Fast kinetic studies on the interaction of cholinergic agonists with the membrane-bound acetylcholine receptor from Torpedo marmorata as revealed by quinacrine fluorescence.

Abstract

Rapid changes of fluorescence intensity take place after fast mixing of quinacrine‐labelled receptorrich membrane fragments from Torpedo marmorata with cholinergic effectors and are recorded in the time range of 1 – 1000 ms, accessible to the stopped‐flow technique. Agonists such as acetylcholine, phenyltrimethylammonium, carbamylcholine, suberyldicholine or choline cause the change, but not antagonists such as d‐tubocurarine, flaxedil or Naja nigricollisα‐toxin. In addition, preincubation of the membrane fragments with N. nigricollisα‐toxin blocks the response to agonists. Ceruleotoxin, a toxin from Bungarus ceruleus venom which blocks the postsynaptic response to acetylcholine at a site distinct from the α‐toxin site, causes a decrease of the apparent amplitude of the fast signal. In the presence of 0.05 mM tetram, an acetylcholinesterase inhibitor, the kinetics of the fast response to acetylcholine do not change; in the second or minute range and in the case of acetylcholine, however, a decrease of fluorescence intensity occurs in the absence of acetylcholinesterase inhibitor showing that the fast fluorescence signal is reversible.Under the present experimental conditions the observed fast kinetics do not vary with the concentration of receptor sites or quinacrine but do vary with the concentration of agonist. The data can be analyzed in terms of the phenomenological reaction scheme , when k2»k3, if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4= (1.5 ± 0.5) s−1, k3= (60 ± 40) s−1, k2≥ 5 × 102 s−1 and k1≥ 107l·mol−1 s−1, K1=k2/k1= (7 ± 3) × 10−5 M, K=K1·k4/k3= (2 ± 1) × 10−6M; for carbamylcholine: k4= (0.2 ± 0.1) s−1, k3= (10 ± 5) s−1, k2× 102 s−1 and k1≥ 3 × 105 s−1, K1= (3 ± 1) × 10−4 M and K= (4 ± 3) × 10−6 M. The values of the apparent dissociation constants determined from the variation of the amplitudes of the fast effect for acetylcholine and carbamylcholine are respectively (1.0 ± 0.5) × 10−6 M and (5 ± 2) × 10−6 M. Some variability in the absolute values of the constants is noticed from one preparation to another and, for a given preparation, changes with age. Sets of kinetic constants have also been determined in the case of choline and suberyldicholine. At 20 °C, the Q10 is about 2.5 in the presence of 0.03 mM carbamylcholine and 1.3 in the presence of 0.01 M carbamylcholine; it is 1.3 with 0.05 M acetylcholine.The data are interpreted in terms of a three‐state model and their physiological significance is discussed.