pH Regulation of Connexin 43: Intermediary Steps

描述（由申请人提供）：连接蛋白是整合的膜蛋白，其寡聚化形成称为间隙连接的细胞间通道。最近，我们已经显示了直接的，pH值依赖的分子内关联之间的羧基末端结构域和Cx43的胞质环的下半部分。该协会的结构基础，动力学和功能后果是该补助金的中心议题。 具体目标1。 建立Cx43羧基端与胞质环结构域相互作用的结构模型。我们的实验表明，在细胞质环和Cx43的CT结构域的组织二级结构的可识别的区域。一旦结构域被结合，结构就可能改变。我们将相关的结构预测与定点诱变和动力学结合试验，使用表面等离子体共振（SPR），以确定关键的结合位点。 具体目标2。 表征CT-CL相互作用在功能性间隙连接斑块形成中的作用。CL区中的选定突变导致转染细胞中可识别的间隙连接斑块的丢失。然而，如果CT结构域也从序列中缺失，则相同的突变体确实形成间隙连接斑块。我们推测，Cx43 CT中的特定序列阻止间隙连接形成，除非被CL结构域中的相应序列所掩盖。 具体目标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。我们建议，CT域的细胞质环的结合介导的连接蛋白43的单通道和“pH门控”行为。结果将与结构预测和结合特征相关（目标1）。这些数据将产生一个整体的图片的功能后果所产生的相互作用的CT与细胞质环。

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.