Rapid kinetics of agonist binding and permeability response analyzed in parallel on acetylcholine receptor rich membranes from Torpedo marmorata

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Abstract
Excitable acetylcholine receptor rich membrane fragments from T. marmorata were used to measure, in parallel, the permeability response to the fluorescent cholinergic agonist Dns-C6-Cho [6-[5-(dimethylamino)-1-naphthalenesulfonamido]hexanoic acid .beta.-(methobromide) ethyl ester] (in the 0.1 .mu.M to millimolar concentration range) characterized by both the initial rate of Li+ transport and the rate of channel closure using the rapid-mixing quench-flow technique and the kinetics of interaction of Dns-C6-Cho with the acetylcholine receptor sites using the rapid-mixing stopped-flow technique. Analysis of the kinetics of Dns-C6-Cho binding in the millisecond to minute time scale leads to the identification of at least 3 conformational states of the acetylcholine receptor: a low-affinity one (.apprx. 50 .mu.M) that can be interconverted in the fraction of a second to a transient state of intermediate affinity (.apprx. 1 .mu.M) followed by the final stabilization, in the second to minute time range, of a state of high affinity (.apprx. 3 nM). Comparison of Dns-C6-Cho binding data with the permeability response to the same agonist shows that the binding to the low-affinity conformation(s) of the acetylcholine receptor sites coincides with the triggering of the permeability increase (or activation) and the transitions to the intermediate- and high-affinity states with the 2-step process of channel closing or desensitization. The data are interpreted in terms of a minimum 4-state allosteric model for the acetylcholine receptor.

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