This proposal will study the structure and function of the gap junction, and its role in intercellular communication. Electron image data will be obtained from frozen-hydrated specimens. Dynamic parts of the channel (connexon) involved in channel gating and in connexon-connexon interaction will be characterized by comparative electron microscopy and X-ray diffraction studies under a range of conditions which alter these flexible components. The domains indentified in the connexon structure will be correlated with the amino acid sequence determined from the recombinant DNA. Communication will also be studied using site- specific antisera generated using synthetic oligopeptides which correspond to defined domains within the amino acid sequence predicted from the liver gap junction 32kD cDNA. These antisera will be used for structural mapping of the topology of the gap junction protein by immunocytochemistry of isolated intact and urea-split gap junctions. The antisera will be tested for physiological activity in the AR4-2J pancreas cell line, and in Xenopus oocytes. Ovarian granulosa cDNA libraries will be made from pregnant mare serum gonadotrophin-stimulated rat ovaries prescreened by Northern analysis to demonstrate positive hybridization with the coding region of liver gap junction cDNA. This library will then be screened with the same probe in order to clone the granulosa gap junction cDNA. Synthetic mRNAs generated from liver, ovarian granulosa, and myocardial gap junction cNDAs will be intracellularly injected into Xenopus oocytes which have been manipulated into contact. In these """"""""reconstructed"""""""" systems, channel properties will be measured in voltage-clamped cells, and differences in liver, heart and granulosa channels measured in the context of an invariant oocyte cytoplasm. These differences in physiology will be explored further by attempting to construct liver/granulosa hybrid gap junctions: junctions with tissue-specific proteins apposed across the gap in asymmetrically-injected oocytes. Site-specific and tissue-specific antisera will be used to provide rigorous control of these experiments.
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