CARDIAC GAP JUNCTIONS--CONTROL OF EXPRESSION AND FUNCTIO

Gap junction channels provide a mechanism for the regulated transport of ions and small molecules from cell to cell. Alterations in gap junction channel activity have been implicated in cardiovascular developmental defects and disorders of cardiac impulse generation and propagation. Gap junction channel proteins are encoded by the connexin multi-gene family, consisting of at least twelve distinct members. Within this family of genes, connexin43 is the most prominent in the cardiovascular system. Connexin43 is post-translationally modified by phosphorylation, and recent evidence of our own and of others has demonstrated that phosphorylation profoundly affects the functional properties of the assembled gap junction channel. Based upon our considerable progress in understanding electrophysiological properties of rodent and human connexin43 channels, the overall goal of this application is to gain mechanistic insight into the relationship between connex43 phosphorylation and channel function. Using a tightly focused combination of cellular electrophysiological and molecular genetic approaches, the Specific Aims of this proposal are to: 1. Undertake detailed electro[physiological studies evaluating the effects of phosphorylating and dephosphorylating agents on human and rodent connexin43 in stably transfected mammalian cells. 2. Examine the role of specific phosphorylatable residues in modifying channel function, through site-directed mutagenesis and structure-function analysis. 3. Transiently express phosphorylation-site mutants in wild-type and connexin43-null neonatal mouse cardiac myocytes and examine the electrophysiological phenotype. 4. Target phosphorylation-site mutants to the hearts of wild-type and connexin43-null mice, and evaluate the junctional channel properties in dissociated cardiac myocytes. We fully expect that this approach, where transfected cell lines are used to identify gating properties and effects of phosphorylation-site mutagenesis, and transiently transfected and transgenic myocytes are used to study the impact of these mutations in the cardiac cell environment, will provide new insight into both mechanisms and consequences of connexin43 phosphorylation and channel function.