The purpose of this research is to understand better the design principles of cell membrane channels and the mechanisms by which they regulate ion flux across membranes. The techniques used will include cryo-electron microscopy and crystallographic methods of three-dimensional structure analysis; systematic efforts will be continued to obtain crystals suitable for x-ray diffraction studies. The findings will furnish three-dimensional framework for relating the extensive data now being obtained from biochemical, pharmacological, genetic and physiological Two of the best characterized channels will be investigated as model systems: (a) The acetylcholine receptor. We plan to extend our electron microscopical analyses of the tubular crystals grown from isolated postsynaptic membranes. Our first steps have been to determine the structure of the receptor at low resolution, and to establish the identity of the individual subunits. Now, by incorporating practical developments in the microscopy and the data processing, we will analyze details of individual subunits on a finer scale, and evaluate different conformational states. Preliminary three-dimensional maps have been obtained revealing differences between resting and desensitized states. Pilot experiments have been initiated to investigate the open state. (b) The gap junction channel. We plan to investigate further the structure and function of the gap junction channel, using the plaques isolated from rat liver and the extensive membrane sheets recently derived by over-expression of the cDNA encoding the human liver polypeptide in yeast. Our first step has been to determine the quaternary structure of this protein. We also found that Ca2+ induces a conformational change, suggesting a plausible mechanism by which the channel opens and closes. Work now aims to describe those parts which are instrumental in facilitating the conformational change. Features of the amino acid sequence appear to fit closely the structural details and have led to a testable model for the folding of the polypeptide chain. The transmembrane portion appears to be a four alpha-helical bundle, which we plan to resolve directly by incorporating improvements in the cryo- electron microscopy technology and utilizing the membranes derived from the yeast.
