STRUCTURE AND DYNAMICS OF CONNEXIN26 GAP JUNCTIONS

描述（由申请人提供）：间隙连接在细胞过程中发挥动态作用，然而，在理解间隙连接蛋白（连接蛋白）如何基于具有刚性和柔性结构域的结构进行调节和门控方面存在基本知识差距。连接蛋白的表达和功能受到高度调节，并且每个同种型的序列赋予分子通过孔的特异性（“选择性渗透”）。连接蛋白六聚体（连接子或半通道）具有由脂质双层限定的三个结构域。两个半通道在其胞外结构域配对以形成细胞间通道。保守的跨膜和胞外结构域是相当刚性的，而胞质结构域是柔性的。细胞质结构域中的序列变异性，特别是在C-末端，允许结合伴侣蛋白独特的每种亚型。在此背景下， 对于区室化结构，我们的中心假设是单体在其刚性结构域中紧密堆积，但胞质结构域的柔性允许在细胞中形成超分子复合物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，通常是由破坏包装或伴侣相互作用的突变引起的。例如，Cx 26突变占高加索人群中约1/2的语前非综合征性耳聋病例，但在所有大陆的人群中均发现病例。拟议的研究探讨这一假设有三个具体目标。(1)研究Cx 26六聚体跨膜区的稳定性，使用已知引起遗传性耳聋的突变。这些实验将与探索通道功能和结构的实验相关联。(2)通过冷冻电子显微镜（cryo-EM）和Cx 50半通道的单颗粒重建确定3D结构。Cx 50细胞间通道在透镜中起关键作用，其功能障碍导致白内障。它具有广泛的有序度较低的胞质结构域，通常无法通过晶体学解析。在这个目标中，单粒子重建是获得大的全长Cx 50半通道结构的最佳技术。(3)创建电子断层扫描体积的遗传标记的Cx43细胞间通道和细胞骨架和支架蛋白在原位，以更好地了解细胞质结构与间隙连接的相互作用。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，在脉管系统和心脏中发挥特别重要的作用。长期目标是以最高分辨率获得全长连接蛋白的更完整描述。该方法是创新的，因为它使用多分辨率成像策略，与通道和半通道的生化和功能分析相协调。这项研究意义重大，因为其结果将有助于确定更好的药物和其他可能改善连接蛋白相关疾病的治疗方法。

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.