Zhang Yanping, Wang Ju, Li Lina, Sun Yurui, Feng Bo
Department of Otolaryngology, Hospital of Chinese PLA, Beijing, China.
Acta Otolaryngol. 2010 Jul;130(7):799-803. doi: 10.3109/00016480903443191.
The three most common GJB2 mutations found in the Chinese populations, c.235delC, c.299-300delAT, and c.176-191de1 (16) bp, cannot form gap junctons (GJs) in the plasma membrane. These mutant proteins were retained in the endoplasmic reticulum (ER), suggesting that ER stress (ERS) and subsequent ERS-induced cell death may be responsible for hearing loss caused by these GJB2 truncation mutations.
The objective of this study was to investigate the subcellular location of the protein products of three GJB2 mutants (c.235de1C, c.299-300delAT, and c.176-191de1 (16) bp) and to explore the deafness mechanism caused by these GJB2 truncation mutations.
Mutant-eGFP fusion protein vectors were constructed by PCR and TA cloning. HEK293 cells were transfected by a liposome-mediated method. Transfected cells were incubated with ER-Tracker and observed under a confocal microscope.
Cells transfected with wild type gave characteristic punctuate patterns of GJs in the cell membrane. In contrast, c.235de1C, c.299-300delAT, and c.176-191de1 (16) bp mutant proteins were found to be trapped in the ER, and were therefore unable to form GJs in the plasma membrane.
在中国人群中发现的三种最常见的GJB2突变，即c.235delC、c.299 - 300delAT和c.176 - 191de1（16）bp，无法在质膜中形成间隙连接（GJs）。这些突变蛋白滞留在内质网（ER）中，这表明内质网应激（ERS）及随后由ERS诱导的细胞死亡可能是这些GJB2截短突变导致听力损失的原因。
本研究的目的是调查三种GJB2突变体（c.235de1C、c.299 - 300delAT和c.176 - 191de1（16）bp）的蛋白质产物的亚细胞定位，并探索这些GJB2截短突变导致耳聋的机制。
通过PCR和TA克隆构建突变体 - eGFP融合蛋白载体。采用脂质体介导的方法转染HEK293细胞。用内质网追踪剂处理转染后的细胞，并在共聚焦显微镜下观察。
转染野生型的细胞在细胞膜上呈现出典型的间隙连接点状模式。相比之下，发现c.235de1C、c.299 - 300delAT和c.176 - 191de1（16）bp突变蛋白被困在内质网中，因此无法在质膜中形成间隙连接。
