Research ArticleSTRUCTURAL BIOLOGY

Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids

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Science Advances  12 Feb 2020:
Vol. 6, no. 7, eaax3157
DOI: 10.1126/sciadv.aax3157
  • Fig. 1 Cryo-EM structure of the undocked INX-6 hemichannel in a nanodisc.

    (A) The 3D reconstruction of a nanodisc-reconstituted WT INX-6 hemichannel viewed parallel to the membrane. The subunit highlighted in blue represents a monomer. The map contour level is 2σ. (B) Octameric (left) and monomeric (right) ribbon models of an undocked hemichannel of WT INX-6 in a nanodisc. A single subunit of the octamer is indicated in dark blue, the extracted monomer is in rainbow colors, and the lipid bilayer boundary is indicated by a gray band. The N-terminal residue is assigned from S16. S16, I52, and G53 are represented in stick style. The four transmembrane helices are indicated as TM1 to TM4. (C) Comparison of the extracellular docking surface of WT INX-6. (Left) Ribbon style model and density map (surface representation in gray) of the nanodisc-reconstituted undocked hemichannel are superimposed. Disordered peptides of the E1 outer lobe and E2 loop are shown as dashed lines, and the flanking residues of L86, S101, N251, and T262 are indicated. (Right) Ribbon style model of a docked junction channel (PDB: 5H1R) (6). The amino acid side chains that participate in the hydrogen bonds are depicted in stick style and are all included in the disordered regions in the undocked hemichannel. The map contour level is 2σ. (D) Sliced views of the Gaussian-filtered 3D density map of the undocked WT INX-6 hemichannel as a vertical cross section (left) and cytoplasmic top view of the slab surface (right). Slab thickness corresponds to the bracket in a side view (left). The surrounding nanodisc densities are shown in magenta, and the double-layer densities that obstruct the pore are shown in green. Newly observed densities X and Y are shown in slate and orange, respectively. The map contour level is 1.5σ. (E) Positional relationship of density Y to TM1 of WT INX-6. Density Y is located within a distance of less than 10 Å from A27, the closest residue in TM1 to density Y. The map contour level is 2σ.

  • Fig. 2 Undocked hemichannel structures of WT INX-6 in detergent and INX-6ΔN in a nanodisc.

    (A) Sliced views of the Gaussian-filtered 3D density map of WT INX-6 in detergent. The surrounding detergent densities are shown in pink, and the N-terminal funnels are shown in red. The ribbon style model is superimposed. The map contour level is 1.5σ. (B) Ribbon style model of a monomeric structure of WT INX-6 in detergent. The assigned residues at the N terminus begin from N12 (shown in stick style), and the I52 and G53 residues are not visible due to disorder. (C) Sliced views of the Gaussian-filtered 3D density map of INX-6ΔN in a nanodisc. The densities corresponding to the nanodisc are shown in magenta, and the ribbon style model is superimposed. The double-layer densities in the pore are shown in green. The thickness of the top view slab is indicated by the bracket in the side view. The map contour level is 1.5σ. (D) Comparison of WT INX-6 to INX-6ΔN in nanodiscs. A subtraction map of INX-6ΔN from WT INX-6 (blue) is superimposed on the structure of INX-6ΔN (yellow), where the represented slab corresponds to the bracket in (C). The map contour level is 3σ.

  • Fig. 3 MD simulation of the undocked INX-6 hemichannels embedded in phospholipids and unassigned densities in cryo-EM maps.

    (A to C) The final models of the three atomic structures obtained by cryo-EM in this work after independently performed MD simulations in POPC for 100 ns [120 ns for (B)]. Slab sections of WT INX-6 in a nanodisc (A), INX-6ΔN in a nanodisc (B), and WT INX-6 in detergent (C) corresponding to the transmembrane domain along with POPC molecules are viewed from the cytoplasmic side. POPC models that are inserted in the space between adjacent subunits are indicated by red circles. (D to F) Unassigned densities (red circles) observed in the space between transmembrane helix bundles of adjacent subunits of WT INX-6 in a nanodisc (D), INX-6ΔN in a nanodisc (E), and WT INX-6 in detergent (F) viewed from the orientation horizontal to the membrane plane.

  • Fig. 4 Functional analysis of INX-6 hemichannels with single Xenopus oocyte voltage clamp.

    (A) Measured hemichannel currents of INX-6 and negative control (H2O) at 50 mV and steady state. Error bars correspond to SE (*P < 0.05 and ***P < 0.001). (B) Representative currents of WT INX-6 (top) and INX-6ΔN2-19 (bottom). The applied membrane potential is increased from −30 to +50 mV in 10-mV steps.

  • Fig. 5 Schematic representation of the N-terminal rearrangement of the INX-6 undocked hemichannel in the lipid bilayer environment.

    (A) INX-6 hemichannel embedded in phospholipids. Instead of the no-funnel configuration, the NTH is rearranged, and possibly the phospholipids enter the pore. The N terminus might be deflected toward the outside of the channel (dotted lines). (B) INX-6 hemichannel surrounded by detergent micelles. NTHs form a funnel configuration with a wide-open pore. The constriction is formed by the E1 inner lobes but may not be the smallest because more than 10 residues at the N terminus are still disordered.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/7/eaax3157/DC1

    Fig. S1. Cryo-EM of the undocked WT INX-6 hemichannel in a nanodisc.

    Fig. S2. Comparison of INX-6 channels between docked and undocked forms focusing on extracellular domains and the pore pathway.

    Fig. S3. Sequence alignment of INX-6 and docking interface residues.

    Fig. S4. Cryo-EM of the undocked WT INX-6 hemichannel in detergent prepared by GraDeR.

    Fig. S5. Cryo-EM of INX-6ΔN in an undocked hemichannel form in a nanodisc.

    Fig. S6. MD simulations of the undocked INX-6 hemichannels.

    Fig. S7. Fluorescent micrographs of INX-6 distribution on Xenopus oocytes.

    Table S1. Summary of data and statistics of the undocked INX-6 hemichannel structure determination.

    Reference (69)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Cryo-EM of the undocked WT INX-6 hemichannel in a nanodisc.
    • Fig. S2. Comparison of INX-6 channels between docked and undocked forms focusing on extracellular domains and the pore pathway.
    • Fig. S3. Sequence alignment of INX-6 and docking interface residues.
    • Fig. S4. Cryo-EM of the undocked WT INX-6 hemichannel in detergent prepared by GraDeR.
    • Fig. S5. Cryo-EM of INX-6ΔN in an undocked hemichannel form in a nanodisc.
    • Fig. S6. MD simulations of the undocked INX-6 hemichannels.
    • Fig. S7. Fluorescent micrographs of INX-6 distribution on Xenopus oocytes.
    • Table S1. Summary of data and statistics of the undocked INX-6 hemichannel structure determination.
    • Reference (69)

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