Mutations in the GJB2 gene encoding transmembrane
protein connexin 26 (Cx26) are the most common cause for
hearing loss worldwide. Cx26 plays a crucial role in the ionic and metabolic homeostasis in the inner ear, indispensable for normal hearing process. Different pathogenic mutations in the GJB2 gene can affect all stages of the Cx26 life cycle and result in nonsyndromic autosomal recessive (DFNB1) or dominant (DFNA3)
deafness and syndromes associating
hearing loss with skin disorders. This study aims to elucidate the functional consequences of a rare GJB2 variant c.516G>C (p.Trp172Cys) found with high frequency in deaf patients from indigenous populations of Southern Siberia (Russia). The substitution c.516G>C leads to the replacement of
tryptophan at a conserved
amino acid position 172 with
cysteine (p.Trp172Cys) in the second extracellular loop of Cx26
protein. We analyzed the subcellular localization of mutant Cx26-p.Trp172Cys
protein by immunocytochemistry and the hemichannels permeability by
dye loading assay. The GJB2 knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different GJB2 variants (wild type and mutations associated with
hearing loss) were established based on knockout cells and used for comparative functional analysis. The impaired trafficking of mutant Cx26-p.Trp172Cys
protein to the plasma membrane and reduced hemichannels permeability support the pathogenic effect of the c.516G>C (p.Trp172Cys) variant and its association with
nonsyndromic hearing loss. Our data contribute to a better understanding of the role of mutations in the second extracellular loop of Cx26
protein in pathogenesis of
deafness.