STRUCTURE, FUNCTION, AND REGULATION OF GAP JUNCTION PROTEINS

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Gap junctions are membrane specializations which permit the direct exchange of ions and small molecules between adjacent cells in many tissues. In myocardium, electrical coupling through gap junctions permits propagation of the action potential and the synchronized contraction of myocyte. During tissue injury, such as the observed in ischemia, an increase in the resistance across junctions may serve to localize damage to the affected site. Thus, gap junctions play a central importance role in intracellular signal transduction an elucidation or their structure and function is of crucial importance to understanding cardiac intercellular communication in both normal and pathophysiologic states. A cDNA encoding the major myocardial gap protein, designated connexin 43 (Cx43), has been identified and there is considerable homology between the extracellular an transmembrane domains of connexins from different tissues. However, the cytoplasmic domains of connexins from different sources are not homologous and may represent unique regulatory sites. In the present proposal, a combination of morphological, biochemical, molecular biological and electrophysical approaches will be used to study gap junction structure and function. Specifically, we will: determine the role of the cytoplasmic domain of Cx43 in regulation of channel function by a variety of second messengers and protein kinase systems; determine the nature of interaction between adjacent cells by probing the extracellular domains of Cx43; and determine some of the sequences important in assembly of functional channels in the membrane. To achieve these goals, both normal, and a series of mutant cDNA's encoding Cx43 will be expressed in the Xenopus laevis oocyte. A variety of electrophysiological, immunochemical and biochemical techniques will be used to evaluate their functional properties.