Lipids intercalate and mediate multi-channel assemblies of connexin-46/50 gap junctions

Gap junction channels enable direct electrical and metabolic exchange between adjacent cells and organize into plaques containing tens to thousands of channels. Although plaque formation modulates junctional conductance, the structural basis for channel–channel organization remains poorly defined. Here, we reconstitute native lens connexin-46/50 (Cx46/50) channels into MSP-based lipid nanodiscs containing multiple channels and determine high-resolution structures of dual-channel assemblies in two configurations. The interfaces lack extensive protein–protein contacts and instead contain ordered lipid densities that define much of the inter-channel interface. These interfaces stabilize discrete lipid populations, including an interstitial lipid near the N-terminal gating domain, suggesting how channel organization may promote lipid occupancy near the pore. We also refine the Cx46/50 single-channel structure to 1.8 Å, providing an internal reference and chemical detail of the stabilized open-state. Together, these results define lipid-rich interfaces as a feature of multi-channel organization and suggest how plaque-like packing may tune gap junction function. Gap junction channels assemble into plaques that coordinate communication across tissues. Here, authors show how neighboring Cx46/50 channels pack within membranes and identify ordered lipids as major components of their interfaces