Gap junctions are low resistance channels that in excitable tissues provide a pathway for impulse propagation and synchronous activation; in inexcitable cells their role is presumably in exchange of metabolites or signalling molecules. Considerable preliminary information is available (largely funded by this grant in prior years) on both short and longterm regulation of gap junctions. We intend to combine techniques of molecular biology, immunology and electrophysiology in order to study expression of gap junction protein and gating of gap junctions between cells of several mammalian tissues. Pairs of isolated adult and cultured neonatal cells as well as various cell lines, will be used as experimental models. In addition we shall use methods of recording from gap junction membranes isolated from rat hepatocytes. An antibody has been obtained which blocks coupling between cell pairs from several tissues and has demonstrated reactivity against gap junction protein. Using monospecific polyclonal antibodies, relative levels of junctional protein will be determined by immuno-precipitation and immunoblot techniques and compared to junctional conductance (gj) under a variety of conditions expected to affect expression. We will also correlate covalent protein modifications (including phosphorylation) on gj, junctional permeability, and their sensitivity to gating stimuli. These experiments should finally begin to answer questions of how gap junctions are regulated and what role they play in propagation of electrical and metabolic activity in physiological and pathological states.
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