STRUCTURE OF BOTH THE LIGAND AND LIPID-DEPENDENT CHANNEL-INACTIVE STATES OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR PROBED BY FTIR SPECTROSCOPY AND HYDROGEN EXCHANGES

FTIR spectra have been recorded both as a function of time and after p rolonged exposure to (H2O)-H-2 buffer in order to study the structural changes that lead to both the ligand- and lipid-dependent channel-ina ctive states of the nicotinic acetylcholine receptor (nAChR). The hydr ogen/deuterium exchange spectra provide insight into both the overall rates and extent of peptide H-1/H-2 exchange and the individual rates and extent to which peptide hydrogens in alpha-helix and beta-sheet co nformations exchange for deuterium. The spectra are also sensitive to the conformation of the polypeptide backbone and thus the secondary st ructure of the nAChR. The various spectral features monitored in the p resence and absence of carbamylcholine and tetracaine are essentially identical, indicating that there are no large net changes in secondary structure in the channel-inactive desensitized state. The various spe ctral features monitored for the nAChR reconstituted into lipid membra nes either with or without cholesterol are very similar, indicating th at cholesterol is not a major structural regulator of the nAChR. Howev er, in the absence of both cholesterol and anionic lipids, there is a slightly enhanced rate of exchange of alpha-helical peptide hydrogens for deuterium that occurs as a result of either an increase in nAChR d ynamics or an increase in the accessibility of transmembrane peptide h ydrogens (H2O)-H-2. The latter may simply be due to an increase in the ''fluidity'' and thus permeability of the lipid bilayers to aqueous s olvent. The results indicate that channel inactivation is due to a ver y subtle change in structure of the nAChR, regardless of whether the i nactive state is stabilized by either prolonged exposure to carbamylch oline or reconstitution into lipid membranes lacking cholesterol and a nionic lipids. The data also illustrate the sensitivity of the amide I band shape to peptide H-1/H-2 exchange. Sample variations in the exte nt of peptide H-1/H-2 exchange can lead to changes in the amide I band that are easily misinterpreted in terms of a change in protein second ary structure.