Mechanisms of Gap Junction Regulation

描述(由申请人提供)：我们工作的长期目标是对缝隙连接调节机制的结构和功能有一个了解。缝隙连接由连接蛋白(CXs)形成，为细胞间的电信号/分子信号的传播提供了一条途径，这是细胞分化、代谢平衡和可兴奋组织中的电耦合所必需的。这种类型的通信允许单个细胞事件同步到整个器官的功能反应中。人类Cx基因中影响细胞偶联的缺陷与多种遗传性疾病(例如，Charcot-Marie-Tooth病和遗传性非综合征性耳聋)有关。小鼠的遗传操作已经证明了CXS在各种器官中的功能重要性。此外，不仅缝隙连接的存在，而且对缝隙连接的适当调控对动态平衡至关重要。例如，细胞内酸化导致所有天然组织和外源表达系统中缝隙连接的关闭。鉴于细胞内酸化是组织缺血的一个主要后果，对缝隙连接的pH依赖调节的研究变得更加重要。酸化诱导的解偶联对缺血区周围组织的保存有影响。因此，我们选择了在心脏、脑和其他组织中表达最广泛的连接蛋白Cx43作为我们的模型系统来研究CXS的结构调控。我们的目标是应用生物物理方法来研究分子内和分子间的相互作用，这些相互作用定义了pH门控过程中Cx43的结构调节。我们假设Cx43羧基末端结构域(CT)作为一个门控“颗粒”，在适当的条件下(如细胞内酸化或磷酸化)，与与孔道相连的“受体”(即Cx43细胞质环；CL)结合并关闭通道。为了研究这一概念，提出了以下具体目标：1)建立CT如何与参与缝隙连接调控的分子伴侣相互作用；2)评估pH对CT和CL结构域的结构影响；3)表征CX亚型之间的细胞质结构域相互作用。这些目的是为了确定CT与分子伙伴和CL相互作用所产生的功能后果，以努力开发定点定向的、特异的缝隙连接通讯调节器，在疾病和缺血性损伤的治疗中具有潜在的意义。

DESCRIPTION (provided by applicant): The long term goal of our work is to gain a structural and functional understanding of the mechanisms of gap junction regulation. Gap junctions, formed of proteins called connexins (Cxs), provide an intercellular pathway for the propagation of electrical/molecular signals, which are necessary for cellular differentiation, metabolic homeostasis, and in excitable tissue, electrical coupling. This type of communication permits individual cell events to synchronize into the functional response of an entire organ. Defects in human Cx genes that affect cell coupling are associated with a variety of inherited disorders (e.g. Charcot-Marie-Tooth disease and hereditary non-syndromic deafness). Genetic manipulations in mice have demonstrated the functional importance of Cxs in a variety of organs. Moreover, not only the presence but also the proper regulation of gap junctions is critical for homeostasis. For example, intracellular acidification leads to closure of gap junctions in all native tissues and exogenous expression systems tested. The study of pH-dependent regulation of gap junctions becomes even more relevant given that intracellular acidification is a major consequence of tissue ischemia. Acidification-induced uncoupling has an impact on the preservation of tissue surrounding the ischemic area. Therefore, we have chosen Cx43, the most widely expressed junction protein in the heart, brain, and other tissues, as our model system to study the structural regulation of Cxs. Our objective is to apply biophysical approaches to investigate intra-and intermolecular interactions that define the structural regulation of Cx43 during pH gating. We hypothesize that the Cx43 carboxyl terminal domain (CT) acts as a gating "particle" that, under the appropriate conditions (e.g. intracellular acidification or phosphorylation), binds to a "receptor" (i.e. Cx43 cytoplasmic loop; CL) affiliated with the pore and closes the channel. The following Specific Aims are proposed to investigate this concept: 1) To establish how the CT interacts with molecular partners that are involved in gap junction regulation; 2) To assess the structural effect of pH on the CT and CL domains; 3) To characterize cytoplasmic domain interactions between Cx isoforms. These Aims are designed to identify the functional consequences resulting from CT interactions with molecular partners and the CL in an effort to develop site-directed, specific modulators of gap junction communication with potential implications in therapeutic treatment of disease and ischemic injury.