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-milli-seconds to seconds time range. Rapid mixing of AcCho and [3H]CPZ with the receptor followed by brief (less than 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 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 dissociation constants of 30 microM, 400 microM, and 300 microM 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.
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002746:Chlorpromazine / Q000378:metabolism*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010777:Photochemistry; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology
D007473:Ion Channels; D011950:Receptors, Cholinergic; D002217:Carbachol; D000109:Acetylcholine; D002746:Chlorpromazine; D014403:Tubocurarine