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    • br Although D reconstruction of an invertebrate gap

    2022-05-26


    Although 3D reconstruction of an invertebrate gap junction channel from native tissue of crayfish was reported in 1991, the negative-staining EM imaging was limited to low-resolution structural analysis [39]. The oligomeric number of innexin channels was believed to be the same as that in connexin, a dodecameric channel [39,40]. The structural studies of innexin channels aiming for high-resolution were initiated a few years ago. A 3D reconstruction of a recombinant innexin channel at 10 Å resolution was obtained from 2D crystals of Ce-INX6 using electron crystallography [41] (Figure 2a). It was demonstrated that Ce-INX6 with an N-terminal deletion construct (Ce-INX6ΔN) is missing amino Nuclear/Cytosol Fractionation Kit sale residues 2 through 19, contains 16 subunits, and therefore has a larger pore diameter than Cx26. This structure revealed four additional densities on the channel pore (Figure 2a), reminiscent of the plug density in the Cx26M34A channel []. While this might be indicative of a loss of functional permeability due to the N-terminal deletion, it was impossible to provide a plausible interpretation for those densities because of the low-resolution (10 Å) and the lack of an available atomic model of innexin [41]. Single-particle cryo-EM is a powerful tool for studying high-resolution structures without crystallization, which allowed us to obtain an atomic model of wild-type Ce-INX6 (Figure 2b) []. GraDeR [42], a method for excluding free detergent micelles by density gradient ultra-centrifugation, was critical for improving the quality of the cryo-EM images, resulting in the high-resolution structural analysis. The structure not only confirmed the hexadecameric subunits of Ce-INX6, but also revealed most of the cytoplasmic domains, which were ambiguous in earlier structures of connexins. A number of helix-turn-helix motifs are formed by the cytoplasmic loop and C-terminus, referred to as the cytoplasmic dome, in a single Ce-INX6 subunit, making up a dome configuration in an octameric hemichannel. In Cx43, nuclear magnetic resonance spectroscopy (NMR) has provided insights into the solution structures of the cytoplasmic loop and carboxyl-terminal domain peptides that contain a couple of alpha helices in a random coil [43,44], which may show a similarity to Ce-INX6. The N-terminal portion carries a short helix and has a funnel conformation (Figure 2c), consistent with the Cx26 X-ray structure by Maeda et al. []. Although the six residues at the N-terminal end are not completely resolved, the 3D density map looks like an open channel because the most constricted region in the pore has a diameter of over 10 Å. Interestingly, a part of the C-terminal loop in the cytoplasmic dome covers the N-terminal funnel, suggesting that it contributes to regulating channel activity (Figure 2d). Ce-INX6 and Cx26 are correlated to each other in the arrangement of the extracellular docking interface with an anti-parallel beta sheet in E2 and a short alpha helix in E1. Whereas only E1 of Ce-INX6 is involved in the interactions between two opposed hemichannels, both E1 and E2 of Cx26 contribute to these interactions with more hydrogen bonds (Figure 2e). The tightness of docking interaction may therefore not be identical to each other. High-resolution structures clearly show that the resolved part of Cx26 is relevant to Ce-INX6 regarding not only the monomeric but also oligomeric structures, despite having different subunit numbers. In connexin, cytoplasmic pH and aminosulfonates are intrinsic modulators of channel function [2,37]. A direct interaction between the C-terminus and the second half of the cytoplasmic loop is implicated for Cx43 [43]. Whether the arrangement of the cytoplasmic domains is analogous between the innexin and connexin gap junction families should be carefully considered, however, because the lengths and sequences of the cytoplasmic loop and C-terminal domains are not consistent. The recent studies of LRRC8 may help to clarify this issue. LRRC8 proteins function as single membrane anion channels and have low sequence similarity with innexins and pannexins [10]. The resolved monomeric and oligomeric arrangements of LRRC8A highly correlate with the hemichannel parts of Cx26 and Ce-INX6, and its cytoplasmic domains comprising helix-turn-helix are common with Ce-INX6 [15, 16, 17]. In this regard, the working model of functional regulation by the cytoplasmic dome of the Ce-INX6 structure (Figure 3b) may be partially shared with LRRC8 family proteins.