Nicotinic acetylcholine receptor probed with a photoactivatable agonist: Improved labeling specificity by addition of Ce-IV/glutathione. Extension tolaser flash photolabeling

The molecular structure of Torpedo marmorata acetylcholine binding sites ha s been investigated previously by photoaffinity labeling. However, besides the nicotine molecule [Middleton et al. (1991) Biochemistry 30, 6987-6997], all other photosensitive probes used for this purpose interacted only with closed receptor states. In the perspective of mapping the functional activ ated state, we synthesized and developed a new photoactivatable agonist of nAChR capable of alkylation of the acetylcholine (ACh) binding sites, as re ported previously [Kotzyba-Hibert et al. (1997) Bioconjugate Chem. 8, 472-4 80]. Here, we describe the setup of experimental conditions that were made in order to optimize the photolabeling reaction and in particular its speci ficity. We found that subsequent addition of the oxidant eerie ion (Ce-IV) and reduced glutathione before the photolabeling step lowered considerably nonspecific labeling (over 90% protection with d-tubocurarine) without affe cting the binding properties of the ACh binding sites. As a consequence, ir radiation at 360 nm for 20 min in these new conditions gave satisfactory co upling yields (7.5%). A general mechanism was proposed to explain the succe ssive reactions occurring and their drastic effect on the specificity of th e labeling reaction. Last, these incubation conditions can be extended to n anosecond pulsed laser photolysis leading to the same specific photoincorpo ration as for usual irradiations (8.5% coupling yield of ACh binding sites, 77% protection with carbamylcholine). Laser flash photocoupling of a diazo cyclohexadienoyl probe on naAChR was achieved for the first time. Taken tog ether, these data indicate that future investigation of the molecular dynam ics of allosteric transitions occurring at the activated ACh binding sites should be possible.