STRUCTURE, FUNCTION & REGULATION OF GAP JUNCTION PROTEIN

我们的中心目标是了解缝隙连接化学调控的分子基础。我们的中心目标是了解缝隙连接化学调控的分子基础及其对细胞行为的影响。该项目自1990年启动以来一直是方案项目赠款的一部分。在下一个资金阶段，我们将专注于四个具体目标：1)开始描述表达Cx40和Cx43的细胞中缝隙连接形成和调控的机制。Cx40和Cx43不形成异型通道。然而，如果两个细胞都表达连接蛋白，就形成了复杂的通道。此外，一些激动剂可以调节一种同工型，但不能调节另一种。我们将确定一个异构体连接蛋白是否与相反的同构体相互作用，如果是的话，通道的调节是否由一种连接蛋白主导。2)测定表达Cx40和Cx43的细胞中缝隙连接的pH敏感性。我们最近证明了截短的Cx40和异源CX43CT之间的“异域相互作用”使通道比野生型Cx40对pH更敏感。因此，我们认为在异构体Cx40-Cx43缝隙结中，异域相互作用导致了pH光栅的协同作用。3)研究连接蛋白CT片段干扰外源同源缝隙连接化学调控的能力。以前的研究表明，向表达Cx43的卵母细胞对注射完整的Cx43CT片段或特定的17肽可以干扰pH门控。在这里，我们将测试这些片段是否可以干扰Cx43或Cx40的其他调控功能。我们还将寻求发现干扰Cx43调控的新多肽。4)确定Cx43CT结构域的过度表达是否会导致细胞生理功能的改变和器官功能的破坏。我们已经建立了一种在巨细胞病毒(CMV)启动子(心脏神经脊靶向)控制下过表达Cx43CT结构域的转基因小鼠。我们假设连接蛋白片段可以在不改变成孔蛋白表达的情况下扰乱组织功能。总体而言，我们关注连接蛋白-连接蛋白相互作用(全长分子之间或连接蛋白和片段之间)如何影响缝隙连接调节。这些研究将有助于更好地理解连接蛋白在健康和疾病中的作用。

Our central goal is to understand the molecular basis for the chemical regulation of gap junction. Our central goal is to understand the molecular basis for the chemical regulation of gap junctions and its implications on cell behavior. This project has been a part of the Program Project Grant since its inception in 1990. For the next funding period, we will focus on four Specific Aims: 1) To begin a characterization of the mechanisms of gap junction formation and regulation in cells expressing Cx40 and Cx43. Cx40 and Cx43 do not form heterotypic channels. Yet, if both cells express both connexins, complex channels are formed. Moreover, some agonists regulate one isotype but not the other. We will determine whether a heteromeric connexin interacts with an opposing homomer and if so, whether the regulation of channel is dominated by one of the connexins. 2) To determine the pH sensitivity of gap junctions in cells expressing Cx40 and Cx43. We recently demonstrated that "hetero- domain interactions" between a truncated Cx40 and the heterologous CX43CT render the channel more pH sensitive than the wild-type Cx40. We therefore propose that in heteromeric Cx40-Cx43 gap junctions, hetero-domain interactions lead to synergism of pH grating. 3) To study the ability of connexin CT fragments to interfere with the chemical regulation of exogenous homomeric gap junctions. Previous studies show that injection of the entire Cx43CT fragment, or of a specific 17mer peptide to Cx43-expressing oocyte pairs can interfere with pH gating. Here, we will test whether these fragments can interfere with other regulatory functions of Cx43, or of Cx40. We will also seek to identify new peptides that interfere with Cx43 regulation. 4) To determine whether over-expression of the Cx43CT domain can lead to functional alterations in cell physiology and to the disruption of organ function. We have developed a transgenic mouse line that over-expressed the Cx43CT domain under the control of the cytomegaloviral (CMV) promoter (cardiac neural crest-targeted). We hypothesize that connexin fragments can disrupt tissue function without altering the expression of pore- forming proteins. Overall, we focus on how connexin-connexin interactions (between full-length molecules or between a connexin and a fragment) affect gap junction regulation. These studies will lead to a better understanding of the role of connexins in health and disease.