Mechanisms of Gap Junction Regulation

描述(申请人提供)：连接蛋白是一种完整的膜蛋白，可通过寡聚作用形成细胞间缝隙连接通道。离子和小分子通过这些通道在细胞间扩散，允许单个细胞事件同步进入整个器官的功能反应。缝隙连接介导了至关重要的过程，如电脉冲传播、细胞生长调节和器官发育。此外，缝隙连接蛋白的突变与各种遗传性疾病有关，包括神经系统疾病、耳聋、白内障、心脏缺陷和皮肤病。虽然关于缝隙连接通道调控中连接蛋白的关键相互作用有相当多的信息，但导致通道关闭和降解的确切机制还没有被确定，涉及的关键辅助蛋白也没有被完全表征。如果要充分了解正常和疾病状态下细胞间通讯的作用，这一信息是至关重要的。我们工作的长期目标是对调节缝隙连接的机制有一个结构性和功能性的了解。该项目的目标是使用多学科的方法来研究在pH介导的门控和降解过程中定义主要心脏缝隙连接蛋白Cx43(Cx43)结构的分子内和分子间相互作用。这项研究的中心假设是，Cx43羧基末端(Cx43CT)残基Y265-A305作为一个主调节域，在适当的条件下(例如细胞内酸化和/或磷酸化)与与孔道相连的“受体”(即Cx43细胞质环(Cx43CL))结合，关闭通道，然后与参与通道降解的分子伙伴结合。PH介导的Cx43调节的研究具有重要意义，因为细胞内酸化导致缝隙连接的关闭和降解，是组织缺血的主要后果。特别是，酸化诱导的Cx43缝隙连接的关闭和降解可能是心肌缺血和梗死时恶性室性心律失常的原因之一。这项拟议研究的基本原理是，更好地理解Cx43调控的结构基础将导致更好的策略来调节因疾病和缺血损伤而改变的缝隙连接通讯。为了研究这一概念，提出了以下具体目标：1)确定c-Src如何介导Cx43缝隙连接的关闭；2)确定参与Cx43降解的分子相互作用；3)识别能够调节连接通讯的分子。

DESCRIPTION (provided by applicant): Connexins are integral membrane proteins that oligomerize to form intercellular gap junction channels. Ions and small molecules diffuse intercellularly through these channels, allowing individual cell events to synchronize into the functional response of an entire organ. Gap junctions mediate vitally important processes such as electrical impulse propagation, regulation of cell growth, and organ development. Moreover, mutations in a gap junction protein are linked to various inherited diseases, including nervous system disorders, deafness, cataracts, heart defects, and skin diseases. While there is considerable information regarding key interactions of connexins in the regulation of gap junction channels, the precise mechanisms that lead to channel closure and degradation have not been defined, nor have the critical accessory proteins involved been fully characterized. This information is pivotal if the role of intercellular communication in normal and diseased states is to be fully understood. The long-term goal of our work is to gain a structural and functional understanding of the mechanisms regulating gap junctions. The objective of this project is to use a multi-disciplinary approach to investigate intra- and intermolecular interactions that define the structure of the major cardiac gap junction protein connexin43 (Cx43) during pH-mediated gating and degradation. The central hypothesis for the proposed research is that Cx43 carboxyl terminal (Cx43CT) residues Y265-A305 act as a master regulatory domain that, under the appropriate conditions (e.g., intracellular acidification and/or phosphorylation), binds to a "receptor" (i.e., Cx43 cytoplasmic loop (Cx43CL)) affiliated with the pore to close the channel and then to molecular partners involved in its degradation. The study of pH-mediated Cx43 regulation is significant because intracellular acidification, which leads to closure and degradation of gap junctions, is a major consequence of tissue ischemia. In particular, acidification-induced closure and degradation of Cx43 gap junctions may be one of the causes for malignant ventricular arrhythmias during myocardial ischemia and infarction. The rationale for the proposed research is that a better understanding of the structural basis of Cx43 regulation will lead to better strategies to modulate gap junction communication that has been altered due to disease and ischemia injury. The following Specific Aims are proposed to investigate this concept: 1) To define how c-Src mediates closure of Cx43 gap junctions, 2) To determine the molecular interactions involved in Cx43 degradation, and 3) To identify molecules that can regulate junctional communication.