Kinetics of agonist-induced intrinsic fluorescence changes in membrane-bound acetylcholine receptor.

Abstract

ACETYLCHOLINE receptor (AChR)-rich membranes derived from fish (Electrophorus and Torpedo) electric organs have been used extensively for in vitro binding and functional studies1. These membrane fragments (microsacs) are particularly suited for physical investigations of AchR–ligand interactions because of their inherently high content of receptor, retained in its natural environment. A number of extrinsic fluorescent probes have been applied to the determination of binding specificity, equilibria and, in a preliminary way, kinetics at the level of the isolated membrane, as well as the neuromuscular junction and purified receptor1–6. We report here the existence of intrinsic protein fluorescence changes, induced in membrane fragments from Torpedo marmorata electroplaques by the natural neurotransmitter acetylcholine. We have used highly sensitive fluorescence kinetic techniques to obtain the concentration dependence and some of the corresponding equilibrium and rate constants for the initial binding and subsequent isomerisation reactions. The latter can be correlated with electrophysiological data and the functional properties of the post-synaptic membrane7–9, particularly with respect to the process of desensitisation, and with other quantitative results obtained with the same microsac preparations10. In particular, our data provide physical evidence for the existence of at least three conformational states of the cholinergic receptor protein in situ, interrelated by reversible kinetic processes.