Mechanisms of Gap Junction Regulation

描述（由申请人提供）：连接蛋白是一种完整的膜蛋白，其寡聚形成细胞间隙连接通道。离子和小分子通过这些通道在细胞间扩散，使单个细胞事件同步成为整个器官的功能反应。间隙连接介导至关重要的过程，如电脉冲传播、细胞生长调节和器官发育。此外，间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。虽然有大量关于连接蛋白在调节间隙连接通道中的关键相互作用的信息，但导致通道关闭和降解的确切机制尚未确定，也没有完全表征所涉及的关键辅助蛋白。如果要充分了解正常和患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获得对调节间隙连接机制的结构和功能理解。该项目的目的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间隙连接蛋白connexin43 （Cx43）结构的分子内和分子间相互作用。本研究的中心假设是，Cx43羧基末端（Cx43CT）残基Y265-A305作为一个主要的调控结构域，在适当的条件下（例如，细胞内酸化和/或磷酸化），与孔附属的“受体”（即Cx43细胞质环（Cx43CL））结合，关闭通道，然后与参与其降解的分子伙伴结合。ph介导的Cx43调节的研究是重要的，因为细胞内酸化导致间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间隙连接的关闭和降解可能是心肌缺血和梗死期间恶性室性心律失常的原因之一。该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由于疾病和缺血损伤而改变的间隙连接通讯。为了研究这一概念，我们提出了以下具体目标：1)定义c-Src如何介导Cx43间隙连接的关闭，2)确定Cx43降解中涉及的分子相互作用，3)确定可以调节连接通信的分子。

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.