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Structure of the connexin 26 gap junction channel at 3.5 Å resolution

Nature volume 458, pages 597–602 (2009)Cite this article

Abstract

Gap junctions consist of arrays of intercellular channels between adjacent cells that permit the exchange of ions and small molecules. Here we report the crystal structure of the gap junction channel formed by human connexin 26 (Cx26, also known as GJB2) at 3.5 Å resolution, and discuss structural determinants of solute transport through the channel. The density map showed the two membrane-spanning hemichannels and the arrangement of the four transmembrane helices of the six protomers forming each hemichannel. The hemichannels feature a positively charged cytoplasmic entrance, a funnel, a negatively charged transmembrane pathway, and an extracellular cavity. The pore is narrowed at the funnel, which is formed by the six amino-terminal helices lining the wall of the channel, which thus determines the molecular size restriction at the channel entrance. The structure of the Cx26 gap junction channel also has implications for the gating of the channel by the transjunctional voltage.

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Figure 1: Overall structure of the Cx26 gap junction channel in ribbon representation.
Figure 2: Stereo view of the Cx26 protomer in ribbon representation.
Figure 3: Molecular architecture of the Cx26 gap junction channel.
Figure 4: Pore structure of the Cx26 gap junction channel.
Figure 5: Structure of the pore funnel.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinate and the structure factor for the reported crystal structure have been deposited with the Protein Data Bank under accession code 2ZW3.

References

  1. Kumar, N. M. & Gilula, N. B. The gap junction communication channel. Cell 84, 381–388 (1996)

    Article  CAS  Google Scholar 

  2. Harris, A. L. Emerging issues of connexin channels: biophysics fills the gap. Q. Rev. Biophys. 34, 325–472 (2001)

    Article  CAS  Google Scholar 

  3. Foote, C. I., Zhou, L., Zhu, X. & Nicholson, B. J. The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for the docking interface of gap junctions. J. Cell Biol. 140, 1187–1197 (1998)

    Article  CAS  Google Scholar 

  4. Levin, M. Gap junctional communication in morphogenesis. Prog. Biophys. Mol. Biol. 94, 186–206 (2007)

    Article  CAS  Google Scholar 

  5. Saez, J. C., Berthoud, V. M., Branes, M. C., Martinez, A. D. & Beyer, E. C. Plasma membrane channels formed by connexins: their regulation and functions. Physiol. Rev. 83, 1359–1400 (2003)

    Article  CAS  Google Scholar 

  6. Kelsell, D. P., Dunlop, J. & Hodgins, M. B. Human diseases: clues to cracking the connexin code? Trends Cell Biol. 11, 2–6 (2001)

    Article  CAS  Google Scholar 

  7. Simon, A. M. & Goodenough, D. A. Diverse functions of vertebrate gap junctions. Trends Cell Biol. 8, 477–483 (1998)

    Article  CAS  Google Scholar 

  8. Unwin, P. N. & Zampighi, G. Structure of the junction between communicating cells. Nature 283, 545–549 (1980)

    Article  ADS  CAS  Google Scholar 

  9. Unwin, P. N. & Ennis, P. D. Two configurations of a channel-forming membrane protein. Nature 307, 609–613 (1984)

    Article  ADS  CAS  Google Scholar 

  10. Unger, V. M., Kumar, N. M., Gilula, N. B. & Yeager, M. Three-dimensional structure of a recombinant gap junction membrane channel. Science 283, 1176–1180 (1999)

    Article  ADS  CAS  Google Scholar 

  11. Fleishman, S. J., Unger, V. M., Yeager, M. & Ben-Tal, N. A C-α model for the transmembrane α helices of gap junction intercellular channels. Mol. Cell 15, 879–888 (2004)

    Article  CAS  Google Scholar 

  12. Oshima, A., Tani, K., Hiroaki, Y., Fujiyoshi, Y. & Sosinsky, G. E. Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule. Proc. Natl Acad. Sci. USA 104, 10034–10039 (2007)

    Article  ADS  CAS  Google Scholar 

  13. Harris, A. L., Spray, D. C. & Bennett, M. V. Kinetic properties of a voltage-dependent junctional conductance. J. Gen. Physiol. 77, 95–117 (1981)

    Article  CAS  Google Scholar 

  14. Verselis, V. K., Ginter, C. S. & Bargiello, T. A. Opposite voltage gating polarities of two closely related connexins. Nature 368, 348–351 (1994)

    Article  ADS  CAS  Google Scholar 

  15. Ebihara, L., Berthoud, V. M. & Beyer, E. C. Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels. Biophys. J. 68, 1796–1803 (1995)

    Article  ADS  CAS  Google Scholar 

  16. Bukauskas, F. F., Bukauskiene, A., Bennett, M. V. & Verselis, V. K. Gating properties of gap junction channels assembled from connexin 43 and connexin 43 fused with green fluorescent protein. Biophys. J. 81, 137–152 (2001)

    Article  CAS  Google Scholar 

  17. Bukauskas, F. F. & Verselis, V. K. Gap junction channel gating. Biochim. Biophys. Acta 1662, 42–60 (2004)

    Article  CAS  Google Scholar 

  18. Muller, D. J., Hand, G. M., Engel, A. & Sosinsky, G. E. Conformational changes in surface structures of isolated connexin 26 gap junctions. EMBO J. 21, 3598–3607 (2002)

    Article  CAS  Google Scholar 

  19. Perkins, G. A., Goodenough, D. A. & Sosinsky, G. E. Formation of the gap junction intercellular channel requires a 30° rotation for interdigitating two apposing connexons. J. Mol. Biol. 277, 171–177 (1998)

    Article  CAS  Google Scholar 

  20. Skerrett, I. M. et al. Identification of amimo acid residues lining the pore of a gap junction channel. J. Cell Biol. 159, 349–360 (2002)

    Article  CAS  Google Scholar 

  21. Zhou, X. W. et al. Identification of a pore lining segment in gap junction hemichannels. Biophys. J. 72, 1946–1953 (1997)

    Article  ADS  CAS  Google Scholar 

  22. Kronengold, J., Trexler, E. B., Bukauskas, F. F., Bargiello, T. A. & Verselis, V. K. Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels. J. Gen. Physiol. 122, 389–405 (2003)

    Article  CAS  Google Scholar 

  23. Suchyna, T. M., Xu, L. X., Gao, F., Fourtner, C. R. & Nicholson, B. J. Identification of a proline residue as a transduction element involved in voltage gating of gap junctions. Nature 365, 847–849 (1993)

    Article  ADS  CAS  Google Scholar 

  24. Laird, D. W. Life cycle of connexins in health and disease. Biochem. J. 394, 527–543 (2006)

    Article  CAS  Google Scholar 

  25. Sheridan, R. P., Levy, R. M. & Salemme, F. R. α-helix dipole model and electrostatic stabilization of 4-α-helical proteins. Proc. Natl Acad. Sci. USA 79, 4545–4549 (1982)

    Article  ADS  CAS  Google Scholar 

  26. Weber, P. A., Chang, H. C., Spaeth, K. E., Nitsche, J. M. & Nicholson, B. J. The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities. Biophys. J. 87, 958–973 (2004)

    Article  CAS  Google Scholar 

  27. Oh, S., Verselis, V. K. & Bargiello, T. A. Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannel. J. Physiol. (Lond.) 586, 2445–2461 (2008)

    Article  CAS  Google Scholar 

  28. Gong, X. Q. & Nicholson, B. J. Size selectivity between gap junction channels composed of different connexins. Cell Commun. Adhes. 8, 187–192 (2001)

    Article  CAS  Google Scholar 

  29. Trexler, E. B., Bukauskas, F. F., Kronengold, J., Bargiello, T. A. & Verselis, V. K. The first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels. Biophys. J. 79, 3036–3051 (2000)

    Article  CAS  Google Scholar 

  30. Purnick, P. E., Benjamin, D. C., Verselis, V. K., Bargiello, T. A. & Dowd, T. L. Structure of the amino terminus of a gap junction protein. Arch. Biochem. Biophys. 381, 181–190 (2000)

    Article  CAS  Google Scholar 

  31. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003)

    Article  ADS  CAS  Google Scholar 

  32. Kelsell, D. P. et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387, 80–83 (1997)

    Article  ADS  CAS  Google Scholar 

  33. Oshima, A., Doi, T., Mitsuoka, K., Maeda, S. & Fujiyoshi, Y. Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function. J. Biol. Chem. 278, 1807–1816 (2003)

    Article  CAS  Google Scholar 

  34. Oshima, A., Tani, K., Hiroaki, Y., Fujiyoshi, Y. & Sosinsky, G. E. Projection structure of a N-terminal deletion mutant of connexin 26 channel with decreased central pore density. Cell Commun. Adhes. 15, 85–93 (2008)

    Article  CAS  Google Scholar 

  35. Purnick, P. E., Oh, S., Abrams, C. K., Verselis, V. K. & Bargiello, T. A. Reversal of the gating polarity of gap junctions by negative charge substitutions in the N-terminus of connexin 32. Biophys. J. 79, 2403–2415 (2000)

    Article  CAS  Google Scholar 

  36. Oh, S., Rivkin, S., Tang, Q., Verselis, V. K. & Bargiello, T. A. Determinants of gating polarity of a connexin 32 hemichannel. Biophys. J. 87, 912–928 (2004)

    Article  CAS  Google Scholar 

  37. Oh, S., Abrams, C. K., Verselis, V. K. & Bargiello, T. A. Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin 32 chimera. J. Gen. Physiol. 116, 13–31 (2000)

    Article  CAS  Google Scholar 

  38. Jan, L. Y. & Jan, Y. N. Structural elements involved in specific K+ channel functions. Annu. Rev. Physiol. 54, 537–555 (1992)

    Article  CAS  Google Scholar 

  39. Trexler, E. B., Bennett, M. V. L., Bargiello, T. A. & Verselis, V. K. Voltage gating and permeation in a gap junction hemichannel. Proc. Natl Acad. Sci. USA 93, 5836–5841 (1996)

    Article  ADS  CAS  Google Scholar 

  40. Peracchia, C. Chemical gating of gap junction channels; roles of calcium, pH and calmodulin. Biochim. Biophys. Acta 1662, 61–80 (2004)

    Article  CAS  Google Scholar 

  41. Delmar, M., Coombs, W., Sorgen, P., Duffy, H. S. & Taffet, S. M. Structural bases for the chemical regulation of connexin43 channels. Cardiovasc. Res. 62, 268–275 (2004)

    Article  CAS  Google Scholar 

  42. Tao, L. & Harris, A. L. 2-Aminoethoxydiphenyl borate directly inhibits channels composed of connexin26 and/or connexin32. Mol. Pharmacol. 71, 570–579 (2007)

    Article  CAS  Google Scholar 

  43. Baker, N. A., Sept, D., Joseph, S. & Holst, M. J. McCammon. J. A. Electrostatics of nanosystems: applications to microtubles and the ribosomes. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    Article  ADS  CAS  Google Scholar 

  44. Stauffer, K. A., Kumar, N. M., Gilula, N. B. & Unwin, N. Isolation and purification of gap junction channels. J. Cell Biol. 115, 141–150 (1991)

    Article  CAS  Google Scholar 

  45. Bellizzi, J. J., Widom, J., Kemp, C. W. & Clardy, J. Producing selenomethionine-labeled proteins with a baculovirus expression vector system. Structure 7, R263–R267 (1999)

    Article  CAS  Google Scholar 

  46. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  47. Collaborative Computational Project 4 The CCP4 suite: Programs for Protein Crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  48. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993)

    Article  CAS  Google Scholar 

  49. Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

    Article  CAS  Google Scholar 

  50. Cowtan, K. An automated procedure for phase improvement by density modification. Joint CCP4 ESF-EACBM Newsletter Protein Crystallogr. 31, 34–38 (1994)

    Google Scholar 

  51. Cowtan, K. D. & Zhang, K. Y. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999)

    Article  CAS  Google Scholar 

  52. Jones, T. A., Zou, J. Y. & Cowan, S. W. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  53. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  54. Brunger, A. T. et al. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  55. Murshudov, G. N., Vagin, A. A. & Dadson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  56. Dodson, E., Kleywegt, G. J. & Wilson, K. Report of a workshop on the use of statistical validators in protein X-ray crystallography. Acta. Crystallogr. D 52, 228–234 (1996)

    Article  CAS  Google Scholar 

  57. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  58. Delano, W. L. The PyMOL Molecular Graphics System. v.0.99 (Delano Scientific, 2006)

    Google Scholar 

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Acknowledgements

We thank T. Tomizaki for help in the diffraction data collection on X06SA at the Swiss Light Source. This work was supported by Grants-in-Aid for Scientific Research (10687101, 16087206 and 18207006) and the GCOE program (A-041) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.T.), the Japan Biological Informatics Consortium (to T.T.), the Strategic Japan-UK Cooperation Program of the Japan Science and Technology Agency (to T.T.), and Grants-in-Aid for Specially Promoted Research (to Y.F.) and the New Energy and Industrial Technology Development Organization (to Y.F.). We thank T. Walz for critical reading of this manuscript.

Author Contributions S.M., S.N., M.S., E.Y. and T.T. performed X-ray structural analysis. S.M., A.O., Y.F. and T.T. wrote the paper.

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Authors and Affiliations

  1. Institute for Protein Research, Osaka University, OLABB, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan ,

    Shoji Maeda, So Nakagawa, Michihiro Suga, Eiki Yamashita & Tomitake Tsukihara

  2. Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan,

    Atsunori Oshima & Yoshinori Fujiyoshi

  3. Picobiology Institute, Graduate School of Life Science, University of Hyogo, Kamigohori, Akoh, Hyogo 678-1297, Japan ,

    Tomitake Tsukihara

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Correspondence to Tomitake Tsukihara.

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Maeda, S., Nakagawa, S., Suga, M. et al. Structure of the connexin 26 gap junction channel at 3.5 Å resolution. Nature 458, 597–602 (2009). https://doi.org/10.1038/nature07869

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