MECHANISMS OF CELL MEMBRANE SIGNALS

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Mechanisms of transmembrane signaling, particularly non-excitable electromagnetic responses of vertebrate cells to external stimuli, are to be studied. Recording of single molecular channel conductances in situ in cell membranes and also as reconstituted into liposomes is to be used to discover and to measure properties of known and anticipated channels. Micropipette gigaohm seals are to be used for whole vesicle recording for direct measurements of currents generated by reconstituted pumps. Electro-optical image enhanced video microscopy with digital image analysis are to be used with flourescent indicators to search for electrogenic responses in nonexcitable cells, to measure spatial distributions of membrane potentials and ligands on surface receptors, and to detect signals associated with surface receptor cross linking, electromigration in applied electrochemical fields and in endocytotic processes. Technical developments designed to enhance the power of fluorescence micrography for investigation of previously inaccessible properties of transmembrane signaling will be pursued. Fluorescent membrane potential pH and calcium ion indicating dyes will be applied to mapping of the spatial distribution of membrane potentials and ion activities in differentiated cells. Large lipid vesicles bearing reconstituted protein channels will be manipulated in order to develop morphologies suitable for whole cell patch recording, as well as our presently available single channel recording. New strategies to take advantage of our simultaneous sensitive optical and electrical recording capability in cell biology will be explored.