The long-sterm objective of this work is to elucideate the ways in which integral membrame proteins control ion movements across the plasma membrane of excitable cells. I plan to study this problem by using voltage clamp techniques with patch electrodes. The solution composition on both the extracellular and intracellular membrane surfaces will be controlles in perfused whole cells as well as in excised membrane patches. Ionic channels wil be studied at three levels: macroscopic currents, single channel currents, and sating currents. Since it is believed that channels open, or 'gate', by means of conformational changes, the kinetics of the opening and closing of channels will be studied. It is hoped that the kinetic behavior will provide information about these conformational chenges, especially with regard to the number of kinetically distinct states, the rates of transitions between states, and the voltage dependence of transition rates. The technique of analysis will involve a Markov-chain representation of the kinetic states, and estimation of the stochastic parameters in the model by use of the maximum likelihood method. There is evidence that ions can regulate their own transport by interactions with the satins processes of ionic channels. One hypothesis is that when a channel is occupied by a permeant ion, it cannot close. This hypothesis will be tested rigorously by detailed studies of ion permeation using absolute rate theory models. Specifically the relationship between ion binding in a channel and open-channel lifetime will be examined. The interaction betwwn membrane lipids and ionic channels will also be studied by altering the lipids in the plasma membranes of excitable cells and studying the effects on the satins and permeation properties of ionic channels. Also the lipid-sensitive probes granicidin, alamethicin, and dipicrylamine will be inserted into membrane pathes to study the lipid asymmetry and surface potential of natural membranes. Finally an attempt will be made to study channels in the cell membrane of bacteria, the long-term goal being the use of genetic techniques to elucidate the ways in which channel proteins are regulated. It is hoped that an understanding of the molecular mechanisms underlying excitability will provide clues into the molecular basis for the many psthological conditions which involve the nervous system and neuromuscular transmission.
