Intercellular communication by the gap junctions is facilitated by a unique macromolecular architecture, in which transmembrane channels directly couple the plasma membranes of neighboring cells. In each membrane, six connexin (Cx) subunits oligomerize to form a hemichannel. Docking between extracellular (EC) domains of two hemichannels forms the complete gap junction intercellular channel. Our current understanding of gap junction structure lacks crucial information about how these channels interact with their native phospholipid environment. To address this gap in knowledge, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual-lipid bilayer closely mimicking a native cell-to-cell junction.