Connexin 43 (Cx43), a constituent protein of gap junctions, is expressed in cardiomyocytes, astrocytes, kidney and liver epithelium, uterine muscle, vascular endothelium and numerous other cell types. Hexamers of Cx43 (connexons) expressed in the plasma membranes constitute hemichannels (GJH) and the association of connexons from neighboring cells forms gap junctions (GJC) that establish cell-to-cell communication. GJC are essential for organ development and function (prominently in the heart) and GJH may be involved in normal cell function, as well as cell injury and death. This makes the function of Cx43 of great importance in health and disease. Phosphorylation of Cx43 has been shown to alter GJC as well as GJH function. The long-term goal of our research is to understand the biophysical, molecular and structural bases of the function and regulation of Cx43 hemichannels and channels. The central hypothesis to be tested in this project is that PKC-mediated phosphorylation of Cx43 produces a large change in conformation that results in association of the C-terminal (CT) domain with other domains, eventually decreasing the effective diameter of the permeation pore, and that partial phosphorylation of Cx43 connexons elicits graded changes in solute permeability.
Our Specific Aims are to: (1) Identify the subdomains and specific amino acid residues involved in the response of Cx43 GJH to phosphorylation by PKC. These studies should define the regions of the molecule that are essential for the functional effects. (2) Determine the movements of the CT domain that result in decreased large-solute permeability in response to phosphorylation. Our general hypothesis is that phosphorylation produces a conformational change in the CTD, causing its association with the IL, and so decreases the cross-sectional area of the permeation pathway. (3) Determine the role of PKC-mediated phosphorylation of individual connexin molecules on the changes in solute permeability of Cx43 GJH. We will use flux measurements and electrophysiology to assess GJH and GJC function, molecular biology techniques, spectroscopy and determinations of changes in intramolecular distances. The latter will be done by resonance energy transfer. These studies are expected to contribute new and significant information on the structure-function relationship of Cx43 GJC and GJH, as well as -because of structural commonality- those of other connexin isoforms.
