The broad aim of this research is to understand better the design principles of ion channels and of the mechanisms by which they regulate the flow of ions across cell membranes. The techniques used will include cryoelectron microscopy and crystallographic methods of structure analysis, which we will develop further to obtain higher resolution information. We are also attempting to grow three-dimensional crystals of ion channels suitable for X-ray diffraction studies. The findings from these structural approaches will furnish a three-dimensional framework for relating the extensive data now being obtained by site-directed mutagenesis experiments combined with physiological study of function. Two of the best characterized channels will be investigated as model systems: (a) The acetylcholine receptor. We will extend our earlier studies of the tubular crystals grown from Torpedo postsynaptic membranes. Our first steps have been to determine the identity of the five transmembrane subunits, the shape of the ion pathway, and the overall quaternary structure. Now, by incorporating practical developments in the microscopy and image processing we plan to obtain a description of the structure at higher resolution (8-9A), sufficient to reveal the precise arrangement of alpha-helices in the transmembrane portion of the channel. We will use this map as a basis for comparison with the structure in the open state, which we are attempting to trap by rapidly freezing the tubes within milliseconds of applying the agonist. (b) The gap junction channel. We will extend earlier studies of gap junctions isolated from rat liver in which we analysed the quaternary structure of this channel and suggested a possible mechanism by which it opens and closes. To this end, we are overexpressing the human liver gene in insect cells with a baculovirus vector and are now purifying the recombinant channels have been obtained, which should be suitable for electron crystallographic studies at higher resolution. The recombinant channels are also being used for three- dimensional crystallization trials; some small microcrystals have been obtained.
