Interaction of the Nicotinic Acetylcholine Receptor with Ligands and Membrane Lipids Studied by Fourier-Transform Infrared Spectroscopy and Photoaffinity Labeling

Monitoring of the amide I band by Fourier-transform infrared spectroscopy (FT-IR) is a valid and flexible approach to monitor changes in the secondary structure of reconstituted Acetylcholine Receptor (AcChR). The continuous exposure of the AcChR to a cholinergic agonist (carbamylcholine), which drives the AcChR into the desensitized state, produces only minor changes in AcChR secondary structure. Nevertheless, carbamylcholine alters the AcChR tertiary or quaternary structure, as indicated by the increased thermal stability of the protein assessed from the temperature-dependence of the infrared spectrum. On the contrary, presence of a competitive cholinergic antagonist (d-tubocurarine) produces no detectable effects on AcChR structure. Cholesterol or the neut-al lipids present in asolectin extracts produce an ordering of the AcChR secondary structure observed by FT-IR and also enable the protein to increase its thermal stability in response to carbamylcholine. These effects of cholesterol or asolectin neutral lipids seem mediated by a direct interaction of all the AcChR subunits with the lipids, as suggested by labeling of the AcChR by a photoactivatable cholesterol analogue. The interaction between the AcChR and the cholesterol analogue is sensitive to AcChR desensitization since the presence of carbamylcholine during photolysis decreases the extent of labeling and alters the labeling stoichiometry in the AcChR subunits. This suggests the occurrence of an agonist-induced change in the arrangement of the transmembrane portion of the desensitized protein, which is consistent with the agonist-induced alteration of the AcChR protein tertiary or quaternary structure detected by FT-IR.