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PNAS 104 (24): 10034-10039

Copyright © 2007 by the National Academy of Sciences.

Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule

Atsunori Oshima{dagger},{ddagger}, Kazutoshi Tani{dagger}, Yoko Hiroaki{dagger},{ddagger}, Yoshinori Fujiyoshi{dagger},{ddagger},§,, and Gina E. Sosinsky,||

{dagger}Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; ||National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0608; {ddagger}Core Research for Evolution Science and Technology (CREST), Japan Science and Technology Agency (JST), Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; and §Japan Biological Information Research Center (JBIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-41-6, Aomi, Koto-ku, Tokyo 135-0064, Japan


Figure 1
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  		Fig. 1. Three-dimensional structure of Cx26 orthorhombic crystals.(A) Molecular packing of Cx26 in the 2D crystal. The gap junction channels are incorporated in three lipid bilayers (Mem-1–Mem-3) with 21 symmetry along Mem-2. (B) View of the Cx26 density map perpendicular to the membrane plane. The three membranes, indicated by gray bars, surround two extracellular gap regions. The map is contoured at 1.0{sigma} (light blue) and 2.4{sigma} (wheat color) above the mean density. The transmembrane {alpha}-helical ribbon model (6) is docked into the density for one of the hemichannels. The four helices are color-coded as in Fig. 2. Two helices D make contact with adjacent gap junction channels (red arrows). (Scale bar, 40 Å.) (C and D) Forty-angstrom-thick sections through the density map parallel to the membrane plane, showing protein embedded in membranes Mem-1 (identical to Mem-3) (C) and Mem-2 (D). Tail ends of two helices D are indicated by red arrows as in B. (Scale bars, 40 Å.)

 

Figure 2
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  		Fig. 2. Structural details of the Cx26 gap junction. The map is contoured as in Fig. 1. (Inset) Twenty-angstrom-thick section perpendicular to the membrane plane through the density map of a hemichannel in Mem-2. This section corresponds to the region enclosed by the white lines shown in A. The arrowhead points to the large density in the pore. The inner cytoplasmic protrusions (white arrows) extend from the cytoplasmic ends of helices B and C. (A–C) Thirty-angstrom-thick slabs through the density map corresponding to the position of the lines shown in Inset. The four helices are labeled A (cyan, A'), B (green, B'), C (yellow), and D (pink) as in the original Cx43 structure (5). The arrowhead and white arrows represent the plug and the inner cytoplasmic protrusions, respectively, as in Inset.

 

Figure 3
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  		Fig. 3. Superimposition of the transmembrane helices of Cx26 (yellow) and Cx43 (red) (6). The arrangement of the helices differs slightly between the two connexins, but all of the corresponding helices more or less overlap with each other. The channel dimensions and the pore diameters are approximately the same in gap junctions formed by Cx26 and Cx43.

 

Figure 4
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  		Fig. 4. Stereoview of the surface structure of Cx26 perpendicular to the membrane plane. The cytoplasmic protrusions are clearly defined. The map is contoured at 1.0{sigma} (solid surface) and at 2.4{sigma} (wheat-colored mesh) above the mean density. Helices B (green) and C (yellow) are color coded as in Fig. 2.

 

Figure 5
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  		Fig. 5. Stereo top view of the Cx26 density map to which a B-factor of –700 was applied. The B-factor was applied to enhance the amplitudes of the high-resolution reflections, revealing six {alpha}-helix-like features in the plug density and protrusions that probably reflect the loops connecting the plug to the surrounding channel wall. The four helices are color-coded A (cyan, A'), B (green, B'), C (yellow), and D (pink) as in Fig. 2.

 

Figure 6
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  		Fig. 6. Hypothesized plug gating mechanism of gap junctions. Each hemichannel (green) can regulate its channel activity autonomously. The gap junction is open only when the plugs (red) in both hemichannels are displaced from the channel constriction formed by the innermost helices C (yellow) toward the cytoplasmic side. The flexible connections of the plug with the channel are shown as red dashed lines.

 

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