Binding Properties of Agonists and Antagonists to Distinct Allosteric States of the Nicotinic Acetylcholine Receptor Are Incompatible with a Concerted Model

Abstract

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein.Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying noncompetitive antagonism at the nAChR. The nicotinic acetylcholine receptor (nAChR)1 from the electric tissue of Torpedo californica is a prototype of the large family of ligand-gated ion channels (1, 2). It is an allosteric protein (3, 4) that can exist in at least three distinct, yet interconvertible conformational states (5): In the resting state the receptor has low affinity for acetylcholine and the ion channel is closed. Binding of two agonist molecules in a positively cooperative manner triggers gating of the intrinsic ion channel, which in turn leads to the permeation of cations. Prolonged agonist exposition induces receptor desensitization (6). In the desensitized conformation the nAChR has an increased affinity for acetylcholine as compared with the resting state, but the ion channel is closed. Each of these allosteric states is characterized by a distinct protein conformation.The structure of the nAChR meets basic prerequisites of allosteric regulatory proteins: First, it is an oligomer formed by five subunits with the stoichiometry ␣ 2 ␤␥␦. Second, given the homology of the subunits (7) and considering the fact that the neuronal ␣7-subunits can form functional homomeric receptors, one can assume that these subunits are arranged in a pseudo-symmetric manner. Indeed, in the pentamer the ␣-helical transmembrane segments M2 of each subunit contribute to a central ion channel (8), whereas the residual segments are oriented toward the plasma membrane or neighboring subunits. Third, the binding sites for agonists and competitive antagonists are located at the interfaces between neighboring subunits, i.e. in the case of Torpedo and of muscle-type nAChR an ␣-subunit and the adjacent ␥...

Competitive Antagonism between the Nicotinic Allosteric Potentiating Ligand Galantamine and Kynurenic Acid at α7* Nicotinic Receptors

“…The finding that KYNA-induced inhibition of choline-evoked whole-cell currents in perforated patches could be reversed after washing of the neurons supported the concept that loss of diffusible intracellular components contributes to the long-lasting effect of KYNA on type IA currents in conventional patch-clamp recordings. Diffusion of ATP-regenerating compounds in conventional patch-clamp recordings is known to stabilize a nonconducting state of the ␣7* nAChRs in hippocampal neurons (Alkondon et al, 1994), and small ligands are known to interact differently with the various conformational states of muscle nAChRs (Krauss et al, 2000). Thus, one could speculate that the interactions of KYNA with its sites on ␣7* nAChRs are dependent on the conformational state of the receptors.…”

Allosteric modifiers of neuronal nicotinic acetylcholine receptors: New methods, new opportunities

“…20 This domain can be viewed as a cation selector in which NCIs bearing a positive charge, usually on an amine moiety, are trapped and directed down the channel by an electrostatic gradient. 32 While NCIs can bind at a variety of sites along the central lumen, it is generally assumed that the non-competitive inhibition is predominately a result of binding at site(s) near or at the V ring, position 15, which essentially plugs the channel and blocks ion flux 33 ( Fig. 1).…”

Non-Competitive Inhibitory Activities of Morphinan and Morphine Derivatives at the α3β4 Neuronal Nicotinic Acetylcholine Receptor Determined Using Nonlinear Chromatography and Chemometric Techniques

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