DESENSITIZATION OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR MAINLY INVOLVES A STRUCTURAL-CHANGE IN SOLVENT-ACCESSIBLE REGIONS OF THE POLYPEPTIDE BACKBONE

The difference between infrared spectra of the nicotinic acetylcholine receptor (nAChR) recorded using the attenuated total reflectance tech nique in the presence and absence of carbamylcholine exhibits a comple x pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon agonist binding and subsequent desensitization. Two relatively intense bands are obse rved in the amide I region of the difference spectra recorded in (H2O) -H-1 buffer near 1655 cm(-1) and 1620 cm(-1) that were previously inte rpreted in terms of either a net conversion of beta-sheet to alpha-hel ix or a reorientation of transmembrane alpha-helix accompanied by a ch ange in structure of beta-sheet and/or turn [Baenziger, J. E., Miller, K. W., & Rothschild, K. J. (1993) Biochemistry 32, 5448-5454], Howeve r, difference spectra recorded in (H2O)-H-2 buffer reveal that these a nd other difference bands in the amide I region undergo downshifts in frequency upon peptide H-1/H-2 exchange that are much larger than the downshifts in frequency that are typically observed for the amide I vi brations of either alpha-helix or beta-sheet. Difference spectra recor ded in (H2O)-H-2 buffer within either minutes or hours of prior exposu re of the nAChR to (H2O)-H-2 exhibit the same amide I difference band shifts that are observed in difference spectra recorded after 3 days p rior exposure of the nAChR to (H2O)-H-2. Most Of the peptides that are involved in both ligand binding and the resting to desensitized confo rmational change and that give rise to bands in the difference spectra therefore exchange their hydrogens for deuterium on the seconds to mi nutes time scale, The frequencies of the difference bands, the magnitu des of the difference band shifts upon peptide H-1/H-2 exchange, and t he rapidity of the hydrogen deuterium exchange kinetics of those struc tures that give rise to amide I bands in the difference spectra all su ggest that the formation of a channel-inactive desensitized state resu lts predominantly from a conformational change in solvent-accessible e xtramembranous regions of the polypeptide backbone as opposed to a lar ge structural perturbation near the ion channel gate. A conformational change in the agonist binding site may be primarily responsible for c hannel inactivation upon desensitization.