A Functional Study of Gap Junction in GJB2 Mutations Associated with Hereditary Hearing Loss / 대한이비인후과학회지

ABSTRACT

BACKGROUND AND OBJECTIVES: GJB2 (Connexin 26), the gene of the gap-junction proteins, was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss (DFNB1). Whereas 35delG was known as the major type mutation in the western countries, 235delC was reported as the specific form of mutation in Asian population. The objective of this study is to identify how two mutations (235 delC, E114G) found in the Korean population affect the function of GJB2 using the molecular biology techniques. MATERIALS AND METHODS: 235delC and E114G types of mutations were cloned in the pcDNA3 vector. HeLa cells were transfected with these cloned vectors by the liposome complex method. 1) The expression and subcellular localization of Cx26 were determined using antibodies against amino acid sequences in the intracellular loop (IL) and N-terminal (NT) portions of Cx26. 2) To analyze functions of the GJB2, we examined the lucifer yellow dye transfer between cells with scrape-loaded technique. We used the wild-type (WT) Cx26 of normal hearing as a positive control, and mock cells as a negative control. RESULTS: The immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctuated patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with the anti-IL antibody but only with the anti-NT antibody slightly around the nucleus regions. In the functional study of GJB2, transfer of lucifer yellow dye into contiguous cells was detected in E114G but not in 235delC. CONCLUSION: The 235delC type of mutation showed loss of their targeting activity on the cell membrane. As a result, the function of gap junction channels were severely deteriorated. With the E114G type mutation, we didn't find any difference when compared with the WT transfected cells. Above data indicate that types of GJB2 mutation are closely related to the status of hearing loss due to altered function of gap junction protein.