Novel mutations in the connexin43 (GJA1) and GJA1 pseudogene may contribute to nonsyndromic hearing loss.

Connexins (CXs), a large family of membrane proteins, are key components of gap junction channels. Among a cohort of patients with nonsyndromic hearing loss, we have recently identified three novel missense mutations in the GJA1 gene and GJA1 pseudogene (rhoGJA1) as likely being causally related to hearing loss. However, the functional alteration of CX43 caused by the mutations of GJA1 and rhoGJA1 gene remains unclear. This study compares the intracellular distribution and assembly of three CX43 mutants expressed in HeLa cells with their wild-type (WT) counterparts and the effects of the mutant proteins on those cells. Localization assay of WT CX43 reveals a typical punctuate fluorescence pattern of a gap junction channel between neighboring expression cells. Additionally, immunoblotting analysis of the transfectants confirms the production of mutant proteins, in which their distributions along appositional membranes are determined using immunofluorescent staining procedures. Furthermore, dye transfer assay results demonstrate that gap junctional intercellular communication is less in HeLa cells carrying mutant GJA1 or rhoGJA1 gene than in WT-expressing cells. The results of this study suggest that the three mutations in GJA1 or rhoGJA1 that we previously reported result in at least partial loss of normal functions carried out by CX43, which may form a basis for the mechanism contributing to hearing loss in patients.