Conformational effects of lipids and local anesthetics on the nicotinic acetylcholine receptor.

Abstract

The mechanisms by which both lipids and local anesthetics modulate the structure and function of the nicotinic acetylcholine receptor (nAChR) from Torpedo have been investigated using the technique of Fourier transform infrared (FTIR) difference spectroscopy. With respect to lipids, the results reveal that reconstitution of the nAChR into a highly fluid egg phosphatidy1choline (EPC) membrane stabilizes the receptor in a channel-inactive desensitized state. The addition of a variety of neutral and/or anionic lipids to the EPC membrane all induce an essentially identical pattern of vibrational changes in the nAChR suggesting that the presence of diverse lipid structures within a reconstituted membrane acts to stabilize the receptor in a channel-competent resting state. In addition, increasing levels of either the neutral lipid, cholesterol (Chol) or the anionic lipid, dioleoylphosphatidic acid (DOPA) within an EPC membrane increasingly stabilizes a larger proportion of nAChR's in a conformation(s) that is (are) capable of undergoing agonist-induced desensitization, although only high levels of DOPA were found to stabilize the receptor in a fully functional resting state. The results have been interpreted in terms of a speculative model that describes the mechanisms by which lipids modulate nAChR function. This model suggests that the composition of the lipid membrane surrounding the nAChR influences the conformational equilibrium of the receptor between the resting, desensitized and a novel intermediate state through an indirect effect on some physical property of the lipid bilayer, possibly bulk fluidity. The model also proposes that anionic lipids, in addition to Proper membrane fluidity, are required to stabilize the nAChR in a fully functional resting conformation. With respect to local anesthetics, the results support the current model of local anesthetic action at the nAChR and suggest that the binding of local anesthetics stabilize the receptor in a desensitized conformation. Contrary to the model, however, the FTIR data suggest that receptor desensitization occurs not through interactions at a distinct noncompetitive blocker site, but solely as a consequence of local anesthetic binding to the neurotransmitter binding sites. The data also suggest that the binding of local anesthetics to the non-competitive blocker site stabilizes the nAChR in an intermediate conformation similar to that stabilized upon reconstitution of the nAChR into EPC membranes containing low concentrations of either Chol or DOPA. Additional studies employing the local anesthetic, proadifen and the hallucinogen, phencyclidine hydrochloride (PCP) support these findings and show conclusively that both local anesthetics and PCP bind to the non-competitive blocker site and stabilize the nAChR in a conformation that is structurally distinct from both the resting and desensitized states.