Structural reorganization of the acetylcholine binding site of the Torpedo nicotinic receptor as revealed by dynamic photoaffinity labeling

We explored the structural changes that occur at the acetylcholine binding site of the Torpedo marmorata nicotinic receptor during activation by the tritiated photoactivatable agonist (diazocyclo-hexadienoylpropyl)trimethylommonium ([H-3]DCTA). We quantified the incorporation, of radioactivity into the receptor subunits as a function of the mixing time of [H-3]DCTA with the receptor by using a rapid-mixing,device adopted with a photochemical quenching system. A saturable increase of the specific photolobeling on the a and gamma subunits was observed with a half-time of about 2 minutes. We further analyzed this photoincorporation either after rapid mixing for 500 ms or after equilibration for 50 minutes. Under these conditions, [H-3]DCTA explored transient state(s) and the stable desensitized state, respectively. Comparative analyses showed that at a probe concentration of 10mum the relative variation of photoincorporation was more pronounced for the gamma subunit (three-to fourfold) than for the a subunit (about twofold). By contrast the relative distribution of radioactivity among a-subunit labeled residues (alpha7yr190, alphaCys192, alphaCysC793, and alphaTyr198) did not change. Altogether, these results reveal that during the course of agonist-induced receptor desensitization, the site-fining peptide loops, which belong to adjacent alpha and gamma subunits, move closer to each other.