Mutations in GJB2 encoding
Connexin 26 (CX26) are associated with
hearing loss and hyperproliferative skin disorders of differing severity including
keratitis-
ichthyosis-
deafness (KID) and
Vohwinkel syndrome. A 6-year-old Caucasian girl who presented with recurrent skin rashes and
sensorineural hearing loss harboured a heterozygous point mutation in GJB2 (c.424T > C; p.F142L). To characterize the impact of CX26F142L on cellular events. Plasmids CX26WT, CX26F142L, CX26G12R (KID) or CX26D66H (Vohwinkel) were transfected into HeLa cells expressing Cx26 or
Cx43 or into HaCaT cells, a model keratinocyte cell line. Confocal microscopy determined
protein localization. MTT assays assessed cell viability in the presence or absence of
carbenoxolone, a
connexin-channel blocker. Co-immunoprecipitation/Western blot analysis determined
Cx43:Cx26 interactions. Quantitative real-time polymerase chain reaction assessed changes in gene expression of ER stress markers.
Dye uptake assays determined
Connexin-channel functionality. F142L and G12R were restricted to perinuclear areas. Collapse of the microtubule network, rescued by co-treatment with
paclitaxel, occurred. ER stress was not involved. Cell viability was reduced in cells expressing F142L and G12R but not D66H. Unlike G12R that forms "leaky" hemichannels, F142L had restricted permeability. Cell viability of F142L and G12R transfected cells was greater in HeLa cells expressing
Cx43 than in native Cx-free HeLa cells. Co-immunoprecipitation suggested a possible interaction between
Cx43 and the three mutations. Expression of CX26F142L and G12R results in microtubule collapse, rescued by interaction with
Cx43. The GJB2 mutations interacted with
Cx43 suggesting that unique
Cx43:Cx26 channels are central to the diverse phenotype of CX26 skin-related
channelopathies.