Functional role of individual protein kinase C isoforms in the modulation of connexin 43 unapposed hemichannels

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The basic protein units that form the permeable channels on the cell surface are called connexins. The opening of connexin channels allow for biological particles to circulate between the interior and the exterior of a cell or between the interiors of two attached cells. In the present study, we are interested in a specific variant of connexins called connexin43 (Cx43). The gating (opening and closing) of Cx43 channels can be regulated. Like many other channels on the cell surface, the primary mean of regulation is achieved by phosphorylation of the channel units. An enzyme, called protein kinase C (PKC), mediates the phosphorylation of Cx43. It acts at precise sites on the Cx43 unit and induces the closure of Cx43-formed channels. There are, at least, twelve identified isoforms of PKC so far. However, the characterization of the functional role of these various isoforms in the regulation of Cx43 channels has largely been limited by the lack of isoform-selective activators and inhibitors. Here, we use a matrix of newly developed PKC isoforms-selective activator and inhibitor peptides, in combination of the state-of-the-art patch clamp technique, to study the effect that various PKC isoforms exert in the regulation of Cx43 channels. This will be achieved by measuring electrical currents recorded from cells expressing Cx43 channels in the presence of various peptides. We will also use molecular biology techniques to eliminate potential phosphorylation sites on Cx43 in order to evaluate their involvement in the regulation of Cx43 channels by PKC. Finally, we will synthesize small protein fragments that mimic the phosphorylation sites on Cx43 and investigate their effects on Cx43 channel properties following activation of PKC.