Structural and Functional Roles of Membrane Lipids in the pH gating of Connexin-46/50 Channels.

Project Summary Connexin 46 and 50 (Cx46/50) gap junctions are critical for maintaining lens transparency, and disruption of proper gap junction function results in the world’s leading cause of blindness: cataract. Cx46/50 gap junctions couple adjacent lens fiber cells together and regulate the lens microcirculatory system, which is responsible for circulating ions, nutrients, and waste products from the oldest to the newest cells. Lens fiber cells rely on anaerobic mechanisms to generate ATP; thus, over time the intracellular pH drops below physiologically neutral conditions. In vitro experiments have shown that Cx46/50 gap junctions are gated under low pH conditions – providing a plausible mechanism contributing to age-related cataract. Preliminary structural data demonstrates that annular membrane lipids can intercalate the inner pore of Cx46/50 under conditions at pH 5.8, and that this pore lipid gating phenomenon is reversible when proteins are returned to pH 7.4. However, it is completely unknown how lipids enter and exit the pore and even if lipids functionally gate Cx46/50 gap junctions in a cellular context. Therefore, the primary goals of this project are to 1) determine the functional role of lipids on Cx46/50 pH gating in a cellular environment and 2) determine the mechanistic pathway of lipid entry into the pore of Cx46/50 gap junctions. For Aim 1, my approach will employ a combination of a chemical biology toolkit together with cellular functional assays to determine the role of lipids in the pH gating of Cx46/50 in a cellular environment. Additionally, suspected pH-sensing amino acid residues will be systematically mutated to determine their role in pH-induced channel closure. For Aim 2, my approach will use a combination of structural biology methods utilizing cryo-electron microscopy together with proteomic approaches to detail the protein-lipid interactions that define the pH-dependent translocation pathway of lipids into the Cx46/50 pore. Success in these studies will clarify critical mechanistic details of how Cx46/50 functionally interact with lipid membranes to respond to intracellular changes in pH. These insights are expected to provide molecular level insights into age-related cataract, with potential broad relevance to understanding how other connexins respond to pH-induced stress conditions.