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 tissues, where they organize into dense plaques containing tens to thousands of channels. Although plaque formation is known to modulate junctional conductance, the structural basis for channel-channel organization within a membrane environment remains poorly defined. Here, we reconstitute native lens connexin-46/50 (Cx46/50) gap junction channels into MSP-based lipid nanodiscs that incorporate multiple channels to create miniature plaque-like complexes suitable for single-particle cryo-electron microscopy (cryo-EM). We determine high-resolution structures of dual-channel assemblies in two distinct configurations and find no ordered protein-protein contacts across the interface. Instead, the inter-channel space is occupied by ordered lipid density, indicating that channel packing in these assemblies is lipid mediated. These channel-channel interfaces stabilize discrete lipid populations, including an interstitial lipid intercalated between subunits and positioned near the N-terminal gating domain, suggesting a route by which channel organization could promote lipid occupancy near the pore. Additionally, we leverage this dataset to refine the Cx46/50 single-channel structure to 1.8 Å resolution, revealing exceptional chemical detail of the pore-lining landscape in the stabilized open-state. Together, these results define principles of lipid-mediated multi-channel organization and suggest how plaque-like packing may tune gap junction function through specific lipid interactions.