In heart, gap junctions serve the crucial role of electrically coupling muscle fibers to ensure rapid propagation of synchronous contractions. However, the identification of multiple connexins in heart (i.e., Cx37,40,43,45,46) clearly indicates a greater complexity. We and others have demonstrated that intracellular channels comprised of some of these connexins show distinctive gating properties and conductance. Heterotypic interactions between connexins can further modify these properties. This diversity of connexins in heart could then confer subtle (and possibly regional) regulation of electrical conductance or could reflect specializations for additional roles such as metabolic coupling or second messenger transmission. We propose to analyze the nature of the specialized properties conferred by these different connexins and their potential role in cardiac function. Initially, the distribution of these different junctional proteins will be established using both in situ hybridization with highly specific nucleotide probes and antibodies raised to peptides from the deduced sequences in complementary studies on rat and bovine heart sections. Concurrent analyses using the Xenopus oocyte expression system will be aimed at defining the specific properties of intercellular channels comprised of different connexins. Using paired Xenopus oocytes injected with cRNAs of the connexins of interest, two major groups of experiments are proposed. First, the gating properties of the various connexins in response to voltage, pH and Ca++ will be determined using dual cell voltage clamps. Possible heteromeric interactions between connexins and their effects on channel properties will also be investigated. Relating these results to the in vivo distribution of connexins may indicate the manner in which gap junctions modulate the flow of current in the heart. A second series of studies will investigate the passage of larger molecules through junctions. These studies have significance for second messenger responses in heart. In the laboratory of Dr. David Triggle, we will develop a graded series of probes which will allow the determination of exclusion limits and selectivity of junctions comprised of each of the cardiac connexins and their potential hybrid forms. By modifying these probes with respect to surface charge and hydrophobicity, and incorporating photoactivatable cross-linking groups, the nature of the residues lining the channel which may contribute to specificity will also be studied. Site-directed mutagenesis of channel residues will further refine our understanding of the structure of these channels and the molecular basis of any selectivity filters that may be detected. These studies will define the properties of intercellular communication in the heart at a molecular level, so that the roles of junctional proteins in electrical conductance and as regulators of excitability can be addressed.
