment of the probe is different for each site. Using a two-site binding model together with published equilibr ium constants to describe the competitive binding of dTC and Dnscho, we rea ch a satisfactory description of the changes in fluorescence lifetimes and propose characteristic fluorescence parameters of the probe bound to each t ype of site. This analysis indicates that Dnscho at the alpha delta site is principally associated with a 8.7 ns lifetime, while it has a 20.2 ns majo r lifetime at the alpha gamma site. Therefore, the observed fluorescence he terogeneity arises in large part from the structural differences of the two binding sites. As a result, this signal can be used to identify the bindin g preferences of competitive ligands of unknown pharmacology.