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

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Abstract
A rapid-mixing photolabeling apparatus was 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 ms to seconds time range. Rapid mixing of AcCho and [3H]CPZ with the receptor followed by brief (< 20 ms) UV irradiation results in the selective labeling of the 4 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 .alpha.-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 Kd of 30, 400 and 300 .mu.M 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. Thus, the agonist-dependent rapid association of [3H]CPZ takes place at the level of a site common to all 5 subunits which lies within the ion channel and becomes accessible when the channel opens.

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