Cryo-EM structure of human Cx31.3/GJC3 connexin hemichannel

Abstract

Connexin family proteins assemble into hexameric channels called hemichannels/connexons, which function as transmembrane channels or dock together to form gap junction intercellular channels (GJIChs). We determined the cryo-electron microscopy structures of human connexin 31.3 (Cx31.3)/GJC3 hemichannels in the presence and absence of calcium ions and with a hearing-loss mutation R15G at 2.3-, 2.5-, and 2.6-Å resolutions, respectively. Compared with available structures of GJICh in open conformation, Cx31.3 hemichannel shows substantial structural changes of highly conserved regions in the connexin family, including opening of calcium ion-binding tunnels, reorganization of salt-bridge networks, exposure of lipid-binding sites, and collocation of amino-terminal helices at the cytoplasmic entrance. We also found that the hemichannel has a pore with a diameter of ~8 Å and selectively transports chloride ions. Our study provides structural insights into the permeant selectivity of Cx31.3 hemichannel.

Fig. 1. Overall structure of Cx31.3 hemichannel with entrance-covering NTHs.

(A and B) The overall structure of Cx31.3 hemichannel. Single-particle reconstruction map (A) and ribbon representations (B) are shown in the same orientations. Six connexins in the hemichannel are shown in different colors, and NTHs are in magenta color in (B). Yellow spheres indicate sulfur atoms in C2. (C) Comparison of NTH conformation in Cx31.3 hemichannel with those of Cx26 and Cx46 GJIChs. Only two interfacing connexins in a hexameric channel are shown for clarity. The accession codes for Cx26 and Cx46 GJICh structures are 2ZW3 and 6MHQ, respectively. NTHs are shown in magenta color. (D) Ribbon presentation of a single Cx31.3 connexin. NTH, TM helices, and ECLs are shown in different colors and are labeled. Cytoplasmic loop (CL) and C-terminal loop (CTL) are represented as gray dashed lines. (E) Intramolecular hydrophobic interactions between NTH and TM2. (F) The solvent-accessible pore radius through Cx31.3 hemichannel pore (gray) is compared with those through Cx26 (orange) and Cx46 (green) GJICh pores. Locations of C2 and the acidic band of Cx31.3 are indicated using an arrow and an asterisk.

Fig. 2. Putative lipid-binding sites on the inner surface of the channel pore.

(A) Surface features of the pore inside are shown by vertical cross section of the hemichannel. The surface electrostatic potential (left) is calculated and colored using Adaptive Poisson-Boltzmann Solver (APBS). The displayed potentials range from −10 (red) to +10 (blue) kT/e. The surface hydrophobicity (right) is represented using PyMOL YRB script. The density maps inside the pore are shown in black meshes. The density blobs predicted to be a phospholipid molecule are encircled by red lines. The locations of the conserved R144 are indicated by cyan lines. (B) Prominently long density chains (red) are shown on the surface of the pore inside. (C) Close-up view of the shorter density chain. R144 and E13 are shown in sticks. A phosphoethanolamine molecule is modeled into the density. The black dotted lines indicate putative salt bridges. (D) Close-up view of the longer density chain. The density chain is located in a long hydrophobic groove formed in the interprotomer interface. The groove-lining residues, which are highly conserved in the connexin family (fig. S1), are shown in sticks and labeled. (E) NTHs in Cx26 and Cx46/50 GJIChs are located on the corresponding surface to the putative lipid-binding sites of Cx31.3 hemichannel. The Cx26 (orange ribbons) and Cx46 (green ribbons) GJICh structures were superposed on the Cx31.3 hemichannel structure (gray surface). Only two neighboring protomers for each channel are shown.

Fig. 3. Anion-selective transport through Cx31.3 hemichannel.

(A and B) Local concentration maps of Cl⁻ (A) and K⁺ (B) ions. Concentration maps were computed using the last 1-μs trajectory without a bias potential. (C and D) Ionic fluxes of Cl⁻ (C) and K⁺ (D) ions under a transmembrane potential of −200 mV. The total ionic currents at −200 mV is −0.166 nA. (E and F) Ionic fluxes of Cl⁻ (E) and K⁺ (F) ions under a transmembrane potential of +200 mV. The total ionic currents at +200 mV is +0.017 nA. (G) Representative current traces of wild-type Cx31.3 hemichannels with indicated ion gradients (in millimolar concentration, cis side//trans side) across the bilayer. Ionic currents were evoked by 500-ms test pulses from −100 to +100 mV, with 10-mV increments. For clarity, currents are displayed in 20-mV increments. Red dashed lines mark zero-current level. (H) Current-voltage relation (I-V) curves in a symmetrical KCl (black circle), 10-fold KCl gradient (blue circle), or asymmetrical cation gradient (green circle). Currents averaged between 290 and 490 ms into the test pulses are normalized to the values at +100 mV. (I) Reversal potentials (V_rev) were determined by null-point measurement in a symmetrical KCl (black circle; n = 7), 10-fold KCl gradient (blue circle; n = 3), or asymmetrical cation conditions (green circle; n = 3). The Nernst potentials for the Cl⁻ (red dashed lines) are displayed in the indicated gradient. Each bar represents the means ± SEM.

Fig. 4. Comparison between wild-type and R15G-mutant hemichannels.

(A) ATP transport assay for wild-type (WT) and R15G-mutant Cx31.3 hemichannels. ATP-loaded liposomes with or without Cx31.3 hemichannels were subjected to desalting column chromatography. Extraliposomal and total ATPs in 10 μl of the eluted liposome fraction were measured by the luciferase assay and represented as white and gray bars, respectively. ATP loading before and Triton X-100 treatment after the desalting process were indicated below. The data are expressed as the means ± SD of the luminescence values from three independent experiments. The asterisks denote P < 0.05 compared between the indicated measurements. (B) Relative amounts of ATPs retained inside proteoliposomes compared with that inside protein-free liposomes. The intraliposomal ATP count was calculated from extraliposomal and total ATP counts in (A). The data are expressed as the means ± SE. (n = 3). The asterisk denotes P < 0.05 compared between the indicated values. (C) WT (gray) and R15G-mutant (yellow) hemichannels were superimposed and shown from cytoplasmic view. The box indicates close-up view of the region around NTHs. The residues showing substantial conformational changes by R15G mutation are represented in sticks. The NTH of the mutant Cx31.3 is a little stretched toward the pore center, compared with that of WT Cx31.3. The black dotted lines indicate salt bridges and hydrogen bonds. The blue lines indicate cation-π interactions. (D) The electrostatic potential of the cytoplasmic surface of WT hemichannel (left) is compared with that of R15G mutant (right). The APBS was used to calculate the electrostatic potential at pH 7. The displayed potentials range from −10 (red) to +10 (blue) kT/e. The location of R15G is indicated.

Fig. 5. The Ca²⁺-binding tunnel.

(A) Water molecules in the Ca²⁺-binding tunnel are shown as red spheres in the ribbon drawings of Cx31.3 hemichannel structure in the absence of Ca²⁺. (B) The tunnel-lining residues in the structurally aligned Ca²⁺-free (gray) and Ca²⁺-bound (cyan) hemichannels are represented in sticks and are labeled. F65, P70, L71, S72, P185, and K188 are not shown for clarity. Six water molecules in the tunnel of the Ca²⁺-free structure are shown in red spheres. Putative calcium ions in the tunnel of the Ca²⁺-bound structure are shown in green spheres. The black arrow indicates the tunnel entrance. (C) Comparison between the density maps of the Ca²⁺-binding tunnel in the presence and absence of calcium ions. Both maps are contoured at σ = 5.3. Two neighboring connexins in the hemichannel are colored pink and orange, respectively. The side chains of the residues are represented in sticks. (D and E) Schematic diagrams of the Ca²⁺-binding tunnel in the absence (D) and presence (E) of calcium ions. All tunnel-lining residues are shown and colored, as in (C). Dashed lines indicate the interactions of the tunnel-lining residues with water molecules (D) or putative calcium ions (E) in the distances equal or less than 4 Å. The interaction distances involving charged residues are labeled. Arrows indicate potential repulsion between calcium ions and basic residues. (F) On the surface representations of Cx31.3 hemichannel, 100% identically conserved residues among 20 human connexins are colored blue, >90% identically or 100% similarly conserved residues are colored cyan, and Cx31.3-specific residues are colored orange. Four of six connexin protomers are shown for clarity.

Fig. 6. Detailed structural comparison of Cx31.3 hemichannel with GJIChs.

(A) The Ca²⁺-binding tunnel is open in Cx31.3 hemichannel (left) but closed in Cx26 [Protein Data Bank (PDB ID), 2ZW3] (middle) and Cx46 (PDB ID, 6MHQ) (right) GJIChs. Cx31.3 hemichannel, Cx26 GJICh, and Cx46 GJICh are colored in gray, orange, and green, respectively. The black dotted lines indicate salt bridges. Red spheres represent water molecules in the tunnel. (B) Salt-bridge networks in and around Ca²⁺-binding tunnels of Ca²⁺-bound Cx31.3 hemichannel (left), Ca²⁺-free Cx31.3 hemichannel (middle), and Cx26 GJICh (right). The salt bridge–forming residues are shown in sticks and labeled. Salt bridges with distances less than 4 Å are indicated by black dotted lines. Transparent gray circles indicate Ca²⁺-binding tunnels. Red and green spheres represent water molecules and putative Ca²⁺-binding positions, respectively. Red two-way arrows represent high flexibility of the indicated residues. The linear and circular salt-bridge networks are highlighted by transparent red marking lines. (C) Structural differences in ECLs and TMDs between Cx31.3 hemichannel (gray) and Cx26 GJICh (orange). The overall structures of Cx31.3 and Cx26 channels were aligned, and two interfacing connexins in each channel are shown. Red arrows indicate substantial conformational shifts of NTHs in Cx26 GJICh compared to Cx31.3 hemichannel. (D) Schematic diagrams represent overall conformational differences between Cx31.3 hemichannel and Cx26 GJICh. Extracellular (top) and side (bottom) views are shown. Circled numbers indicate TM helices. Green circles indicate Ca²⁺-binding sites shown in the current Cx31.3 structure and the Ca²⁺-bound Cx26 GJICh structure (PDB ID, 5ER7).