Electron cryo-crystallography of a recombinant cardiac gap junction channel

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin43 (Cx43; α₁), the principal cardiac gap junction protein. The projection map at 7 Å resolution revealed that each 30 kDa connexin subunit has a transmembrane α-helix that lines the aqueous pore and a second α-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by ∼ 30°. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of ∼7.5 Å and a vertical resolution of ∼ 25 Å. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an α-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated α₁Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of α₁Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.