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

在细胞表面形成可渗透通道的基本蛋白质单位称为连接蛋白。连接蛋白通道的开放允许生物颗粒在细胞的内部和外部之间或在两个附着细胞的内部之间循环。在本研究中，我们感兴趣的连接蛋白的一个特定的变体称为连接蛋白43（Cx43）。Cx43通道的门控（打开和关闭）可以调节。与细胞表面上的许多其他通道一样，调节的主要手段是通过通道单位的磷酸化来实现的。 一种称为蛋白激酶C（PKC）的酶介导Cx43的磷酸化。它作用于Cx43单位的精确位点，并诱导Cx43形成的通道关闭。迄今为止，至少有12种已鉴定的PKC亚型。然而，这些不同亚型在调节Cx43通道中的功能作用的表征在很大程度上受到缺乏亚型选择性激活剂和抑制剂的限制。在这里，我们使用新开发的PKC亚型选择性激活剂和抑制剂肽的矩阵，结合国家的最先进的膜片钳技术，研究各种PKC亚型发挥的作用，在调节Cx43通道。这将通过测量在各种肽存在下从表达Cx43通道的细胞记录的电流来实现。我们还将使用分子生物学技术来消除Cx43上潜在的磷酸化位点，以评估它们参与PKC对Cx43通道的调节。最后，我们将合成模拟Cx43上磷酸化位点的小蛋白片段，并研究它们在PKC激活后对Cx43通道特性的影响。

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.