We propose a multidisciplinary approach for the study of the functional organization of cardiac gap junctions at the molecular level. The protein has been isolated and a partial amino acid sequence obtained. We have also isolated undegraded heart total poly(A)+ RNA with which to build libraries. Purified heart gap junctions containing the native protein as well as synthetic peptides modeled on the sequence will be used to produce antibodies. With these and synthetic oligonucleotide probes based on the known amino acid sequence, we will search an expression library for clones expressing the junction protein. Further protein sequencing will yield data with which to prepare other oligonucleotides and site-directed antibodies. Clone(s) containing cDNA encoding the full length of the gap junction protein will be sequenced. The deduced amino acid sequence will then serve to build models of the organization of the junction protein in the membranes. A combination of specific proteolytic cleavages and reactivity with site-directed antibodies will be used to test these models. The models will permit us to examine physiologically the role of different regions of the molecule in cell-cell signaling and exchanges. In vitro mutagenesis should allow us to explore the consequences of modifications to the junction protein at specific locations. This work should provide data permitting the rational design of approaches to the treatment of heart diseases in which defects in conduction may be involved.
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