A Functional Study of Gap Junction in GJB2 Mutations Associated with Hereditary Hearing Loss
유전성 난청과 동반된 GJB2 유전자 변이의 세포간극에 대한 기능적 연구
정, 연훈; 유, 상준; 이, 준호; 박, 홍준
Taehan Ibi Inhukwa Hakhoe chi, 44(3):239-245, 2001
Taehan Ibi Inhukwa Hakhoe chi; Journal of the Korean Otolaryngological Society; 대한이비인후과학회지
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
countries, 235delC was reported as the specific form of mutation in Asian population. The objective of this study is to identify how two mutations (235delC, E114G) found in the Korean population affect the function of GJB2 using the molecular
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
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