A novel mutation in the connexin 29 gene may contribute to nonsyndromic hearing loss.
Connexins (Cxs) are homologous four-transmembrane domain proteins and constitute the major components of gap junctions. Among a cohort of patients with nonsyndromic hearing loss, we recently identified a novel missense mutation, E269D, in the GJC3 gene encoding connexin 29 (Cx29), as being causally related to hearing loss. The functional alteration of Cx29 caused by the mutant GJC3 gene, however, remains unknown. This study compared the intracellular distribution and assembly of mutant Cx29 (Cx29E269D) with that of the wild-type Cx29 (Cx29WT) in HeLa cells and the effect the mutant protein had on those cells. Cx29TW showed continuous staining along apposed cell membranes in the fluorescent localization assay. In contrast, the p.E269D missense mutation resulted in accumulation of the Cx29 mutant protein in the endoplasmic reticulum (ER) rather than in the cytoplasmic membrane. Co-expression of Cx29WT and Cx29E269D proteins by a bi-directional tet-on expression system demonstrated that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the formation of the gap junction. Based on these findings, we suggest that Cx29E269D has a dominant negative effect on the formation and function of the gap junction. These results provide a novel molecular explanation for the role Cx29 plays in the development of hearing loss.
Hong
Hui-Mei
HM
Department of BioMedical Sciences, Chung Shan Medical University, Taichung, Taiwan, ROC.
Yang
Jiann-Jou
JJ
Su
Ching-Chyuan
CC
Chang
Juan-Yu
JY
Li
Tung-Cheng
TC
Li
Shuan-Yow
SY
D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D017630:Connexins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D034381:Hearing Loss / Q000235:genetics* / Q000473:pathology; D006801:Humans; D008856:Microscopy, Fluorescence; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017630:Connexins; C464674:GJC3 protein, human; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins