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Annotations
20. Burendei B, Shinozaki R, Watanabe M, Terada T, Tani K, Fujiyoshi Y, Oshima A: Cryo-EM
structures of undocked innexin-6 hemichannels in phospholipids. Sci Adv 2020, 6, eaax3157 doi:
10.1126/sciadv.aax3157.
●● This work shows the first high-resolution structure of an undocked INX-6 gap junction hemichannel
in a nanodisc determined by cryo-EM. The pore of the channel is blocked by double-layer densities
along with N-terminal rearrangement suggesting a conformational change in the N-terminal domains
in the presence and absence of phospholipids.
19. Myers JB, Haddad BG, O'Neill SE, Chorev DS, Yoshioka CC, Robinson CV, Zuckerman DM,
Reichow SL: Structure of native lens connexin 46/50 intercellular channels by cryo-EM. Nature
2018, 564:372–377.
●● The cryo-EM structures of Cx46/50 isolated from native sheep lens are reported. Intriguingly, highresolution structure determination was achieved even though a single channel contains mixed
connexins of Cx46 and Cx50. The N-terminus is reasonably oriented in the pore as hydrophobic side
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chains face the pore wall.
34. Kasuya G, Nakane T, Yokoyama T, Jia Y, Inoue M, Watanabe K, Nakamura R, Nishizawa T,
Kusakizako T, Tsutsumi A, et al: Cryo-EM structures of the human volume-regulated anion
channel LRRC8. Nat Struct Mol Biol 2018, 25:797–804. doi: 10.1038/s41594-018-0109-6.
●● This work reports a cryo-EM structure of human LRRC8A. Interestingly, a C3 hexameric channel
with a different amount of space between the adjacent transmembrane bundles was determined. The
asymmetric feature of compact and relaxed forms may be useful for understanding the molecular
mechanism of the LRRC8A channel gating.
45. Kern DM, Oh S, Hite RK, Brohawn SG: Cryo-EM structures of the DCPIB-inhibited volumeregulated anion channel LRRC8A in lipid nanodiscs. Elife 2019, 8:e42636.
The cryo-EM structure of mouse LRRC8A in complex with DCPIB, an anionic inhibitor, in lipid
nanodiscs is reported. The structure suggests a mechanism of channel inhibition by DCPIB along with
lipid molecules, where DCPIB stays like a cork in a bottle. Six-fold rotational symmetry has been
applied.
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Figure legends
Figure 1
Electrostatic surface potential distribution of three connexin gap junction channels.
The pore surfaces of Cx50 (A) [19●●], Cx46 (B) [19●●], and Cx26 (C) [12] are shown. Negative
potentials are colored in red, and positive potentials are in blue. The contour level is from –10 kT/e to +10
kT/e.
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Figure 2
Structures of the N-terminal domains of Cx46 and Cx26
(A) The Cx46 pore wall viewed from inside the pore [19●●]. The N-terminal helix (NTH) of Cx46
(magenta) is distributed in the pocket generated by TM1, TM2 (green), and adjacent subunit TM1 (cyan),
where the hydrophobic face of NTH is buried. The nearby hydrophobic side chains are depicted as a stick
model.
(B) The X-ray structure of Cx26 shows the six NTHs forming a pore funnel stabilized by a circular
network of hydrogen bonds between Asp2 and Thr5 [12]. The hydrogen bonds are shown as red dashed
lines. NTH of each subunit is color coded. The pore wall of Cx26 is shown as a surface representation.
(C) Crystallographic temperature factor distribution of Cx26 [12]. A gap junction channel (left) and a
monomer (right) of Cx26 are shown with colors representing the range between 70 Å2 (blue) and 220 Å2
(red).
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Figure 3
Cryo-EM structures of undocked INX-6 hemichannels [20●●]
(A~C) The three-dimensional structures of undocked INX-6 hemichannels of a nanodisc-reconstituted
wild-type INX-6 (A), wild-type INX-6 in detergent (B), and N-terminal deleted INX-6 in a nanodisc (C).
The nanodisc densities are colored in magenta, and the double-layer pore obstructing densities are shown
in green. Densities, probably corresponding to the N-terminus, are shown in slate and orange,
respectively. (D) Schematic representation of the N-terminal rearrangement of the INX-6 undocked
hemichannel in the lipid bilayer environment. Upon reconstitution in phospholipids, the N-terminus might
be deflected toward the cytoplasmic side of the channel from a funnel. It remains uncertain if the Nterminus can be deflected outside the channel. Labeling is as follows; C-dome: cytoplasmic dome, E1:
first extracellular loop, E2: second extracellular loop, TM1~TM4: transmembrane helix 1 to 4, NTH: Nterminal helix. (E) Unassigned densities are observed in the space between adjacent transmembrane helix
bundles (orange ellipse).
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