He Li-Qiang, Liu Yu, Cai Fang, Tan Zhi-Ping, Pan Qian, Liang De-Sheng, Long Zhi-Gao, Wu Ling-Qian, Huang Liang-Qun, Dai He-Ping, Xia Kun, Xia Jia-Hui, Zhang Zhuo-Hua
National Laboratory of Medical Genetics, Central South University, Changsha 410078, China.
Acta Biochim Biophys Sin (Shanghai). 2005 Aug;37(8):547-54. doi: 10.1111/j.1745-7270.2005.00080.x.
Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV), hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD, 141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I141V and 652del12). Immunoblotting analysis showed that 12 mutated Cx31s, like WT-Cx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) caused cell death in HeLa cells. However, the five hearing impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, disease-associated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.
连接蛋白31（Cx31）突变与可变型红斑角皮症（EKV）、听力障碍和周围神经病变相关；然而，Cx31突变体的病理机制仍不清楚。本研究分析了11种与疾病相关的Cx31变体和1种与疾病无关的Cx31变体，并比较了它们在HeLa细胞中的细胞内分布和组装情况以及对这些细胞的影响。荧光定位分析显示，在表达隐性EKV相关突变体（L34P）和4种听力障碍相关突变体（66delD、141delI、R180X和E183K）的细胞中未形成间隙连接斑，在5种EKV相关显性突变体（G12R、G12D、R42P、C86S和F137L）的细胞中斑形成显著减少，而在另外两种突变体（I141V和652del12）的细胞中无明显变化。免疫印迹分析表明，12种突变的Cx31s与野生型Cx31一样，能够形成Triton X-100不溶性复合物；然而，转染的HeLa细胞中Triton X-100不溶性复合物的量在不同的Cx31突变体之间有所不同。此外，5种EKV相关显性突变体（G12R、G12D、R42P、C86S和F137L）的表达导致HeLa细胞死亡。然而，5种听力障碍相关突变体未诱导细胞死亡。上述结果表明，与疾病相关的突变体获得了不同的有害功能。总之，与疾病相关的Cx31突变体在不同水平上损害正常间隙连接的形成，与Cx31突变相关的疾病可能是由Cx31突变体的异常组装、运输和代谢引起的。
