STRUCTURE AND DYNAMICS OF CONNEXIN26 GAP JUNCTIONS

描述(申请人提供)：缝隙连接在细胞过程中扮演着动态的角色，然而，在理解缝隙连接蛋白是如何基于具有刚性和柔性结构域的结构来调节和门控方面存在基本的知识缺口。连接蛋白的表达和功能受到高度调控，每个亚型的序列赋予分子通过孔道的特异性(“渗透选择性”)。连接蛋白六聚体(连接蛋白或半通道)有三个结构域，由脂双层定义。两个半通道在它们的胞外区配对，形成一个细胞间通道。保守的跨膜和胞外结构域相当坚硬，而细胞质结构域是灵活的。胞质结构域的序列可变性，特别是在C-末端，允许每个异构体特有的配对蛋白结合。在这一背景下 通过划分结构，我们的中心假设是单体紧密地堆积在其刚性结构域中，但细胞质结构域中的灵活性允许在细胞中形成超分子复合体以及控制磷酸化和门控的蛋白质的结合。连接蛋白病是一种遗传性人类疾病，通常是由突变引起的，这些突变往往会扰乱包装或伴侣之间的相互作用。例如，在高加索人群中，Cx26突变约占语前非综合征耳聋病例的1/2，但在所有大陆的人群中都有病例。拟议的研究探讨了这一假设，有三个具体目的。(1)利用已知可导致遗传性耳聋的突变，研究Cx26六聚体跨膜区的稳定性。这些实验将与探索通道功能和结构的实验相关联。(2)用冷冻电子显微镜(Cryo-EM)和单粒子重建技术确定CX50半管的三维结构。CX50细胞间通道在晶状体中起重要作用，其功能障碍导致白内障。它具有广泛的、不太有序的细胞质结构域，通常不能通过结晶学来解析。在这一目标中，单粒子重建是获得大尺寸全长CX50半通道结构的最佳技术。(3)原位创建基因标记的Cx43细胞间通道、细胞骨架和支架蛋白的电子断层扫描体积，以更好地了解与缝隙连接相互作用的细胞质结构。Cx43含有细胞骨架成分和支架蛋白ZO-1的结合域。它广泛存在于大多数器官系统中，在血管和心脏中起着特别重要的作用。长期的目标是以最高的分辨率获得全长连接蛋白的更完整的描述。这种方法是创新的，因为它使用了多分辨率成像策略，并协调了对通道和半通道的生化和功能分析。这项拟议的研究意义重大，因为结果将有助于定义更好的药物和其他治疗方法，潜在地改善连接蛋白相关疾病。

DESCRIPTION (provided by applicant): Gap junctions play dynamic roles in cellular processes, however, there is a fundamental knowledge gap in understanding how gap junction proteins, the connexins, are regulated and gated based on a structure that has rigid and flexible domains. Connexin expression and function are highly regulated and the sequence of each isoform imparts specificity ("permselectivity") to which molecules pass through the pore. The connexin hexamer (connexon or hemichannel) have three domains defined by the lipid bilayer. Two hemichannels pair at their extracellular domains to form an intercellular channel. The conserved transmembrane and extracellular domains are fairly rigid while the cytoplasmic domain is flexible. The sequence variability in the cytoplasmic domains, particularly in the C-terminus, allows for binding of partner proteins unique to each isoform. Within the context of this compartmentalized structure, our central hypothesis is that the monomer is tightly packed in its rigid domains, but flexibility in the cytoplasmic domains permit supra-molecular complexes to be formed in cells as well as binding of proteins controlling phosphorylation and gating. Connexin-opathies, hereditary human diseases, are often caused by mutations that often disrupt packing or partner interactions. For example, Cx26 mutations account for ~1/2 of cases of pre-lingual non-syndromic deafness in Caucasian populations but cases are found in populations across all continents. The proposed studies explore this hypothesis with three specific aims. (1) To investigate the stability of the transmembrane region of the Cx26 hexamer using mutations known to cause heredity deafness. These experiments will be correlated with ones probing channel function and structure. (2) To determine the 3D structure by cryo-electron microscopy (cryo-EM) and single particle reconstruction of Cx50 hemichannels. Cx50 intercellular channels serve critical functions in lens and its dysfunction leads to cataracts. It has extensive less ordered cytoplasmic domains typically not resolvable by crystallography. In this aim, single particle reconstruction is the best technique to obtain a structure of the large, full-length Cx50 hemichannel. (3) To create electron tomographic volumes of genetically labeled Cx43 intercellular channels and cytoskeletal and scaffolding proteins in situ to better understand the cytoplasmic architecture interacting with a gap junction. Cx43 contains binding domains for cytoskeletal components and the scaffolding protein, ZO-1. It is widespread through most organ systems with particularly important roles in vasculature and heart. The long-term goal is to obtain a more complete depiction of full-length connexins at the highest resolution obtainable. The approach is innovative because it uses a multi-resolution imaging strategy coordinated with biochemical and functional analyses of channels and hemichannels. The proposed research is significant because results will be useful in defining better drugs and other therapeutics that potentially ameliorate connexin-related diseases.