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Probing the Reorganization of the Nicotinic Acetylcholine Receptor during Desensitization by Time-Resolved Covalent Labeling Using [³H]AC5, a Photoactivatable Agonist

Laboratoire de Chimie Bioorganique, Unité Mixte de Recherche (UMR) 7514 Centre National de la Recherche Scientifique (CNRS) (A.M., F.K.-H. M.G.) and Laboratoire de Bioinformatique du Médicament, UMR 7081 CNRS (J.R.), Faculté de Pharmacie, Université Louis Pasteur Strasbourg, Illkirch, France; and Département de Neurosciences, Centre Médical Universitaire, Faculté de Médecine, Geneva, Switzerland (S.B., D.B.)

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. [³H]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 (EC₅₀ = 1.2 µM). Photoaffinity labeling of this receptor in the desensitized state showed a predominant specific labeling of and subunits, whereas the 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 [³H]AC5 (17 ms–1.25 h). During agonist-induced desensitization, specific labeling increased substantially, with similar time constants for and subunits (0.016 s^–1), whereas labeling of the subunit remained relatively low. Therefore, the repartition of radioactivity shifted during desensitization from a weak but predominant labeling of the and subunits toward a substantial labeling of and 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.

Address correspondence to: Dr. Alexandre Mourot, Max Planck Institut für Biophysik, Max-von-Laue str.3, 60 438 Frankfurt am Main, Germany. E-mail: alexandre.mourot{at}mpibp-frankfurt.mpg.de