Mechanisms of Gap Junction Regulation

描述（由申请人提供）：连接蛋白是整合的膜蛋白，其寡聚化形成细胞间间隙连接通道。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到整个器官的功能反应中。缝隙连接介导电脉冲传播、细胞生长调节和器官发育等重要过程。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。虽然有相当多的信息，在间隙连接通道的调节连接蛋白的关键相互作用，导致通道关闭和降解的精确机制尚未被定义，也没有充分的特点，涉及的关键辅助蛋白。如果要充分理解细胞间通讯在正常和疾病状态中的作用，这些信息是至关重要的。我们工作的长期目标是获得调节间隙连接的机制的结构和功能的理解。本项目的目的是使用多学科的方法来调查内和分子间的相互作用，定义的结构的主要心脏间隙连接蛋白连接蛋白43（Cx43）在pH值介导的门控和降解。所提出的研究的中心假设是Cx43羧基末端（Cx43 CT）残基Y265-A305作为主调节结构域，在适当的条件下（例如，细胞内酸化和/或磷酸化），结合到“受体”（即，Cx43胞质环（Cx43 CL））与孔相连以关闭通道，然后与参与其降解的分子伴侣相连。pH介导的Cx43调节的研究是有意义的，因为细胞内酸化，导致关闭和降解的间隙连接，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期间恶性室性心律失常的原因之一。拟议研究的基本原理是，更好地了解Cx43调节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。本论文的主要目的是：1）明确c-Src如何介导Cx43间隙连接的闭合; 2）确定Cx43降解过程中的分子相互作用; 3）鉴定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.