Connexin31显性听力下降相关突变体研究  

Defective Membrane Localization of Dominant Hearing Impairment Associated Connexin31 Mutants

作　　者：谭志平[1] 刘宇[1] 蔡芳[1] 潘乾[1] 贺立强 黄亮群[1] 戴和平[1] 夏昆[1] 夏家辉[1] 张灼华[1] 

机构地区：[1]中南大学医学遗传学国家重点实验室,长沙410078

出　　处：《生物化学与生物物理进展》2005年第5期403-407,共5页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展规划项目(973)(2001CB510302);国家自然科学基金(30370737;39970372)~~

摘　　要：Cx31突变可以导致常染色体显性听力下降、常染色体隐性听力下降、周围神经疾病伴听力丧失,以及变性红皮肤病角化病,其导致不同疾病的机理一直是研究的重点.利用定点突变技术(site-directed mutagenesis,SDM)构建connexin31显性听力下降相关突变体Cx31R180X,Cx31E183K并插入到真核表达载体pEGFP-N1,转染HeLa细胞,转染Cx31R180X-pEGFP-N1和Cx31E183K-pEGFP-N1Cx31突变体质粒的HeLa细胞质膜上没有出现斑块状染色和聚集现象,分别用内质网染料conA和高尔基体染料WGA进行免疫荧光染色,结果显示Cx31显性听力下降相关突变体蛋白主要分布在细胞质内,且大部分定位在内质网和高尔基体上.同时分别用抗Cx31和抗GFP抗体进行蛋白质印迹检测,证实Cx31R180X,Cx31E183K在转染的HeLa细胞中都有表达.研究发现Connexin31显性耳聋相关突变体Cx31R180X,Cx31E183K不能正常地形成间隙连接通道,这与connexin31 EKV(变性红皮肤病角化病)相关突变体能够运输到细胞膜上形成间隙连接通道的报道不相同,提示connexin31不同部位突变导致不同疾病的致病机理可能不一样,从而为解释“one connexin two diseases”提供分子水平的依据.Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Connexin31 (Cx31) is an important member of connexin 0 family. Mutations in Cx31 are associated with erythrokeratoderma variabilis (EKV), hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Using Site-directed mutagenesis (SDM) to construct two dominant hearing impairment associated Cx31 mutant expression plasmids, R180X-pEGFP-N1 and E183K- pEGFP-N1. A number of functional analysis were described to investigate the effect of two dominant hearing impairment-associated Cx31 mutants, R180X and E183K, on connexon trafficking. The majority of the mutant R180X and E183K were cytoplasmic, they can not traffic to normal intracellular localization and can not form functional gap junction channels. But not observed with wild-type Cx31. This may provide an insight into the pathological mechanism of Cx31 mutants.

关 键 词：cx31 定点突变技术 突变体 绿色荧光蛋白 

分 类 号：Q61[生物学—生物物理学]