Characterization of the Transient Agonist-Triggered State of the Acetylcholine Receptor Rapidly Labeled by the Noncompetitive Blocker [³H]Chlorpromazine: Additional Evidence for the Open Channel Conformation

The kinetics of covalent labeling of the α, β, γ, and δ chains of the acetylcholine receptor (AcChR) from Torpedo marmorata by the noncompetitive blocker [³H]chlorpromazine ([³H]CPZ) are investigated by using rapid mixing photolabeling techniques. In an initial study [Heidmann, T., & Changeux, J. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1897–1901], it was shown that the rate of [³H]CPZ labeling increases 100-1000-fold upon simultaneous addition of nicotinic agonists to the AcChR and that prior addition of these agonists abolishes the effect. The data were interpreted in terms of the rapid labeling of the transient active state of the AcChR where the ion channel is in its open configuration. This interpretation was recently challenged [Cox, R. N., Kaldany, R. R. J., Di Paola, M., & Karlin, A. (1985) J. Biol. Chem. 260, 7186–7193] on the ground of studies with a different noncompetitive blocker, [³H]quinacrine azide, and the suggestion was made that this compound labels the rapidly desensitized closed channel conformation of the AcChR. In this paper it is shown that the rate of rapid labeling of the AcChR by [³H]CPZ decreases to negligible values upon exposure of the AcChR to nicotinic agonists, in the 100-500-ms time range. The absolute values of the rate constants of this decrease (10–15 s^-1 for saturating concentrations of acetylcholine and carbamoylcholine) and their variation with agonist concentration (apparent dissociation constants of 40 μM and 0.4 mM for acetylcholine and carbamoylcholine, respectively) are those expected for the rapid desensitization of the AcChR. In addition, it is shown that at variance with what is observed with [³H]quinacrine azide, the rate of [³H]CPZ rapid labeling increases linearly with CPZ concentration, without saturation. The data support the conclusion that, as previously suggested, the state of the AcChR that [³H]CPZ rapidly labels is not the rapidly desensitized but the active conformation where the ion channel is open and to which [³H]CPZ binds in a diffusion-controlled manner.