Role of Connexin43 in Heart Function

描述(由申请人提供)：我们的长期目标是：a)确定心脏缝隙连接调节的分子机制，b)确定这一调节对心脏功能的影响。我们以前的研究集中在连接蛋白43(Cx43)的结构基础上，连接蛋白43是最丰富的心脏缝隙连接蛋白。利用外源表达系统，我们已经证明了Cx43(Cx43CT)的羧基末端区域作为调节域。在这里，我们提出：a)Cx43CT结构域的完整性对于调节天然心脏缝隙连接是必不可少的；b)这种调节在缺血事件后特定的形态和电生理变化中起关键作用。心脏缝隙连接调节的研究由于缺乏合适的实验模型而受到阻碍，在该模型中CT结构域的功能可以直接和特异性地改变。最近，Willecke实验室开发了一种“敲除/敲入”小鼠品系，其中野生型Cx43的基因编码被一种缺少大部分CT结构域的截断形式所取代。这些小鼠向我们展示了第一个直接证明Cx43CT在心脏生理学中的作用的生物系统。具体地说，我们将描述Cx43CT结构域在以下方面的作用：1)心脏缝隙连接的生物物理特性和pH门控。2.低pH值或全脑缺血时Cx43的内化。3.成年小鼠脑室对低pH和缺血反应的电生理行为。最后，我们将确定CT片段在活着的有机体环境中作为一个独立结构域的能力。总体而言，这些研究将为我们理解心脏缝隙连接调节的分子机制以及它们在健康和疾病中对心脏功能的作用提供基础数据。

DESCRIPTION (provided by applicant): Our long-term goals are: a) to characterize the molecular mechanisms of cardiac gap junction regulation and b) to identify the consequences of this regulation on the function of the heart. Our previous studies have centered on the structural bases for the regulation of Connexin43 (Cx43), the most abundant cardiac gap junction protein. Using exogenous expression systems, we have shown that the carboxyl terminal region of Cx43 (Cx43CT) acts as a regulatory domain. Here, we propose that: a) the integrity of the Cx43CT domain is essential for the regulation of native cardiac gap junctions and b) this regulation plays a key role in specific morphological and electrophysiological changes that follow an ischemic event. Studies on cardiac gap junction regulation have been hindered by the lack of an appropriate experimental model where the function of the CT domain can be directly and specifically altered. Recently, the Willecke laboratory developed a "knock-out/knock-in" mouse line where the gene coding for wild-type Cx43 was replaced with a truncated form that lacks most of the CT domain. These mice present us with the first biological system to document directly the role of Cx43CT in the physiology of the heart. Specifically, we will characterize the role of the Cx43CT domain on: 1) the biophysical properties and pH gating of cardiac gap junctions. 2. The internalization of Cx43 in response to low intracellular pH (pHi) or global ischemia. 3. The electrophysiological behavior of adult murine ventricle in response to low pHj and ischemia. Finally, we will determine the ability of the CT fragment to act as an independent domain within the environment of a living organism. Overall these studies will offer data fundamental to our understanding of the molecular mechanisms of regulation of cardiac gap junctions and their role in heart function in health and disease.