Effect of lipid environment on the structure and organization of connexin-46/50 gap junction channels

项目摘要 晶状体缝隙连接通道由连接蛋白-46/50(Cx46/50)组成，是正常发育所必需的 以及维持哺乳动物眼睛晶状体的透明度。当通道功能被继承扰乱时 突变或累积的翻译后修饰在我们的一生中，白内障都会发生。在 天然晶状体膜环境，Cx46/50缝隙连接天然存在，磷脂和胆固醇 构图是高度动态的，在我们的一生中都会发生显著的变化。功能实验： 其他连接蛋白亚型(Cx32、Cx26)已经证明这些通道的通透性是敏感的 到独特的磷脂类型和胆固醇含量。但是，因为没有高分辨率的 脂双层中任意缝隙连接通道的结构，膜组成的机制(S) 对缝隙连接的结构和功能的影响完全不清楚。此外，由于缝隙连接 占主导地位的是细胞膜上的一种更高级的组装(称为缝隙连接斑块)，它完全 未知蛋白质-蛋白质或蛋白质-脂-蛋白质相互作用如何促进超分子组织 斑块形成/重塑。为了解决这些知识上的差距，本提案的主要目标是 1)解开不同膜组分对晶状体结构和功能的影响 缝隙连接Cx46/50；和2)表征密切的蛋白质-蛋白质和蛋白质-脂肪相互作用 在缝隙连接斑块的背景下稳定和组织几个通道。主要方法是 为了达到这些目标，我们采用了单粒子冷冻电子显微镜和脂质纳米盘相结合的方法。 技术和体外囊泡通透性功能研究。这些目标的成功有望直接 影响我们理解与年龄相关的膜脂环境变化如何促进年龄- 缝隙连接组织和通道活动的相关变化。从这些研究中得到的见解可能是广泛的 适用于了解缝隙连接蛋白相关疾病可能如何受 组织特有的脂质环境(例如，白内障形成、动脉粥样硬化、中风和癌症)。

Project Summary The lens gap junction channels, composed of connexin-46/50 (Cx46/50), are essential to proper development and maintenance of transparency in the mammalian eye lens. When channel function is disrupted by inherited mutations or accumulated posttranslational modifications throughout our lifetimes, cataracts occur. In the native lens membrane environment that Cx46/50 gap junctions naturally exist, phospholipid and cholesterol composition is highly dynamic and changes markedly throughout our lifetimes. Functional experiments with other connexin isoforms (Cx32, Cx26) have demonstrated that the permeability of these channels is sensitive to unique phospholipid types and cholesterol content. However, because there are no high-resolution structures of any gap junction channels in a lipid bilayer, the mechanism(s) by which membrane composition influences the structure and function of gap junctions is totally unclear. Additionally, since gap junctions predominate in the cell membrane as a higher-order assembly (known as gap junctional plaques), it is totally unknown how protein-protein or protein-lipid-protein interactions contribute to the supra-molecular organization of plaque formation/remodeling. To address these gaps in knowledge, the primary objective of this proposal are: 1) to deconvolute the influence of distinct membrane components on the structure and function of the lens gap junctions Cx46/50; and 2) to characterize the intimate protein-protein and protein-lipid interactions that stabilize and organize several channels in the context of gap junctional plaques. The primary method employed in pursuit of these objectives is single particle cryo-electron microscopy, coupled with lipid nanodisc technologies and in vitro vesicle-permeability functional studies. Success in these aims are expected to directly impact our understanding how age-related changes in the membrane lipid environment contribute to age- related changes in gap junction organization and channel activity. Insights from these studies may be broadly applicable to understanding how connexin-related diseases may be influenced by associated changes in the tissue-specific lipid environment (e.g., cataract formation, atherosclerosis, stroke and cancers).