PHYSIOLOGY OF RECONSTITUTED GAP JUNCTION CHANNELS

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The gap junction is the locus of direct transfer of ions and small molecules from cell to cell. It is composed of an array of protein molecules in the plasma membranes of two abutting cells which form an aqueous channel connecting the cytoplasms of the cells. On the basis of intriguing circumstantial evidence, it is widely held that the intercellular communication mediated by junctional channels must be important in the normal development and mature function of many tissues. Recently it has become clear that the conductance and permeability of the junctional channel can be dynamically regulated by voltage, pH, calcium ions and cyclic nucleotides. Therefore information about how the channel functions, how it is gated, and what goes through it is likely to be of interest beyond its biophysical aspects. The proposal is directed toward the study of junctional channels from rat liver reconstituted in planar lipid bilayers. It builds on (1) past work which characterized the gating properties of junctional channels in cellular membranes using electrophysiological methods, and (2) preliminary experiments in which channels with many of the properties of junctional channels were incorporated into planar bilayers, with isolated gap junctions as the starting material. It is proposed to (a) improve the efficiency of the reconstitution, (b) to study the gating and selectivity of the reconstituted junctional channels, specifically regarding voltage, pH and osmotic effects (c) to test antisera against junctional protein for functional effects on reconstituted channels, ad (d) to incorporate into lipid bilayers the products of in vitro translation of RNA coding for junctional protein. Through such studies of channel physiology, one hopes to understand the mechanisms by which cells can modulate this form of intercellular communication. Coupling by way of gap junctions is so widespread that elucidation of this process will undoubtedly have profound effects in many areas of cellular and developmental biology.