Structure of human Cx43 gap junction channel.

a-b, Cryo-EM density map and model of Cx43 GJC solved by cryo-EM at 2.26 Å resolution. The individual Cx43 monomers in each HC within the GJC are coloured blue, pink, grey, green, salmon and orange. Grey densities correspond to the detergent micelle and the bound sterol-like molecules. c, The position of a single Cx43 monomer (orange) within a GJC (represented as a surface of juxtaposed Cx43 monomers in two distinct membrane regions, white and grey). d, Alignment of the monomers of Cx43, Cx26 (PDB ID: 2zw3), Cx46 (PDB ID: 7jkc) and Cx31.3 (PDB ID: 6l3t) shows the distinct orientations of the N-terminal domain (“NTD”) helix. Individual TM domains, extracellular loops 1 and 2 (“ECL1-2”), relative positions of the intracellular loop (“ICL”) and the C-terminus (“C-term”) are indicated. e-f, Same as a-b, for the Cx43 HC in MSP2N2 nanodiscs at 3.98 Å resolution. g, Alignment of three indicated structures shows that the conformation of the gating region is highly conserved. The models are shown as ribbons; one of the aligned monomers in each of the structures is represented as cartoon, to clearly indicate the monomer boundaries.

The Cx43 gate adopts a closed conformation.

a-b, Analysis of the pore opening dimensions using HOLE reveals a constriction of the pore in the putative gate region. Only a central opening and one of the six peripheral openings within one HC of Cx43 GJC in digitonin is shown. The distance is calculated from the center of the GJC pore to a point outside of the channel. Central and peripheral openings are coloured blue and cyan, respectively. c-d, Comparison of the Cx43 in digitonin with the Cx31.3 HC shows that the peripheral opening is created by the particular arrangement of the NTD and by adjustment of the TM2. e-f, The distinct NTD/TM2 arrangement results in a single pore opening in the structure of Cx31.3 (indicated with an asterisk, e), contrary to Cx43 (f). g, A view of the Cx43 gating region from the cytosol reveals the location of the “lipid-N” molecules stabilizing the NTD arrangement, shown as isolated densities (cyan). h, A slab view of the gating region parallel to the membrane plane shows the relative arrangement of the lipid-N and the intra-pore lipid densities (“lipid-1” and lipid-2”; orange).

Extracellular domain of Cx43 GJC and HC.

a-b, The lateral views of Cx43 GJC in digitonin, indicating the positions of the extracellular loops 1 and 2. c, Views of the extracellular loops ECL1 (top) and ECL2 (bottom), for Cx43 (left), Cx46, (middle; PDB ID: 7jkc), and Cx26 (right; PDB ID: 2zw3). The residues of one monomer in each of the structures, directly involved in junction formation, are coloured by element with carbon atoms as orange/yellow (Cx43), brown/pink (Cx46) and teal/cyan (Cx26). The residues of the neighbouring connexin monomers within 4 Å distance are shown coloured with white carbon atoms. Dotted lines indicate electrostatic contacts, calculated in PyMol. d, Sequence alignment of the complete ECL1 and ECL2 regions of the three proteins shown in c. The black lines indicate the GJC interface residues, as shown in c. e, Alignment of the Cx43 GJC structures in digitonin micelles and in nanodiscs. Cα atoms of the interface residues shown in C are represented as spheres (the ribbon and spheres of Cx43 in nanodiscs is coloured black). f, Same as e, for a comparison between Cx43 GJC in digitonin and Cx43 HC in nanodiscs. The arrow indicates the movement of the two loops that accompanies GJC formation. The ribbon and spheres of Cx43 HC are light grey. The grey numbers in brackets indicate displacement of selected Cα atoms (residues P59, Q58, P193, P194).

Locations of the disease-linked mutations in the Cx43 GJC structure.

a, Disease-linked mutations mapped on the sequence of Cx43. The amino acid residues proximal to the intrapore lipid densities (“Pore lipid sites”) are shown as white dots. The residues at the GJC interface are shown as yellow dots (“ECD”). The residues of the putative gating region (“Gate region”) are shown as blue (NTD), green (TM1) and violet (TM2) dots. The sequence elements not resolved in our 3D reconstruction are indicated with a dashed line. b, Two sterol-like density elements (lipid-1 and -2) are located within the pore region of Cx43 GJC (white). The indicated disease-linked mutations are located within close distance of the pore lipid densities. c, A view of the extracellular loops forming the junction, indicating two residues known to be linked with ODDD, P59 and H194 (side chains shown as sticks; one of the monomers in the GJC is coloured yellow). d, A view of the gating region with the highlighted disease-linked mutations, colored as in a. e, A similar representation of the disease-linked mutations, with Cα atoms shown as spheres, illustrating the contribution of the mutants to gate formation.

Molecular dynamics simulations of the Cx43 GJC and HC.

a, Cartoon representation of the double-bilayer system including the GJC (cartoon) and ions (vdw spheres). Lipidic membrane and water residues have been removed for clarity. The direction of the applied constant electric field, Ez, is indicated with an arrow. b, Ion density (ρ) profiles (average and fluctuations) along the diffusion axis of the GJC coloured in red for anions and blue for cations, for the simulated applied transjunctional voltages. c, The solvent-accessible radius (and fluctuations during MD) along the diffusion axis of the GJC. The dotted lines correspond to the minimum radius and the position of the NTD regions. The surface representation of Cx43 is coloured according to the calculated electrostatic potential; the slab view shows the properties of the pore. d, Average free energy experienced (at least two simulation replicates per panel) by the K+ (blue) and Cl- (red) while permeating the pore with different applied transmembrane potential (from right to left: 0 mV, -100 mV, -200 mV and - 500 mV). The dotted lines correspond to the maximum free energy barrier for anions (red) and cations (blue). e, Cartoon representation of the lipid-bound system including the HC (cartoon) and ions (vdw spheres). Lipidic membrane and water residues have been removed for clarity. The direction of the applied constant electric field, Ez, is indicated with an arrow. f, Same as c, for the Cx43 HC. g, Ion density (ρ) profiles (average and fluctuations) along the diffusion axis of the HC coloured in red for anions and blue for cations, for the simulated applied transmembrane voltages.

A structure-based view of connexin gating states.

a, The side view of a fully open gate, as observed in Cx26 GJC (PDB ID: 2zw3); the gate-forming regions of the protein, NTD (red) and TM2 (blue), are shown as cylinders. Similar arrangement of the gating elements has been observed in Cx46/Cx50. This conformation of the gate is permeable to a wide range of substrates, including small molecules and ions. b, The semi-permeable central gate is featured in the structure of Cx31.3 HC (PDB ID: 6l3t). In this conformation the gate is likely selective for ions (Cx31.3 has been shown to have a preference for anions), based on the dimensions of the gate. c, The putative closed state is featured in the Cx43 structures (here, the Cx43 in digitonin is shown as an example). d-f, Same as a-c, viewed from the cytosolic side of the channel. The gray surface corresponds to the NTD and TM2 regions; the white surface corresponds to the rest of the protein; lipid-N molecules in f are shown as black spheres, using a model of DHEA manually placed into the six NTD lipid site regions for illustration purposes (the exact identity of the lipid-N molecule is unknown).