Rapid Kinetics of Agonist Binding and Permeability Response Analyzed in Parallel on Acetylcholine Receptor Rich Membranes from Torpedo marmorata

Thierry Heidmann, Julius Bernhardt, Jean Pierre Changeux, Julius Bernhardt, Eberhard Neumann

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Abstract

Excitable acetylcholine receptor rich membrane fragments from Torpedo marmorata have been used to measure, in parallel, (1) the permeability response to the fluorescent cholinergic agonist Dns-C6-Cho (in the 0.1 µM 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 (2) 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 three conformational states of the acetylcholine receptor: a “low-affinity” one (∼50 µM) that can be interconverted in the fraction of a second to a transient state of “intermediate affinity” (∼1 µM), followed by the final stabilization, in the second to minute time range, of a state of “high affinity” (∼3 nM). Comparison of Dns-C6-Cho binding data with the permeability response to the same agonist demonstrates 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 two-step process of channel closing—or “desensitization”. The data are interpreted in terms of a minimum four-state “allosteric” model for the acetylcholine receptor.

Original languageEnglish
Pages (from-to)5452-5459
Number of pages8
JournalBiochemistry
Volume22
Issue number23
DOIs
Publication statusPublished - 1 Jan 1983
Externally publishedYes

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