The Astrocyte Nexus: CX43-Protein Interactions

描述（由申请人提供）：星形胶质细胞之间的长距离细胞间通讯在很大程度上由间隙连接通道介导，离子和代谢物通过间隙连接通道直接从一个细胞传递到下一个细胞。星形胶质细胞间隙连接的孔主要由差距连接蛋白连接蛋白43（Cx43）形成。最近的证据表明，其他蛋白质与缝隙连接处的Cx43相关，形成了一个大分子复合物，我们称之为Nexus。我们推测，Nexus组件可以调节差距连接通道的特性，也可以在星形胶质细胞内的细胞内信号转导和Cx43的运输中发挥作用，所有这些都是维持整个大脑中耦合星形胶质细胞网络的关键。在支持的最后一段时间，我们发现，无论是内部和分子间的相互作用发生与Cx43，量化了在不同的pH值和磷酸化条件下的几个相互作用的亲和力，并使用NMR来解决相关的Cx43胞质结构域的结构，并确定如何结合后的结构变化。我们还确定了Cx43的新的结合伙伴，并已开始研究如何与结合伙伴的相互作用影响Cx43间隙连接通道的功能和这种蛋白质的细胞内运输和从膜。因此，星形胶质细胞中的Nexus复合物是动态的，由于局部条件和局部配体浓度而改变结合伴侣亲和力。我们现在建议扩展这些研究，以确定细胞骨架蛋白与Cx43的相互作用是如何与星形胶质细胞网络的快速和逐渐重塑相关联的。拟议的研究使用跨学科的方法与新的差距连接领域的几种技术，严格探索新的概念，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。因此，这些研究有望对缝隙连接在神经系统和其他地方的作用产生新的认识。

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.