Abstract
Gap junctions (GJs) are intercellular channels associated with cell–cell communication. Connexin 26 (Cx26) encoded by the GJB2 gene forms GJs of the inner ear, and mutations of GJB2 cause congenital hearing loss that can be syndromic or non-syndromic. It is difficult to predict pathogenic effects using only genetic analysis. Using ionic and biochemical coupling tests, we evaluated the pathogenic effects of Cx26 variants using computational analyses to predict structural abnormalities. For seven out of ten variants, we predicted the variation would result in a loss of GJ function, whereas the others would completely fail to form GJs. Functional studies demonstrated that, although all variants were able to function normally as hetero-oligomeric GJ channels, six variants (p.E47K, p.E47Q, p.H100L, p.H100Y, p.R127L, and p.M195L) did not function normally as homo-oligomeric GJ channels. Interestingly, GJs composed of the Cx26 variant p.R127H were able to function normally, even as homo-oligomeric GJ channels. This study demonstrates the particular location and property of an amino acid are more important mainly than the domain where they belong in the formation and function of GJ, and will provide information that is useful for the accurate diagnosis of hearing loss.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- Cx26:
-
Connexin 26
- GJs:
-
Gap junctions
- TM:
-
Transmembrane domains
- EL:
-
Extracellular loop domain
- CL:
-
Cytoplasmic loop domain
- ER:
-
Endoplasmic reticulum
- LY:
-
Lucifer yellow
- PI:
-
Propidium Iodide
References
Antoniadi T, Gronskov K, Sand A, Pampanos A, Brondum-Nielsen K, Petersen MB (2000) Mutation analysis of the GJB2 (connexin 26) gene by DGGE in Greek patients with sensorineural deafness. Hum Mutat 16:7–12
Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921
Bruzzone R, White TW, Paul DL (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 238:1–27
Chan DK, Chang KW (2014) GJB2-associated hearing loss: systematic review of worldwide prevalence, genotype, and auditory phenotype. Laryngoscope 124:E34–E53
Chan DK, Schrijver I, Chang KW (2010) Connexin-26-associated deafness: phenotypic variability and progression of hearing loss. Genet Med 12:174–181
Choi SY, Park HJ, Lee KY, Dinh EH, Chang Q, Ahmad S, Lee SH, Bok J, Lin X, Kim UK (2009) Different functional consequences of two missense mutations in the GJB2 gene associated with non-syndromic hearing loss. Hum Mutat 30:E716–E727
Choi SY, Lee KY, Kim HJ, Kim HK, Chang Q, Park HJ, Jeon CJ, Lin X, Bok J, Kim UK (2011) Functional evaluation of GJB2 variants in nonsyndromic hearing loss. Mol Med 17:550–556
Cohn ES, Kelley PM, Fowler TW, Gorga MP, Lefkowitz DM, Kuehn HJ, Schaefer GB, Gobar LS, Hahn FJ, Harris DJ, Kimberling WJ (1999) Clinical studies of families with hearing loss attributable to mutations in the connexin 26 gene (GJB2/DFNB1). Pediatrics 103:546–550
Dai P, Yu F, Han B, Liu X, Wang G, Li Q, Yuan Y, Liu X, Huang D, Kang D, Zhang X, Yuan H, Yao K, Hao J, He J, He Y, Wang Y, Ye Q, Yu Y, Lin H, Liu L, Deng W, Zhu X, You Y, Cui J, Hou N, Xu X, Zhang J, Tang L, Song R, Lin Y, Sun S, Zhang R, Wu H, Ma Y, Zhu S, Wu BL, Han D, Wong LJ (2009) GJB2 mutation spectrum in 2,063 Chinese patients with nonsyndromic hearing impairment. J Transl Med 7:26
D’Andrea P, Veronesi V, Bicego M, Melchionda S, Zelante L, Di Iorio E, Bruzzone R, Gasparini P (2002) Hearing loss: frequency and functional studies of the most common connexin26 alleles. Biochem Biophys Res Commun 296:685–691
Das S, Smith TD, Sarma JD, Ritzenthaler JD, Maza J, Kaplan BE, Cunningham LA, Suaud L, Hubbard MJ, Rubenstein RC, Koval M (2009) ERp29 restricts Connexin43 oligomerization in the endoplasmic reticulum. Mol Biol Cell 20:2593–2604
del Castillo FJ, del Castillo I (2011) The DFNB1 subtype of autosomal recessive non-syndromic hearing impairment. Front Biosci (Landmark Ed) 16:3252–3274
DeLano WL (2006) The PyMOL Molecular Graphics System. Version 0.99 edn. Delano Scientific, New York
Diez JA, Ahmad S, Evans WH (1999) Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions. Eur J Biochem 262:142–148
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132
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
Gardner P, Oitmaa E, Messner A, Hoefsloot L, Metspalu A, Schrijver I (2006) Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow-up. Pediatrics 118:985–994
Gerido DA, White TW (2004) Connexin disorders of the ear, skin, and lens. Biochim Biophys Acta 1662:159–170
Haas K, Sin WC, Javaherian A, Li Z, Cline HT (2001) Single-cell electroporation for gene transfer in vivo. Neuron 29:583–591
Han SH, Park HJ, Kang EJ, Ryu JS, Lee A, Yang YH, Lee KR (2008) Carrier frequency of GJB2 (connexin-26) mutations causing inherited deafness in the Korean population. J Hum Genet 53:1022–1028
Harris AL (2001) Emerging issues of connexin channels: biophysics fills the gap. Q Rev Biophys 34:325–472
Hua VB, Chang AB, Tchieu JH, Kumar NM, Nielsen PA, Saier MH Jr (2003) Sequence and phylogenetic analyses of 4 TMS junctional proteins of animals: connexins, innexins, claudins and occludins. J Membr Biol 194:59–76
Jiang JX, Gu S (2005) Gap junction- and hemichannel-independent actions of connexins. Biochim Biophys Acta 1711:208–214
Kenna MA, Feldman HA, Neault MW, Frangulov A, Wu BL, Fligor B, Rehm HL (2010) Audiologic phenotype and progression in GJB2 (Connexin 26) hearing loss. Arch Otolaryngol Head Neck Surg 136:81–87
Koval M (2006) Pathways and control of connexin oligomerization. Trends Cell Biol 16:159–166
Lai-Cheong JE, Arita K, McGrath JA (2007) Genetic diseases of junctions. J Invest Dermatol 127:2713–2725
Maeda S, Nakagawa S, Suga M, Yamashita E, Oshima A, Fujiyoshi Y, Tsukihara T (2009) Structure of the connexin 26 gap junction channel at 3.5 A resolution. Nature 458:597–602
Mani RS, Ganapathy A, Jalvi R, Srikumari Srisailapathy CR, Malhotra V, Chadha S, Agarwal A, Ramesh A, Rangasayee RR, Anand A (2009) Functional consequences of novel connexin 26 mutations associated with hereditary hearing loss. Eur J Hum Genet 17:502–509
Martin PE, Coleman SL, Casalotti SO, Forge A, Evans WH (1999) Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness. Hum Mol Genet 8:2369–2376
Maza J, Mateescu M, Das Sarma J, Koval M (2003) Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras. Cell Commun Adhes 10:319–322
Maza J, Das Sarma J, Koval M (2005) Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum. J Biol Chem 280:21115–21121
Müller DJ, Hand GM, Engel A, Sosinsky GE (2002) Conformational changes in surface structures of isolated connexin 26 gap junctions. EMBO J 21(14):3598–3607
Murgia A, Orzan E, Polli R, Martella M, Vinanzi C, Leonardi E, Arslan E, Zacchello F (1999) Cx26 deafness: mutation analysis and clinical variability. J Med Genet 36:829–832
Piazza V, Beltramello M, Menniti M, Colao E, Malatesta P, Argento R, Chiarella G, Gallo LV, Catalano M, Perrotti N, Mammano F, Cassandro E (2005) Functional analysis of R75Q mutation in the gene coding for Connexin 26 identified in a family with nonsyndromic hearing loss. Clin Genet 68:161–166
Pollak A, Skorka A, Mueller-Malesinska M, Kostrzewa G, Kisiel B, Waligora J, Krajewski P, Oldak M, Korniszewski L, Skarzynski H, Ploski R (2007) M34T and V37I mutations in GJB2 associated hearing impairment: evidence for pathogenicity and reduced penetrance. Am J Med Genet A 143A:2534–2543
Prasad S, Cucci RA, Green GE, Smith RJ (2000) Genetic testing for hereditary hearing loss: connexin 26 (GJB2) allele variants and two novel deafness-causing mutations (R32C and 645-648delTAGA). Hum Mutat 16:502–508
Rabionet R, Zelante L, Lopez-Bigas N, D’Agruma L, Melchionda S, Restagno G, Arbones ML, Gasparini P, Estivill X (2000) Molecular basis of childhood deafness resulting from mutations in the GJB2 (connexin 26) gene. Hum Genet 106:40–44
Ramchander PV, Nandur VU, Dwarakanath K, Vishnupriya SV, Padma T (2005) Prevalence of Cx26 (GJB2) gene mutations causing recessive nonsyndromic hearing impairment in India. Int J Hum Genet 5:241–246
Richard G (2005) Connexin disorders of the skin. Clin Dermatol 23:23–32
Saez JC, Retamal MA, Basilio D, Bukauskas FF, Bennett MV (2005) Connexin-based gap junction hemichannels: gating mechanisms. Biochim Biophys Acta 1711:215–224
Snoeckx RL, Huygen PL, Feldmann D, Marlin S, Denoyelle F, Waligora J, Mueller-Malesinska M, Pollak A, Ploski R, Murgia A, Orzan E, Castorina P, Ambrosetti U, Nowakowska-Szyrwinska E, Bal J, Wiszniewski W, Janecke AR, Nekahm-Heis D, Seeman P, Bendova O, Kenna MA, Frangulov A, Rehm HL, Tekin M, Incesulu A, Dahl HH, du Sart D, Jenkins L, Lucas D, Bitner-Glindzicz M, Avraham KB, Brownstein Z, del Castillo I, Moreno F, Blin N, Pfister M, Sziklai I, Toth T, Kelley PM, Cohn ES, Van Maldergem L, Hilbert P, Roux AF, Mondain M, Hoefsloot LH, Cremers CW, Lopponen T, Lopponen H, Parving A, Gronskov K, Schrijver I, Roberson J, Gualandi F, Martini A, Lina-Granade G, Pallares-Ruiz N, Correia C, Fialho G, Cryns K, Hilgert N, Van de Heyning P, Nishimura CJ, Smith RJ, Van Camp G (2005) GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet 77:945–957
Sohl G, Willecke K (2004) Gap junctions and the connexin protein family. Cardiovasc Res 62:228–232
Sohl G, Nielsen PA, 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 Commun Adhes 10:27–36
Sosinsky G (1995) Mixing of connexins in gap junction membrane channels. Proc Natl Acad Sci USA 92:9210–9214
Sosinsky GE, Nicholson BJ (2005) Structural organization of gap junction channels. Biochim Biophys Acta 1711:99–125
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
Tang HY, Fang P, Ward PA, Schmitt E, Darilek S, Manolidis S, Oghalai JS, Roa BB, Alford RL (2006) DNA sequence analysis of GJB2, encoding connexin 26: observations from a population of hearing impaired cases and variable carrier rates, complex genotypes, and ethnic stratification of alleles among controls. Am J Med Genet A 140:2401–2415
Thimm J, Mechler A, Lin H, Rhee S, Lal R (2005) Calcium-dependent open/closed conformations and interfacial energy maps of reconstituted hemichannels. J Biol Chem 280(11):10646–10654
van Steensel MA (2004) Gap junction diseases of the skin. Am J Med Genet C Semin Med Genet 131C:12–19
Yilmaz A (2014) Bioinformatic analysis of GJB2 gene missense mutations. Cell Biochem Biophys 71:1623–1642
Acknowledgments
The authors thank KIHIRA Kiyohito for protein structure analysis and prediction. This work was supported by the Korea Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI12C1004 to UKK), the Bio and Medical Technology Development Program of the NRF, funded by the Ministry of Science, ICT and Future Planning (2014M3A9D5073865 to UKK), and the TJ Park Science Fellowship funded by the POSCO Foundation of Korea (S. K. Oh).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no competing financial interests.
Additional information
H. R. Kim, S.-K. Oh, E.-S. Lee and S.-Y. Choi contributed equally.
Electronic supplementary material
Below is the link to the electronic supplementary material.
439_2015_1625_MOESM2_ESM.doc
Fig. S1. Subcellular localization of mutants that do not form GJs by immunocytochemistry. The red signal indicates mCherry-tagged Cx26-MT and the green signal indicates ER detected by anti-KDEL antibody. The expressions of p.S85Y, p.F191L, and p.F195V are concordant with the signal of KDEL; as a result, these the Cx26 protein mutants accumulate in the ER.(DOC 1628 kb)
439_2015_1625_MOESM3_ESM.doc
Fig. S2. Formation of seven hetero-oligomeric mutant GJ channels for ionic coupling test in HeLa cells. The red signal indicates mCherry-tagged Cx26-WT, and the green signal indicates each EGFP-tagged Cx26 mutant; the arrows indicate the gap junction between cells. Cx26-WT and Cx26-mutant both formed gap junctions, and the GJ are almost identical and are in the same location. The cells definitely formed hetero-oligomeric gap junctions composed of Cx26-WT and Cx26-mutant. Cx26-WT-mCherry and Cx26-E47K-EGFP (A), Cx26-E47Q-EGFP (B), Cx26-H100L-EGFP (C), Cx26-H100Y-EGFP (D), Cx26-R127H-EGFP (E), Cx26-R127L-EGFP (F), or Cx26-M195L-EGFP (G).(DOC 2381 kb)
439_2015_1625_MOESM4_ESM.doc
Fig. S3. LY permeability of GJs composed of seven different homo-oligomeric/hetero-oligomeric mutant connexin channels in HeLa cells. Individual cells expressing GJs were injected with LY, and the intercellular diffusion of the dye was monitored 2 min after injection. Cell #1 was injected with LY, and cell #2 formed a GJ with cell #1. The dye was transferred through a GJ composed of Cx26-WT-mCherry (A). Cells transfected with Cx26-M195L-mCherry (B), Cx26-E46K-mCherry (C), Cx26-E47Q-mCherry (D), Cx26-H100L-mCherry (E), Cx26-H100Y-mCherry (F), Cx26-R127H-mCherry (G), and Cx26-R127L-mCherry (H) were uncoupled. The cells were transfected for hetero-oligomeric GJ channels with Cx26-WT-mCherry and Cx26-E47K-EGFP (I), Cx26-E47Q-EGFP (J), Cx26-H100L-EGFP (K), Cx26-H100Y-EGFP (L), Cx26-R127H-EGFP (M), Cx26-R127L-EGFP (N), or Cx26-M195L-EGFP (O).(DOC 2108 kb)
439_2015_1625_MOESM5_ESM.doc
Fig. S4. Percentage of control and transfected HeLa cells loaded with PI dye through hemichannels. Untransfected cells and cells transfected with EGFP vector used as a negative control did not show PI dye loading. PI dye was loaded in almost all cells transfected with Cx26-WT but in almost none of the cells transfected with the 7 variants (Cx26-E47K, E47Q, H100L, H100Y, R127H, R127L and M195L).(DOC 125 kb)
Rights and permissions
About this article
Cite this article
Kim, H.R., Oh, SK., Lee, ES. et al. The pathological effects of connexin 26 variants related to hearing loss by in silico and in vitro analysis. Hum Genet 135, 287–298 (2016). https://doi.org/10.1007/s00439-015-1625-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00439-015-1625-7