george eisenman, md - US grants

Continuation of Grant GM 24749 is requested for experimental and theoretical studies on the selective ion permeation and gating through artificial peptide channels across lipid bilayer membranes and biological channels in excised patches of cell membranes to provide a foundation for understanding, at a molecular level, the ion selective permeation mechanisms of nerve. For artificial channels, we propose to continue studies on selectivity among monovalent cations for the Gramicidin A channel, extending these to theoretical investigations of multi-site, multi-barrier single filing models and experiments designed to distinguish between alternative models. These studies should extend the basis laid by Hille and Armstrong for using cations as "probes" of the structure of the Na ion and K ion channels of nerve, extending this to the selectivity filters as well as the bindin sites. For biological channels, we propose to characterize ion permeation and gating mechanisms of Na and acetylcholine-activated channels of nerve and muscle membranes using the excised patch technique. We will study details of the ion selectivity of the channels and construct barrier models consistent with the experimental observations. We will particularly emphasize effects on divalent cations on the permeability and open-lifetime.

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.

The International Union for Pure and Applied biophysics will hold its General Assembly and 10th International Congress in Vancouver, Canada, July 29-August 3, 1990. It is proposed that the Biophysical Society administer a travel grant program offering a limited number of travel awards to qualified U.S. scientists who may require such assistance, with preference to young investigators and invited participants. The U.S. National Committee for the International Union of Pure and Applied Biophysics (USNC/IUPAB) of the National Academy of Sciences will announce the award program, distribute applications, and receive the completed applications. Responsibility for screening travel grant applications will be vested in a Committee of the Biophysical Society chosen by the Council of the Society or its Executive Committee from a slate of candidates provided by the USNC/IUPAB. The Biophysical Society and the USNC/IUPAB feel that qualified U.S. scientists should be assisted to attend this Congress to facilitate exchange of recent significant developments and dissemination of biophysical research data long before it could appear in journal form. In order to facilitate the maximum number of U.S. scientists in receiving travel awards, the Biophysical Society has agreed to manage this program without assessing an overhead charge.

The International Union for Pure and Applied Biophysics will hold its 10th International Biophysics Congress and General Assembly in Vancouver, Canada, July 29 to August 3, 1990. The meeting offers a broad program of symposia on topics of current biophysical interest such as: 1) membrane organization and function; 2) macromolecular structure function recognition; 3) biophysical techniques; and 4) cell and bioenergetics. Scientists of international reputation have been selected to serve as invited participants. In addition, poster sessions will provide for the latest research results to be discussed in detail. This meeting provides the opportunity for U.S. scientists to meet outstanding foreign scientists, to present their work to an international audience and to, perhaps, begin important international collaborations.