The Astrocyte Nexus: CX43-Protein Interactions

描述（申请人提供）：星形胶质细胞之间的长距离细胞间通讯在很大程度上是由间隙连接通道介导的，离子和代谢物通过间隙连接通道直接从一个细胞传递到另一个细胞。星形胶质细胞缝隙连接孔主要由缝隙连接蛋白connexin43 （Cx43）形成。最近的证据表明，其他蛋白质在间隙连接处与Cx43相关，形成我们称之为Nexus的大分子复合物。我们假设Nexus成分可能调节间隙连接通道的特性，也可能在星形胶质细胞内的细胞内信号转导和Cx43的运输中起作用，所有这些对于维持整个大脑中偶联星形胶质细胞的网络至关重要。在最后一段时间的支持中，我们发现Cx43在分子内和分子间都发生了相互作用，我们量化了不同pH和磷酸化条件下几种相互作用的亲和性，并使用NMR解决了相关Cx43细胞质结构域的结构，并确定了结构在结合后的变化。我们还发现了Cx43的新结合伙伴，并开始研究与结合伙伴的相互作用如何影响Cx43间隙连接通道的功能以及该蛋白在细胞内进出膜的运输。因此，星形胶质细胞中的Nexus复合物是动态的，由于局部条件和配体的局部浓度而改变结合伙伴的亲和力。我们现在建议扩展这些研究，以确定细胞骨架蛋白与Cx43的相互作用如何与星形胶质细胞网络的快速和渐进重塑相关联。本研究采用跨学科的方法，结合间隙连接领域的几种新技术，严格探索连接蛋白-细胞骨架相互作用是星形胶质细胞间隙连接功能的主要决定因素这一新概念。因此，这些研究有望对间隙连接在神经系统和其他地方所起的作用产生新的见解。

DESCRIPTION (provided by applicant): Long range intercellular communication among astrocytes is in large part mediated by gap junction channels, through which ions and metabolites pass directly from one cell to the next. The pore of astrocyte gap junctions is formed primarily of the gap junction protein connexin43 (Cx43). Recent evidence indicates that other proteins are associated with Cx43 at gap junctions, forming a macromolecular complex that we have termed the Nexus. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction and trafficking of Cx43 within the astrocyte, all of which are critical for maintaining the network of coupled astrocytes throughout the brain. During the last period of support, we found that both intra- and intermolecular interactions occur with Cx43, have quantified the affinities of several interactions under different pH and phosphorylation conditions and used NMR to solve structures of relevant Cx43 cytoplasmic domains and to determine how structures change upon binding. We have also identified new binding partners for Cx43 and have begun to examine how interaction with binding partners affects function of Cx43 gap junction channels and the intracellular trafficking of this protein to and from the membrane. The Nexus complex in astrocytes is thus dynamic, changing binding partner affinities due to local conditions and local concentrations of ligands. We now propose to extend these studies to determine how interactions of cytoskeletal proteins with Cx43 is linked to both rapid and gradual remodeling of the astrocyte network. The proposed studies use an interdisciplinary approach with several techniques that are new to the gap junction field to rigorously explore the novel concept that connexin-cytoskeletal interaction is a major determinant of gap junction function in astrocytes. As such, these studies are expected to lead to novel insight of roles that gap junctions play in the nervous system and elsewhere.