Probing the reorganization of the nicotinic acetylcholine receptor during desensitization by time-resolved covalent labeling using [3H]AC5, a photoactivatable agonist.

The structural reorganizations occurring on the nicotinic acetylcholine receptor (nAChR) during activation and subsequent desensitization have been investigated through time-resolved photoaffinity labeling using a photoactivatable nicotinic agonist. [(3)H]AC5 is a photosensitive nicotinic probe with high affinity for the desensitized state of the Torpedo marmorata receptor (K(D) = 5 nM) that displays full agonist activity on the Torpedo californica receptor expressed in oocytes (EC(50) = 1.2 microM). Photoaffinity labeling of this receptor in the desensitized state showed a predominant specific labeling of gamma and delta subunits, whereas the alpha subunit was barely labeled. Using a stopped-flow device combined with a flash photolysis quenching system, we investigated the covalent mapping of the subunits as a function of incubation time of the receptor with [(3)H]AC5 (17 ms-1.25 h). During agonist-induced desensitization, specific labeling increased substantially, with similar time constants for gamma and delta subunits (0.016 s(-1)), whereas labeling of the alpha subunit remained relatively low. Therefore, the repartition of radioactivity shifted during desensitization from a weak but predominant labeling of the alpha and gamma subunits toward a substantial labeling of gamma and delta subunits. The observed time-dependent labeling pattern together with AC5 docking into a homology model of the T. californica nAChR suggest a subunit reorganization during agonist-induced desensitization, leading to a tightly packed arrangement that corresponds to a stable high affinity state for agonists.