[go: up one dir, main page]
Skip to main content
SpringerLink
Log in

Mechanism of Two Novel Human GJC3 Missense Mutations in Causing Non-Syndromic Hearing Loss

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

Connexins (CXs), as a component of gap junction channel, are homologous four transmembrane-domain proteins, with numerous studies confirming their auditory functions. Among a cohort of patients having incurred non-syndromic hearing loss, we identified two novel missense mutations, p.R15G and p.L23H, in the GJC3 gene encoding CX30.2/CX31.3, as causally related to hearing loss in previous study. However, the functional alteration of CX30.2/CX31.3 caused by the mutant GJC3 gene remains unknown. In this study, we compared the intracellular distribution of mutant CX30.2/CX31.3 (p.R15G and p.L23H) with the wild-type (WT) protein in HeLa cells and the effect of the mutant protein had on those cells. Analytical results indicated that p.R15G and p.L23H mutant exhibited continuous staining along apposed cell membranes in the fluorescent localization assay, which is the same with the WT. Moreover, ATP release (hemichannel function) is less in HeLa cells carrying mutant GJC3 genes than those of WT expressing cells. We believe that although p.R15G and p.L23H mutants do not decrease the trafficking of CX proteins, mutations in GJC3 genes result in a loss of hemichannel function of CX30.2/CX31.3 protein, possibly causing hearing loss. Results of this study provide a novel molecular explanation for the role of GJC3 in hearing loss.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Bitner-Glindzicz, M. (2002). Hereditary deafness and phenotyping in humans. British Medical Bulletin, 63, 73–94.

    Article  PubMed  CAS  Google Scholar 

  2. Morton, N. E. (1991). Genetic epidemiology of hearing impairment. Annals of the New York Academy of Sciences, 630, 16–31.

    Article  PubMed  CAS  Google Scholar 

  3. Gorlin, R. J. T. H., & Cohen, M. M. J. (1995). Hereditary hearing loss and its syndromes. Oxford: Oxford University Press.

    Google Scholar 

  4. Petit, C., Levilliers, J., & Hardelin, J. P. (2001). Molecular genetics of hearing loss. Annual Review of Genetics, 35, 589–646.

    Article  PubMed  CAS  Google Scholar 

  5. Willems, P. J. (2000). Genetic causes of hearing loss. New England Journal of Medicine, 342, 1101–1109.

    Article  PubMed  CAS  Google Scholar 

  6. Resendes, B. L., Williamson, R. E., & Morton, C. C. (2001). At the speed of sound: gene discovery in the auditory system. American Journal of Human Genetics, 69, 923–935.

    Article  PubMed  CAS  Google Scholar 

  7. Morton, C. C. (2002). Genetics, genomics and gene discovery in the auditory system. Human Molecular Genetics, 11, 1229–1240.

    Article  PubMed  CAS  Google Scholar 

  8. Lautermann, J., ten Cate, W. J., Altenhoff, P., Grummer, R., Traub, O., Frank, H., et al. (1998). Expression of the gap-junction connexins 26 and 30 in the rat cochlea. Cell and Tissue Research, 294, 415–420.

    Article  PubMed  CAS  Google Scholar 

  9. Forge, A., Becker, D., Casalotti, S., Edwards, J., Evans, W. H., Lench, N., et al. (1999). Gap junctions and connexin expression in the inner ear. Novartis Foundation Symposium, 219, 134–150.

    PubMed  CAS  Google Scholar 

  10. Kelley, P. M., Abe, S., Askew, J. W., Smith, S. D., Usami, S., & Kimberling, W. J. (1999). Human connexin 30 (GJB6), a candidate gene for nonsyndromic hearing loss: molecular cloning, tissue-specific expression, and assignment to chromosome 13q12. Genomics, 62, 172–176.

    Article  PubMed  CAS  Google Scholar 

  11. Xia, A. P., Ikeda, K., Katori, Y., Oshima, T., Kikuchi, T., & Takasaka, T. (2000). Expression of connexin 31 in the developing mouse cochlea. NeuroReport, 11, 2449–2453.

    Article  PubMed  CAS  Google Scholar 

  12. Liu, X. Z., Xia, X. J., Adams, J., Chen, Z. Y., Welch, K. O., Tekin, M., et al. (2001). Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness. Human Molecular Genetics, 10, 2945–2951.

    Article  PubMed  CAS  Google Scholar 

  13. Yang, J. J., Huang, S. H., Chou, K. H., Liao, P. J., Su, C. C., & Li, S. Y. (2007). Identification of mutations in members of the connexin gene family as a cause of nonsyndromic deafness in Taiwan. Audiology and Neurootology, 12, 198–208.

    Article  CAS  Google Scholar 

  14. Yang, J. J., Wang, W. H., Lin, Y. C., Weng, H. H., Yang, J. T., Hwang, C. F., et al. (2010). Prospective variants screening of connexin genes in children with hearing impairment: genotype/phenotype correlation. Human Genetics, 128, 303–313.

    Article  PubMed  CAS  Google Scholar 

  15. Wang, W. H., Yang, J. J., Lin, Y. C., Yang, J. T., Chan, C. H., & Li, S. Y. (2010). Identification of novel variants in the Cx29 gene of nonsyndromic hearing loss patients using buccal cells and RFLP method. Audiology and Neurootology, 15, 81–87.

    Article  CAS  Google Scholar 

  16. Spicer, S. S., & Schulte, B. A. (1996). The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency. Hearing Research, 100, 80–100.

    Article  PubMed  CAS  Google Scholar 

  17. Spicer, S. S., & Schulte, B. A. (1998). Evidence for a medial K + recycling pathway from inner hair cells. Hearing Research, 118, 1–12.

    Article  PubMed  CAS  Google Scholar 

  18. Denoyelle, F., Lina-Granade, G., Plauchu, H., Bruzzone, R., Chaib, H., Levi-Acobas, F., et al. (1998). Connexin 26 gene linked to a dominant deafness. Nature, 393, 319–320.

    Article  PubMed  CAS  Google Scholar 

  19. Kelley, P. M., Harris, D. J., Comer, B. C., Askew, J. W., Fowler, T., Smith, S. D., et al. (1998). Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. American Journal of Human Genetics, 62, 792–799.

    Article  PubMed  CAS  Google Scholar 

  20. Xia, J. H., Liu, C. Y., Tang, B. S., Pan, Q., Huang, L., Dai, H. P., et al. (1998). Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment. Nature Genetics, 20, 370–373.

    Article  PubMed  CAS  Google Scholar 

  21. Grifa, A., Wagner, C. A., D’Ambrosio, L., Melchionda, S., Bernardi, F., Lopez-Bigas, N., et al. (1999). Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nature Genetics, 23, 16–18.

    PubMed  CAS  Google Scholar 

  22. Hamelmann, C., Amedofu, G. K., Albrecht, K., Muntau, B., Gelhaus, A., Brobby, G. W., et al. (2001). Pattern of connexin 26 (GJB2) mutations causing sensorineural hearing impairment in Ghana. Human Mutation, 18, 84–85.

    Article  PubMed  CAS  Google Scholar 

  23. Pallares-Ruiz, N., Blanchet, P., Mondain, M., Claustres, M., & Roux, A. F. (2002). A large deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect? European Journal of Human Genetics, 10, 72–76.

    Article  PubMed  CAS  Google Scholar 

  24. Makowski, L., Caspar, D. L., Phillips, W. C., Baker, T. S., & Goodenough, D. A. (1984). Gap junction structures. VI. Variation and conservation in connexon conformation and packing. Biophysical Journal, 45, 208–218.

    Article  PubMed  CAS  Google Scholar 

  25. Willecke, K., Eiberger, J., Degen, J., Eckardt, D., Romualdi, A., Guldenagel, M., et al. (2002). Structural and functional diversity of connexin genes in the mouse and human genome. Biological chemistry, 383, 725–737.

    Article  PubMed  CAS  Google Scholar 

  26. Bruzzone, R., White, T. W., & Paul, D. L. (1996). Connections with connexins: the molecular basis of direct intercellular signaling. European Journal of Biochemistry, 238, 1–27.

    Article  PubMed  CAS  Google Scholar 

  27. Steel, K. P., Barkway, C., & Bock, G. R. (1987). Strial dysfunction in mice with cochleo-saccular abnormalities. Hearing Research, 27, 11–26.

    Article  PubMed  CAS  Google Scholar 

  28. Sohl, G., Eiberger, J., Jung, Y. T., Kozak, C. A., & Willecke, K. (2001). The mouse gap junction gene connexin29 is highly expressed in sciatic nerve and regulated during brain development. Biological chemistry, 382, 973–978.

    Article  PubMed  CAS  Google Scholar 

  29. Sohl, G., Nielsen, P. A., Eiberger, J., & Willecke, K. (2003). Expression profiles of the novel human connexin genes hCx30.2, hCx40.1, and hCx62 differ from their putative mouse orthologues. Cell Communication and Adhesion, 10, 27–36.

    Article  PubMed  Google Scholar 

  30. Altevogt, B. M., Kleopa, K. A., Postma, F. R., Scherer, S. S., & Paul, D. L. (2002). Connexin29 is uniquely distributed within myelinating glial cells of the central and peripheral nervous systems. Journal of Neuroscience, 22, 6458–6470.

    PubMed  CAS  Google Scholar 

  31. Ahmad, S., Chen, S., Sun, J., & Lin, X. (2003). Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochemical and Biophysical Research Communications, 307, 362–368.

    Article  PubMed  CAS  Google Scholar 

  32. Yang, J. J., Liao, P. J., Su, C. C., & Li, S. Y. (2005). Expression patterns of connexin 29 (GJE1) in mouse and rat cochlea. Biochemical and Biophysical Research Communications, 338, 723–728.

    Article  PubMed  CAS  Google Scholar 

  33. Tang, W., Zhang, Y., Chang, Q., Ahmad, S., Dahlke, I., Yi, H., et al. (2006). Connexin29 is highly expressed in cochlear Schwann cells, and it is required for the normal development and function of the auditory nerve of mice. Journal of Neuroscience, 26, 1991–1999.

    Article  PubMed  CAS  Google Scholar 

  34. Hong, H. M., Yang, J. J., Su, C. C., Chang, J. Y., Li, T. C., & Li, S. Y. (2010). A novel mutation in the connexin 29 gene may contribute to nonsyndromic hearing loss. Human Genetics, 127, 191–199.

    Article  PubMed  CAS  Google Scholar 

  35. Berezin, C., Glaser, F., Rosenberg, J., Paz, I., Pupko, T., Fariselli, P., et al. (2004). ConSeq: the identification of functionally and structurally important residues in protein sequences. Bioinformatics, 20, 1322–1324.

    Article  PubMed  CAS  Google Scholar 

  36. Liang, W. G., Su, C. C., Nian, J. H., Chiang, A. S., Li, S. Y., & Yang, J. J. (2011). Human connexin 30.2/31.3 (GJC3) does not form functional gap junction channels but causes enhanced ATP release in HeLa cells. Cell Biochemistry and Biophysics, 61, 189–197.

    Article  PubMed  CAS  Google Scholar 

  37. George, C. H., Martin, P. E., & Evans, W. H. (1998). Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins. Biochemical and Biophysical Research Communications, 247, 785–789.

    Article  PubMed  CAS  Google Scholar 

  38. Rubin, J. B., Verselis, V. K., Bennett, M. V., & Bargiello, T. A. (1992). A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32. Proceedings of the National Academy of Sciences of the United States of America, 89, 3820–3824.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  40. Maeda, S., Nakagawa, S., Suga, M., Yamashita, E., Oshima, A., Fujiyoshi, Y., et al. (2009). Structure of the connexin 26 gap junction channel at 3.5 A resolution. Nature, 458, 597–602.

    Article  PubMed  CAS  Google Scholar 

  41. Nakagawa, S., Maeda, S., & Tsukihara, T. (2010). Structural and functional studies of gap junction channels. Current Opinion in Structural Biology, 20, 423–430.

    Article  PubMed  CAS  Google Scholar 

  42. Purnick, P. E., Benjamin, D. C., Verselis, V. K., Bargiello, T. A., & Dowd, T. L. (2000). Structure of the amino terminus of a gap junction protein. Archives of Biochemistry and Biophysics, 381, 181–190.

    Article  PubMed  CAS  Google Scholar 

  43. Cruciani, V., & Mikalsen, S. O. (2006). The vertebrate connexin family. Cellular and Molecular Life Sciences, 63, 1125–1140.

    Article  PubMed  CAS  Google Scholar 

  44. Beyer, E. C., & Berthoud, V. M. (2009). The family of connexin gene. In A. Harris & D. Locke (Eds.), Connexin: A guide (pp. 3–26). Springer: Humana Press.

    Chapter  Google Scholar 

  45. Oshima, A., Tani, K., Hiroaki, Y., Fujiyoshi, Y., & Sosinsky, G. E. (2007). Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule. Proceedings of the National Academy of Sciences of the United States of America, 104, 10034–10039.

    Article  PubMed  CAS  Google Scholar 

  46. Oshima, A., Tani, K., Toloue, M. M., Hiroaki, Y., Smock, A., Inukai, S., et al. (2011). Asymmetric N-terminal rearrangements in connexin26 gap junction channels. Journal of Molecular Biology, 405, 724–735.

    Article  PubMed  CAS  Google Scholar 

  47. Torok, K., Stauffer, K., & Evans, W. H. (1997). Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains. Biochemical Journal, 326, 479–483.

    PubMed  CAS  Google Scholar 

  48. Zhou, X. W., Pfahnl, A., Werner, R., Hudder, A., Llanes, A., Luebke, A., et al. (1997). Identification of a pore lining segment in gap junction hemichannels. Biophysical Journal, 72, 1946–1953.

    Article  PubMed  CAS  Google Scholar 

  49. Leube, R. E. (1995). The topogenic fate of the polytopic transmembrane proteins, synaptophysin and connexin, is determined by their membrane-spanning domains. Journal of Cell Science, 108, 883–894.

    PubMed  CAS  Google Scholar 

  50. Zhao, H. B., Yu, N., & Fleming, C. R. (2005). Gap junctional hemichannel-mediated ATP release and hearing controls in the inner ear. PNAS, 102, 18724–18729.

    Article  PubMed  CAS  Google Scholar 

  51. Mammano, F., Bortolozzi, M., Ortolano, S., & Anselmi, F. (2007). Ca2 + signaling in the inner ear. Physiology, 22, 131–144.

    Article  PubMed  CAS  Google Scholar 

  52. Vlajkovic, S. M., Thorne, P. R., Housley, G. D., Munoz, D. J., & Kendrick, I. S. (1998). Ecto-nucleotidases terminate purinergic signaling in the cochlear endolymphatic compartment. NeuroReport, 9, 1559–1565.

    PubMed  CAS  Google Scholar 

  53. Housley, G. D., Greenwood, D., & Ashmore, J. F. (1992). Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea. Proceedings of the Royal Society of London. Series B: Biological Sciences, 249, 265–273.

    Article  PubMed  CAS  Google Scholar 

  54. Housley, G. D. (2000). Physiological effects of extracellular nucleotides in the inner ear. Clinical and Experimental Pharmacology and Physiology, 27, 575–580.

    Article  PubMed  CAS  Google Scholar 

  55. Parker, M. S., Margarett, S., Onyenekwu, N. N., & Bobbin, R. P. (2003). Localization of the P2Y4 receptor in the guinea pig organ of Corti. Journal of the American Academy of Audiology, 10, 286–295.

    Google Scholar 

Download references

Acknowledgments

This work is supported by the Chung Shan Medical University, Tian-Sheng Memorial Hospital (CSMU-TSMH-098-001) and National Science Council, Republic of China (NSC98-2320-B-040-016-MY3). Ted Knoy is appreciated for his editorial assistance.

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan, ROC

    Shuan-Yow Li, Jhih-Hao Nian, Wei-Guang Liang & Jiann-Jou Yang

  2. Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC

    Shuan-Yow Li & Jiann-Jou Yang

  3. Department of Cardiovascular Surgery, Tian-Sheng Memorial Hospital, Tong Kang, Pin-Tong, Taiwan, ROC

    Ching-Chyuan Su

  4. Department of Ophthalmology, Chi-Mei Medical Center, Liou-Ying, Tainan, Taiwan, ROC

    Yung-Chang Yen

  5. Department of Nursing, Min Hwei College of Health Care Management, Tainan, Taiwan, ROC

    Yung-Chang Yen

Authors
  1. Ching-Chyuan Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuan-Yow Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yung-Chang Yen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jhih-Hao Nian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei-Guang Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jiann-Jou Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jiann-Jou Yang.

Additional information

Ching-Chyuan Su and Shuan-Yow Li contributed equally to this study.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplemental Fig. 1

Dye transfer after scrape loading HeLa cells that stably express CX30.2/CX31.3. Digital fluorescence images of HeLa cells that stably express CX30.2/CX31.3 (a) and mock HeLa cell (b). Cells were incubated in Lucifer Yellow (LY; 443 Da, −2 charge), propidium iodide (PI; 415 Da, +2 charge), neurobiotin (NB–Cl; 287 Da, +1 charge) or biotin ethylenediamine (NB–Br; 297 Da, uncharge). Rhodamine-dextran (10,000 Da) was the negative control. The wounded cells took up the dye in all cases, but no dye was transferred from wounded parental cells to neighboring cells (TIFF 2953 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Su, CC., Li, SY., Yen, YC. et al. Mechanism of Two Novel Human GJC3 Missense Mutations in Causing Non-Syndromic Hearing Loss. Cell Biochem Biophys 66, 277–286 (2013). https://doi.org/10.1007/s12013-012-9481-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-012-9481-8

Keywords

Search

Navigation