Gap junctions provide aqueous channels between cells which are permeable to ions and small molecules. These junctions transmit impulses between excitable cells, but are common in many inexcitable tissues where their role is ill defined. In the latter, they may transmit signalling molecules and mediate sharing of metabolites. Gap junction proteins, now termed connexins, have been shown to comprise a family of several members encoded by distinct, but homologous genes. Specific nucleotide probes and antibodies are allowing classification of which connexin is expressed by which cell, and permit analysis of regulation at the levels of transcription, translation and degradation. Gating of single gap junction channels can be detected in appropriate cells. This Program Project, an extension of our previous Program, will use these new probes and techniques to study gap junctions in respect to connexin type, gating, regulation and function. Project 1 Physiological Properties and Regulation of Gap Junctions in Non-neuronal Cells will utilize several tissues in which these questions have been little studied. Preliminary data suggest functionally significant modulation. Project 2 Gap Junctions In the Nervous System will study neurons and glia in culture, in particular under conditions in which gap junctions are regulated by hormones and trophic factors. Regional differences and specificity of junction formation are major foci. Project 3 Exogenous of Expression of Gap Junction Proteins will utilize exogenous mRNAs, naturally occurring and modified, for expression in Xenopus oocytes and transfection into cell lines. How molecular modification changes junctional properties is a major focus as is alteration in cell behavior by specifically introducing or blocking expression of gap junctions. A third facet will be introduction of normal or modified proteins into artificial bilayers for study of single channel properties. Project 4 Pinealocyte Gap Junctions: Characterization, Regulation and Function extends to these cells the same techniques as applied in Projects I and 2. Pinealocytes uniquely express a particular connexin in isolation, and although they are neurosecretory, they appear not to employ exocytosis; thus the role of their gap junctions in secretion may be unusual. Project 5 Classical and Molecular Genetic Approach to Gap Junctions will develop antibody probes for gap junctions in Drosophila and C. elegans as tools in subsequent analysis. These gap junctions, often inaccessible in situ, will be studied by incorporation into planar bilayers or by expression in Xenopus oocytes. New strategies are proposed for isolating gap junction genes. Core A Administrative provides vital research services. Core B Cytological provides an essential morphological link between electrophysiological measurements of coupling and biochemical determination of protein levels. Immunocytochemistry with LM resolution often can quantify incidence of gap junctions, and gap junction antigen can be localized with EM resolution.
