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Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

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Abstract

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels.

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References

  • Attal J, Theron MC, Houdebine LM (1999) The optimal use of IRES (internal ribosome entry site) in expression vectors. Genet Anal 15:161–165

    PubMed  CAS  Google Scholar 

  • Bao X, Chen Y, Reuss L, Altenberg GA (2004) Functional expression in Xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43. J Biol Chem 279:9689–9692

    Article  PubMed  CAS  Google Scholar 

  • Bao X, Lee SC, Reuss L, Altenberg GA (2007) Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC. Proc Natl Acad Sci USA 104:4919–4924

    Article  PubMed  CAS  Google Scholar 

  • Bennett MVL, Verselis VK (1992) Biophysics of gap junctions. Semin Cell Biol 3:29–47

    Article  PubMed  CAS  Google Scholar 

  • Bruzzone S, Guida L, Zocchi E, Franco L, De Flora A (2001) Connexin 43 hemichannels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells. FASEB J 15:10–12

    PubMed  CAS  Google Scholar 

  • Dahl G, Werner R, Levine E, Rabadan-Diehl C (1992) Mutational analysis of gap junction formation. Biophys J 62:172–180

    Article  PubMed  CAS  Google Scholar 

  • De Vuyst E, Decrock E, De Bock M, Yamasaki H, Naus CC, Evans WH, Leybaert L (2007) Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor. Mol Biol Cell 18:34–46

    Article  PubMed  CAS  Google Scholar 

  • Duffy HS, Sorgen PL, Girvin ME, O’Donnell P, Coombs W, Taffet SM, Delmar M, Spray DC (2002) pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains. J Biol Chem 277:36706–36714

    Article  PubMed  CAS  Google Scholar 

  • Evans WH, De Vuyst E, Leybaert L (2006) The gap junction cellular internet: connexin hemichannels enter the signalling limelight. Biochem J 397:1–14

    Article  PubMed  CAS  Google Scholar 

  • Fleishman SJ, Unger VM, Yeager M, Ben-Tal N (2004) A Calpha model for the transmembrane alpha helices of gap junction intercellular channels. Mol Cell 15:879–888

    Article  PubMed  CAS  Google Scholar 

  • Foote CI, Zhou L, Zhu X, Nicholson BJ (1998) 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

    Article  PubMed  CAS  Google Scholar 

  • Gerido DA, Derosa AM, Richard G, White TW (2007) Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness. Am J Physiol Cell Physiol 293:337–345

    Article  CAS  Google Scholar 

  • Hanemann CO, Bergmann C, Senderek J, Zerres K, Sperfeld AD (2003) Transient, recurrent, white matter lesions in X-linked Charcot-Marie-Tooth disease with novel connexin 32 mutation. Arch Neurol 60:605–609

    Article  PubMed  Google Scholar 

  • Hertlein B, Butterweck A, Haubrich S, Willecke K, Traub O (1998) Phosphorylated carboxy terminal serine residues stabilize the mouse gap junction protein connexin45 against degradation. J Membr Biol 162:247–257

    Article  PubMed  CAS  Google Scholar 

  • Hu X, Ma M, Dahl G (2006) Conductance of connexin hemichannels segregates with the first transmembrane segment. Biophys J 90:140–150

    Article  PubMed  CAS  Google Scholar 

  • Lai A, Le D-N, Paznekas WA, Gifford WD, Wang Jabs E, Charles AC (2006) Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells. J Cell Sci 119: 532–541

    Article  PubMed  CAS  Google Scholar 

  • Laird DW, Castillo M, Kasprzak L (1995) Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat mammary tumor cells. J Cell Biol 131:1193–1203

    Article  PubMed  CAS  Google Scholar 

  • Lampe PD, Cooper CD, King TJ, Burt JM (2006) Analysis of connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. J Cell Sci 119:3435–3442

    Article  PubMed  CAS  Google Scholar 

  • Lin Liang SG, de Miguel M, Gomez-Hernandez JM, Glass JD, Scherer SS, Mintz M, Barrio LC, Fischbeck KH (2005) Severe neuropathy with leaky connexin32 hemichannels. Ann Neurol 57:749–754

    Article  CAS  Google Scholar 

  • Martin PE, Blundell G, Ahmad S, Errington RJ, Evans WH (2001) Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels. J Cell Sci 114:3845–3855

    PubMed  CAS  Google Scholar 

  • Oh SY, Dupont E, Madhukar BV, Briand JP, Chang CC, Beyer E, Trosko JE (1993) Characterization of gap junctional communication-deficient mutants of a rat liver epithalial cell line. Eur J Cell Biol 60:250–255

    PubMed  CAS  Google Scholar 

  • Paznekas WA, Boyadjiev SA, Shapiro RE, et al. (2003) Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet 72:408–418

    Article  PubMed  CAS  Google Scholar 

  • Saez JC, Retamal MA, Basilio D, Bukauskas FF, Bennett MV (2005) Connexin-based gap junction hemichannels: gating mechanisms. Biochim Biophys Acta 1711:215–224

    Article  PubMed  CAS  Google Scholar 

  • Soehl G, Willecke K (2003) An update on connexin genes and their nomenclature in mouse and man. Cell Commun Adhes 10:173–180

    Article  CAS  Google Scholar 

  • Spray DC, Ye ZC, Ransom BR (2006) Functional connexin “hemichannels”: a critical appraisal. Glia 547:758–773

    Article  Google Scholar 

  • Stong BC, Chang Q, Ahmad S, Lin X (2006) A novel mechanism for connexin 26 mutation linked deafness: cell death caused by leaky gap junction hemichannels. Laryngoscope 116:2205–2210

    Article  PubMed  CAS  Google Scholar 

  • Stout CE, Costantin JL, Naus CCG, Charles AC (2002) Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem 12:10482–10488

    Article  CAS  Google Scholar 

  • Wilgenbus KK, Kirkpatrick CJ, Knuechel R, Willecke K, Traub O (1992) Expression of Cx26, Cx32 and Cx43 gap junction proteins in normal and neoplastic human tissues. Int J Cancer 51:522–529

    Article  PubMed  CAS  Google Scholar 

  • Yotsumoto S, Hashiguchi T, Chen X, Ohtake N, Tomitaka A, Akamatsu H, Matsunaga K, Shiraishi S, Miura H, Adachi J, Kanzaki T (2003) Novel mutations in GJB2 encoding connexin-26 in Japanese patients with keratitis-ichthyosis-deafness syndrome. Br J Dermatol 148:649–653

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

This work was supported by a grant of the German Research Association through SFB 645, project B2 (to K. W.). We thank Dr. Elmar Endl (Institute of Molecular Medicine, University of Bonn) for advice on the use of the FACS instrument.

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Correspondence to Klaus Willecke.

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Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.

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Dobrowolski, R., Sommershof, A. & Willecke, K. Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels. J Membrane Biol 219, 9–17 (2007). https://doi.org/10.1007/s00232-007-9055-7

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