The major objectives of this proposal are to measure the electrostatic potential at the surface of cellular membranes and to relate the measured values of the potential to the structure of the charged residues at the membrane surface. Most of the charge at the surface of cellular membranes is due to sialic acid residues in sialoglycolipids (gangliosides) and sialoglycoproteins. The sialic acid residues can extend 10-100 A from the surface of the cell, which reduces their effect on the surface potential; for example, the magnitude of the zeta potential of erythrocytes is approximately one-third of the value calculated from the total surface charge density using the Gouy-Chapman-Stern theory. The effect of the "delocalization" of charge on the surface potential will be investigated in phospholipid membranes reconstituted with purified gangliosides (GM1 GDa and GT1), where the sialic acid residues can extend up to 15A from the surface, and purified glycophorin A (the major sialoglycoprotein in the erythrocyte membrane), where the sialic acid residues can extend up to 100 A from the surface. The techniques developed for these model systems will then be used to measure the electrostatic potential at the extracellular face of the erythrocyte ghost membrane. 31P and 1H NMR techniques will be used to measure the electrostatic potential at specific sites on the membrane surface, e.g., the phosphodiester group and the choline trimethylammonium group: these techniques take advantage of the effects of very low concentrations of paramagnetic divalent cations on the NMR signals from these groups. The structure of saccharide residues at the surface of membranes will be investigated using phosphatidyl choline membranes reconstituted with GM1 specifically deuterated in the terminal galactose residue. 2H NMR techniques will be used to study the extent and the rate of internal motion in the saccharide residues, and neutron diffraction techniques will be used to determine how far the saccharide residues extend from the surface of the membranes. The surface potential measurements and the structural measurements will provide the first experimental test of three recent theories that attempt to relate the electrostatic potential to the structure of the charged residues at the cell surface. The long-term goal of this research is to understand quantitatively the role of electrostatic surface potentials in cellular functions; e.g., the effect of surface potentials on the ionic conductances of membranes of excitable pells, and the role of surface potentials in cell-cell interactions.