pH Regulation of Connexin 43: Intermediary Steps

描述(申请人提供)：连接蛋白是一种完整的膜蛋白，通过寡聚形成称为缝隙连接的细胞间通道。最近，我们已经证明了Cx43的羧基末端结构域和细胞质环的后半部分之间存在直接的、依赖于pH的分子内联系。这种联系的结构基础、动力学和功能后果是这笔赠款的中心议题。 具体目的1.建立Cx43羧基末端与胞质环区相互作用的结构模型。我们的实验表明，在Cx43的细胞质环和CT结构域中，存在可识别的有组织的二级结构区域。一旦结构域被绑定，结构可能会发生变化。我们将使用表面等离子共振(SPR)将结构预测与定点突变和动力学结合分析相关联，以确定关键的结合位点。 具体目的2.研究CT-CL相互作用在功能性缝隙连接斑块形成中的作用。CL区域的选择性突变会导致转基因细胞中可识别的缝隙连接斑块的丢失。然而，如果CT结构域也从序列中删除，相同的突变体确实会形成缝隙连接斑块。我们假设Cx43CT中的特定序列可以防止缝隙连接的形成，除非被CL结构域中的相应序列掩盖。 具体目的3.确定CT-CL相互作用是否参与决定Cx43的单通道特性和pH门控行为。我们认为细胞质环与CT结构域的结合介导了连接蛋白43的单通道和“pH门控”行为。结果将与结构预测和结合谱相关联(目标1)。这些数据将产生CT与细胞质环相互作用所产生的功能后果的全局图景。

DESCRIPTION (provided by applicant): Connexins are integral membrane proteins that oligomerize to form intercellular channels called gap junctions. Recently, we have shown direct, pH-dependent intramolecular association between the carboxyl terminal domain and the second half of the cytoplasmic loop of Cx43. The structural bases, kinetics and functional consequences of this association are the central topics of this grant. Specific Aim 1. To generate a structural model of the interaction between the carboxyl terminal and the cytoplasmic loop domains of Cx43. Our experiments show identifiable regions of organized secondary structure in the cytoplasmic loop and in the CT domains of Cx43. The structures are likely to change once the domains are bound. We will correlate structural predictions with site-directed mutagenesis and kinetic binding assays using surface plasmon resonance (SPR) to identify the crucial binding sites. Specific Aim 2. To characterize the role of the CT-CL interaction in the formation of functional gap junction plaques. Selected mutations in the CL region cause the loss of identifiable gap junction plaques in transfected cells. However, the same mutant does form gap junction plaques if the CT domain is also deleted from the sequence. We hypothesize that a specific sequence in Cx43CT prevents gap junction formation unless masked by a corresponding sequence in the CL domain. Specific Aim 3. To establish whether the CT-CL interaction is involved in determining the single channel properties and pH gating behavior of Cx43. We propose that binding of the cytoplasmic loop to the CT domain mediates the single channel and "pH gating" behavior of Connexin 43. Results will be correlated with structural predictions and binding profiles (Aim 1). These data will yield a global picture of the functional consequences resulting from the interaction of the CT with the cytoplasmic loop.