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

项目摘要 透镜间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透镜的透明度。当通道功能被继承的 突变或积累的翻译后修饰在我们的一生中，白内障发生。在 天然透镜膜环境，Cx46/50间隙连接天然存在，磷脂和胆固醇 组成是高度动态的，并且在我们的一生中显著变化。功能实验， 其他连接蛋白亚型（Cx 32，Cx 26）已经证明这些通道的通透性是敏感的 独特的磷脂类型和胆固醇含量。然而，由于没有高分辨率 脂质双层中任何间隙连接通道的结构，膜组成 影响缝隙连接的结构和功能是完全不清楚的。此外，由于间隙连接 在细胞膜中占主导地位的高级组件（称为间隙连接斑块），它完全是 未知蛋白质-蛋白质或蛋白质-脂质-蛋白质相互作用如何有助于超分子组织 斑块形成/重塑。为了弥补这些知识差距，本提案的主要目标是 是：1）去卷积不同膜组分对透镜的结构和功能的影响 缝隙连接Cx46/50;和2）表征密切的蛋白质-蛋白质和蛋白质-脂质相互作用， 在缝隙连接斑块的情况下稳定和组织几个通道。的主要方法 在追求这些目标时采用的是单粒子冷冻电子显微镜，加上脂质纳米盘， 技术和体外囊泡通透性功能研究。这些目标的成功预期将直接 影响我们对膜脂环境中与年龄相关的变化如何影响年龄的理解- 缝隙连接组织和通道活动的相关变化。这些研究的见解可能广泛地 适用于理解连接蛋白相关疾病如何受到连接蛋白相关改变的影响。 组织特异性脂质环境（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).