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 nigricollis cx-toxin. In addition, preincubation of the membrane fragments with N . nigricollis a-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 a-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 A + B AB 3 A*B, when k, % k,, if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4=

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

“…Early rapid mixing experiments using snake 3 H-labeled ␣-toxin as a probe and receptorrich membranes from T. marmorata revealed changes in conformation that took seconds to reach a high affinity state, possibly desensitized, from a low affinity resting state (87). Consistent findings were subsequently reported using muscle cells (88) and Torpedo membranes (89,90,107,108). A refined kinetic analysis of the binding interaction of the fluorescent nicotinic agonist dansyl-C6-choline with receptor-rich membranes (91,92,109) and correlation with the in vitro measurement of ion transport through the ion channel (93) resulted in the demonstration of allosteric transitions between several conformational states: a resting closed channel state (R) stabilized by snake ␣-toxin and nicotinic antagonists; an active transient open channel state with low affinity for ACh and nicotinic agonists (A), and at least one desensitized and slowly accessible refractory state (D) with high affinity for both agonists and antagonists (in addition to a fast desensitized state (I)) (Fig.…”

Reconstitution of a Functional Acetylcholine Receptor

“…It was proposed that the 66000-M, chain was the subunit responsible for this dimerization [3,39,56,79]. In our hands, the result of the reconstitution was never shown to depend on the fraction of receptor protein in a dimeric state.…”

“…In a previous series of experiments [16] performed with the receptor-rich membranes [2], it was shown that, after equilibration with the fluorescent local anesthetic quinacrine, carbamylcholine triggered an increase of quinacrine fluorescence emission under conditions of energy transfer from proteins (see also [39]) ; moreover, histrionicotoxin caused a decrease of the fluorescence signal both in the presence and in the absence of carbamylcholine. These experiments have been repeated with the 'reconstituted' acetylcholine receptor (Fig.…”

“…Binding [8, 36-381 and spectroscopic [39] experiments have lead to the distinction of two main categories of pharmacologically significant sites on the receptor-rich membranes : the acetylcholine receptor site and the binding site for non-competitive blocking agents. Aminated local anesthetics and histrionicotoxin enhance the equilibrium binding of cholinergic agonists [8,37,38,40,41] and accelerate the slow transition of the receptor towards its high-affinity state for agonists (reviewed in [41,42]).…”

“…Aminated local anesthetics and histrionicotoxin enhance the equilibrium binding of cholinergic agonists [8,37,38,40,41] and accelerate the slow transition of the receptor towards its high-affinity state for agonists (reviewed in [41,42]). Conversely, cholinergic agonists potentiate the binding of the noncompetitive blockers [38,39] to their high-affinity site. Conclusively, allosteric interactions are established between these two classes of sites and may serve as a reliable index to monitor the functional state of the acetylcholine receptor [43 -451.…”