Kinetics of agonist-induced intrinsic fluorescence changes in the Torpedo acetylcholine receptor

Abstract

The nicotinic acetylcholine receptor from Torpedo electric organs is a ligand-gated ion channel that undergoes conformational transitions for activation and/or desensitization. Earlier work suggested that intrinsic fluorescence changes of the receptor monitors kinetic transitions toward the high-affinity, desensitized state. Here, using highly purified membrane preparations to minimize contaminating fluorescence, we examined kinetic mechanisms of the receptor as monitored by its intrinsic fluorescence. Fluorescence changes were specific to the receptor as they were blocked by alpha-bungarotoxin and were induced by agonists, but not by the antagonist hexamethonium. Acetylcholine, carbamylcholine and suberyldicholine showed only one kinetic phase with relatively fast rates (t(1/2) = 0.2-1.2 s). Effective dissociation constants were at least an order of magnitude higher than the high affinity, equilibrium binding constants for these agonists. A semirigid agonist isoarecolone-methiodide, whose activation constant was approximately 3-fold lower than acetylcholine, induced an additional slow phase (t(1/2) = 4.5-9 s) with apparent rates that increased and then decreased in a concentration dependent manner, revealing a branched mechanism for conformational transitions. We propose that the intrinsic fluorescence changes of the receptor describe a process(es) toward a fast desensitization state prior to the formation of the high affinity state.

A novel pharmacological activity of caffeine in the cholinergic system

“…a-BTX alone induced no change in the K D of CrV for the R state, suggesting that the blockage of both ACh binding sites by this toxin is not detectable by CrV. Using the measurement of the AChR intrinsic fluorescence as a strategy to monitor AChR conformational changes towards its desensitized state, Kawai and Raftery (2010) (Kawai and Raftery, 2010) reported that in the presence of a-BTX alone no significant AChR intrinsic fluorescence changes were observed, in agreement with our results. Furthermore, analyzing the pattern of incorporation of the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[ 125 I] iodophenyl)diazirine ([ 125 I]TID) into AChR, Moore and McCarthy (1994) (Moore and McCarthy, 1994) concluded that a-BTX increased/stabilized the amount of AChR in the R state.…”