The broad aim of this research is to understand better the design principles of ion channels and of the mechanisms by which they regulate ion transport across cell membranes. The techniques used will include electron crystallographic methods of structure determination, which will be developed further to analyze rapid conformational changes and to obtain higher resolution information. The applicant will also attempt to grow three-dimensional crystals of ion channels suitable for X-ray diffraction studies. The finding from these structural approaches will furnish three-dimensional frameworks for relating the extensive data now being obtained by site-directed mutagenesis experiments combined with electro-physiological study of function, and should provide direct insight into how the channels work. Three well characterized channels will be investigated: (a) The acetylcholine receptor. Dr. Unwin will extend his studies of the tubular crystals grown from Torpedo postsynaptic membranes, from which the receptor structure has now been determined at a resolution of 9 A in both the closed- and (very recently) open-channel forms. He will determine the structure of the receptor at 9 A resolution in the desensitized state, to derive a complete description of the conformational changes occurring at this resolution. At the same time, by further developments in the microscopy and image processing, he will work towards obtaining a more detailed description of the structure in the closed-channel form (resolution: 4-5 A). (b) The gap junction channel. Dr. Unwin will extend earlier studies of gap junctions isolated from rat liver in which he analyzed the quaternary structure of this channel and suggested a possible mechanisms by which it opens and closes. Recent developments in image processing methods, applied to two-dimensional sheets, are enabling him now to explore their three- dimensional structures at 9 A resolution, rather than 18 A resolution as was done previously. As an alternative approach, he is also overexpressing the human liver gene in insect cells with a baculovirus vector and is now purifying the recombinant channels to homogeneity in milligram quantities. Extensive crystalline sheets of the channels have been obtained, which may eventually be suitable for crystallographic studies at higher resolution. (c) Voltage-gated potassium channels. A study of voltage-gated Shaker potassium channels has been initiated with the long-term goal of understanding the structural changes that take place in response to changes in voltage, in analogy with the experiments being performed on the ligand-gated ion channels. The applicant is interested to know if the voltage-gated channels open and close by similar, or completely unrelated, mechanisms.
