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

单个蛋白激酶 C 亚型在连接蛋白 43 未并列半通道调节中的功能作用

• 批准号: 341794-2007
• 负责人: Baroudi, Ghayath
• 金额: $ 1.38万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=429865
• 关键词: Functional; role; individual; protein; kinase

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.

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。连接蛋白通道的打开允许生物颗粒在细胞的内部和外部之间或两个附着细胞的内部之间循环。在目前的研究中，我们对连接蛋白的一种特定变体connexin43 （Cx43）感兴趣。Cx43通道的门控（开闭）是可以调节的。像细胞表面的许多其他通道一样，调节的主要手段是通过通道单元的磷酸化来实现的。一种叫做蛋白激酶C （PKC）的酶介导Cx43的磷酸化。它作用于Cx43单元上的精确位点，诱导Cx43形成的通道关闭。到目前为止，至少有12种已确定的PKC亚型。然而，由于缺乏同种异构体选择性激活剂和抑制剂，这些不同同种异构体在Cx43通道调节中的功能作用的表征在很大程度上受到限制。在这里，我们使用新开发的PKC异构体-选择性激活剂和抑制剂肽的基质，结合最先进的膜片钳技术，研究各种PKC异构体在Cx43通道调节中的作用。这将通过测量在各种多肽存在下表达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。