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).