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 non-competitive antagonism at the nAChR.
Highlights
The nicotinic acetylcholine receptor1 from the electric tissue of Torpedo californica is a prototype of the large family of ligand-gated ion channels [1, 2]
In our experiments, treatment of nAChRrich membranes in the absence of agonist with the non-desensitizing cross-linker glutardialdehyde resulted in a population of low affinity binding sites (KD ϭ 0.7 Ϯ 0.2 M) for acetylcholine (Fig. 1A, left panel), whereas after cross-linking in the presence of carbachol binding sites with high affinity for ace
In the conformation resembling the resting state, a minor population (25–35%) of high affinity binding sites was observed, which was explained by a preformed equilibrium existing between the different states of the nicotinic acetylcholine receptor (nAChR) in the absence of ligand
Summary
Materials—Liquid nitrogen-frozen tissue from Torpedo californica was supplied by C. As for [3H]ACh binding assays, the centrifugation technique was used to determine binding of increasing concentrations of NTII to receptor-rich membranes in the native, the desensitized, and the resting state. The affinity state of nAChR-rich membranes was determined after cross-linking with the homo-bifunctional reagent glutardialdehyde (A) in the absence (left panel, open squares) and in the presence of the agonists carbamoylcholine (100 M; left panel, solid squares) or (Ϫ)-nicotine (20 M; right panel, open squares); (B) in the presence of the competitive antagonists gallamine (50 M; left panel) and hexamethonium (100 M; right panel); and (C) in the presence of the non-competitive antagonists ethidium (10 M; left panel) and TPMPϩ (50 M; right panel). Control curves of nAChR-rich membranes fixed in the presence of 100 M carbamoylcholine (solid squares) are shown for determining the maximal number of binding sites. The apparent dissociation constant of non-fluorescent compounds was determined in fluorescence titrations as described by Bixel et al [30]
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