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

• Published in: Molecular pharmacology Vol. 69; no. 2; pp. 452 - 461
• Main Authors: Mourot, Alexandre, Rodrigo, Jordi, Kotzyba-Hibert, Florence, Bertrand, Sonia, Bertrand, Daniel, Goeldner, Maurice
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.02.2006
• Subjects: Animals; Diazonium Compounds - chemistry; Diazonium Compounds - pharmacology; Models, Molecular; Nicotinic Agonists - chemistry; Nicotinic Agonists - pharmacology; Oocytes - drug effects; Phenylurea Compounds - chemistry; Phenylurea Compounds - pharmacology; Photoaffinity Labels - chemistry; Protein Conformation; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - drug effects; Torpedo; Torpedo californica; Torpedo marmorata
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.105.017566

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 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 [ 3 H]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.