Dominant connexin26 mutants associated with human hearing loss have trans-dominant effects on connexin30
Introduction
Gap junctions allow the direct passage of ions and small molecules (typically < 1000 Da) between adjacent cells, and are thought to have diverse functions, including the propagation of electrical signals, metabolic cooperation, spatial buffering of ions, growth control, and cellular differentiation (Bruzzone, 1996, Harris, 2001). They are formed by two apposed hemichannels (or connexons); a complete channel is formed when one hemichannel docks with a compatible hemichannel on an apposed cell membrane. Each hemichannel is composed of six compatible connexin molecules - a large family of highly conserved proteins, named according to their predicted molecular mass (Willecke et al., 2002). Individual hemichannels can be composed of one (homomeric) or more than one (heteromeric) type of connexin. Similarly, channels can be composed of hemichannels of the same (homotypic) or different (heterotypic) connexins (Kumar and Gilula, 1996, White and Bruzzone, 1996). Any two compatible connexins can theoretically form 196 different channels (Brink et al., 1997) whose biophysical properties (such as permeability and gating) may be different from their homomeric homotypic counterparts(Brink et al., 1997).
Mutations in GJB2, GJB6, and GJB3, the genes that encode the human gap junction proteins connexin26 (Cx26), Cx30, and Cx31, respectively, cause hearing loss (Estivill, 1998, Grifa, 1999, Kelsell, 1997, Xia, 1998). GJB2 mutations are the most common cause of hereditary non-syndromic hearing loss (NSHL), with over 90 recessive mutations reported (http://davinci.crg.es/deafness/). Dominant mutations in GJB2 also cause hearing loss, either in isolation (non-syndromic) or as part of a syndrome associated with various skin disorders. Recessive GJB2 mutations likely cause simple loss of function, whereas dominant GJB2 mutations likely cause gain of function, including dominant-negative effects on wild type Cx26 and/or Cx30 (Marziano et al., 2003) because haplotype insufficiency of GJB2 does not cause hearing loss based on the observation that the heterozygous parents of deaf children (who have homozygous GJB2 mutations) do not themselves have hearing loss.
Cx26 and Cx30 are the major gap junction proteins expressed in cochlea with broadly overlapping but not identical distributions (Ahmad, 2003, Forge, 2002, Jagger and Forge, 2006, Kikuchi, 1995, Lautermann, 1998, Liu and Zhao, 2008, Sun, 2005, Zhao and Yu, 2006). They have been co-immunoprecipitated from mouse cochlear homogenates and transfected cells co-expressing them (Ahmad, 2003, Di, 2005, Forge, 2003, Sun, 2005, Yum, 2007), indicating that they form hybrid heteromeric/heterotypic gap junction channels. Here, we investigated whether similar interactions occur between Cx30 and nine dominant Cx26 mutants (Fig. S1) - six (W44C, W44S, R143Q, D179N, R184Q and C202F) that cause NSHL and three (G59A, R75Q and R75W) that cause syndromic hearing loss (SHL) associated with palmoplantar keratoderma (PPK). All mutants co-localized and co-immunoprecipitated with Cx30, and 8/9 had different degrees of trans-dominant effects on the function of Cx30; providing further evidence that these dominant effects contribute to the pathogenesis of hearing loss.
Section snippets
All 9 dominant Cx26 mutants form gap junction-like plaques
To investigate the biology of GJB2 mutations, we expressed wild type GJB2 and 9 different dominant GJB2 mutations (Fig. S1) by transfection into communication-incompetent HeLa cells, followed by bulk-selection. Immunostaining with a rabbit antiserum against the C-terminus of Cx26 demonstrated gap junction plaques (GJPs) on apposing cell membranes for cells expressing wild type Cx26 and all 9 (W44C, W44S, G59A, R75Q, R75W, R143Q, D179N, R184Q, C202F) mutants (Fig. 1). A monoclonal antibody
Characterization of dominant Cx26 mutants
Up to 30 different dominant mutations in GJB2 have been reported; 10 are associated with NSHL, and the others cause SHL with various skin diseases. The expression pattern in mammalian cells was investigated for 13 of these mutants (Common, 2003, de Zwart-Storm, 2008, Haack, 2006, Martin, 1999, Marziano, 2003, Matos, 2008, Oshima, 2003, Piazza, 2005, Richard, 2002, Stong, 2006, Thomas, 2004), but most of these studies primarily focused on mutations causing SHL, and majority of the mutants
Mutant Cx26 expression constructs
A plasmid containing human GJB2 (kindly provided by Dr. Bruce Nicholson) was amplified by PCR using oligonucleotide primers designed to include the open reading frame (ORF) and incorporate a 5′ NheI site and a 3′ BamHI site, and the PCR product was ligated into pIRESneo3, pIRESpuro3 and/or the pIRES2-DsRed bicistronic vector (Orthmann-Murphy et al., 2007) as previously described (Yum et al., 2007). The GJB2 mutations were introduced into the ORF of human GJB2 cDNA by PCR site-directed
Acknowledgments
We thank Dr. Bruce Nicholson for providing the human Cx26 cDNA, Dr. Klaus Willecke for the HeLa cells, Dr. Charles Abrams for the pIRES2-DsRed vector, Dr. Hajime Takano for his advice, Drs. Jennifer Orthmann-Murphy and David Kelsell for comments, and Jennifer Faerber and Dr. Tom Tenhave for statistical analysis. This work was supported by NIH grants KO8DC005394 (to S.W.Y.), RO1 NS55284 (to S.S.S.), and subcontract and NIH P30 NS047321, which supports the Center for Dynamic Imaging of Nervous
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