Topography of the Acetylcholine Receptor Revealed by Fluorescence Energy Transfer

Abstract

Fluorescence spectroscopy provides a valuable means by which the sites of ligand binding on the acetylcholine receptor can be better defined and related to overall receptor structure. The approach carries the inherent advantage that conformation can be monitored on a functional receptor. Also, fluorescence polarization, lifetimes, quantum yields, and spectral characteristics all yield information on molecular motion, the environment of the site occupied by the ligand and intersite distances on the receptor. We have employed these techniques with fluorescent α-toxins, agonists, reversible antagonists, and noncompetitive inhibitors to describe the topography of the receptor. Fluorescent α-toxins, conjugated at lysine 23, provide the initial frame of reference, and it was found that the α-toxin molecules bind towards the outer perimeter of the receptor. To further localize the agonist/antagonist sites of binding, dansylhexanoylcholine and decidium were employed as ligands. Fluorescence energy transfer between these ligands and the lysine 23-labeled α-toxin again show a substantial intersite distance separating the fluorophores. This is consistent with the agonist/antagonist binding site also being removed from the central axis of the molecule. Finally, ethidium has been employed as a ligand for the noncompetitive inhibitor binding site. Ethidium’s specificity has been established by direct binding measurements, competition with other noncompetitive inhibitors and fluorescence lifetime analysis. We observe a stoichiometry of one ethidium site per receptor where the binding is enhanced by agonist and is completely dissociated by phencyclidine or histrionicotoxin. Lifetime estimates, polarization measurements and wavelength dependences all indicate that ethidium is rigidly immobilized in a hydrophobic crevice. These findings suggest that a likely site for ethidium binding is between hydrophobic segments spanning the membrane portion of the channel rather than associating with in a water-lined channel. Energy transfer measurements also reveal distances between the agonist sites and noncompetitive inhibitor site that are consistent with this locus of binding.