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. [3H]AC5 is a photosensitive nicotinic probe with high affinity for the desensitized state of the Torpedo marmorata receptor (KD = 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 γ and δ subunits, whereas the α 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 γ and δ subunits (0.016 s–1), whereas labeling of the α subunit remained relatively low. Therefore, the repartition of radioactivity shifted during desensitization from a weak but predominant labeling of the α and γ subunits toward a substantial labeling of γ and δ 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.

Journal article

Probing the Reorganization of the Nicotinic Acetylcholine Receptor during Desensitization by Time-Resolved Covalent Labeling Using [3H]AC5, a Photoactivatable Agonist, Molecular Pharmacology 2006 69 (2), 452-461