Impaired membrane targeting and aberrant cellular localization of human Cx26 mutants associated with inherited recessive hearing loss.
CONCLUSIONS
This study demonstrated that five Cx26 mutations (R32H, S199F, 572delT, 631-632delGT, and Y155X) affect gap junction (GJ) functions by causing impaired membrane targeting and aberrant cellular localization, and one mutation (R165W) leads to a constriction of the channel pore with no dye coupling.


OBJECTIVE
To investigate the pathogenetic roles of six recessive Cx26 mutations (p.R32H, p.R165W, p.S199F, c.572delT, c.631-632delGT, and p.Y155X), which have not been functionally analyzed in vitro.


METHODS
The six mutants and wild-type Cx26 (wtCx26) were cloned into the EcoRI and SalI sites of pEGFP-N1 vector. We transfected the seven constructs into HeLa cells, followed by analysis of their protein expression using the western blot method, study of the protein localizations and gap junction-plaques on the cytomembrane under confocal microscopy, and assessment of the dye coupling of the mutated GJ channels by intercellular dye transfer experiment.


RESULTS
p.R165W targeted the cytomembrane and formed GJ-like structures in adjacent HeLa cells, causing null dye coupling. The mutants (p.R32H, p.S199F, c.572delT, c.631-632delGT, and p.Y155X) failed to reach the cell surface, and perfectly co-localized with endoplasmic reticulum (ER) throughout the cells.
Xiao
Zian
Z
Department of Otolaryngology and Head-Neck Surgery, Second Xiangya Hospital of Central South University, Changsha, China. xiaozian@sina.com
Yang
Zhongchun
Z
Liu
Xuezhong
X
Xie
Dinghua
D
D000483:Alleles; D015153:Blotting, Western; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D004252:DNA Mutational Analysis; D003638:Deafness / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000235:genetics; D017629:Gap Junctions / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005808:Genes, Recessive / Q000235:genetics*; D006056:Golgi Apparatus / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26