The gated connexon channels of gap junction membranes regulate the intercellular communication essential for differentiation and other integrated tissue activities. The cytoplasmic portion of the connexon appears to be flexible, while the transmembrane domain has a relatively invariant structure. Gap junctions are composed of a family of related proteins called connexins which can localize in the same junctional plaque. Determination of the sequences of the connexin proteins and correlation of information on antibody binding, proteolytic cleavage and chemical reactivity have led to a model where the C- and N-termini are located at the cytoplasmic surface and four alpha-helical segments of the protein cross the membrane. Objectives of this proposal are: 1.To obtain electron micrographs of complete and split gap junctions either with antibody or chemical labels or subjected to partial proteolysis. The positions of these labels/protein modifications will be correlated with the amino acid sequence to develop an improved model of gap junction structure. 2.To use the Scanning Transmission Electron Microscope at Brookhaven to record dark field micrographs of gap junction connexon arrays in order to determine the mass of the connexon and the distribution of Connexin32 and Connexin26 in connexons. 3.To obtain a low-resolution three-dimensional reconstruction of the flexible cytoplasmic domains from thin section data. 4.To continue effort to develop better-ordered specimens and improve embedding media for high resolution (> 10 alpha) electron microscopic structure determination.
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