Time-resolved photolabeling by the noncompetitive blocker chlorpromazine of the acetylcholine receptor in its transiently open and closed ion channel conformations

A rapid-mixing photolabeling apparatus is developed to resolve the kinetics of association of the noncompetitive channel blocker [³H]chlorpromazine (CPZ) with the membrane-bound acetylcholine (AcCho) receptor from Torpedo marmorata and to photolabel its subunits in the 100-milliseconds to seconds time range. Rapid mixing of AcCho and [³H]CPZ with the receptor followed by brief (<20 msec) UV irradiation results in the selective labeling of the four chains of the AcCho receptor, according to a rapid bimolecular association process close to diffusion-controlled. Rapid association is not observed with the competitive antagonists d-tubocurarine or flaxedil or the snake venom α-toxins. Its initial rate increases with agonist concentration, with maxima of 0.6 for carbamoylcholine and 0.2 for phenyltrimethylammonium taking 1 for AcCho, with apparent dissociation constants of 30 μM, 400 μM, and 300 μM for AcCho, carbamoylcholine, and phenyltrimethylammonium, respectively, and with sigmoid shape (Hill coefficients of 1.1-1.3). Under conditions in which the receptor 'desensitizes' and the ionic channel closes (preincubation with AcCho), rapid [³H]CPZ association decreases in parallel. It is concluded that the agonist-dependent rapid association of [³H]CPZ takes place at the level of a site common to all five subunits, which lies within the ion channel and becomes accessible when the channel opens.