Characterization of a mouse Cx50 mutation associated with the No2 mouse cataract.
Characterization of a mouse Cx50 mutation associated with the No2 mouse cataract.
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DOI：
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发表时间：
1999-07
影响因子：
4.4
通讯作者：
X. Xu;L. Ebihara
中科院分区：
文献类型：
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作者：
X. Xu;L. Ebihara
最近，小鼠连接蛋白50 （Cx50）基因的错义突变与核不透明2 （No2）小鼠白内障有关。这种错义突变（D47A）导致Cx50第一个胞外结构域第47个氨基酸位置的天冬氨酸与丙氨酸取代。为了更好地了解Cx50在先天性白内障发病中的作用，我们研究了D47A突变在爪蟾卵细胞表达系统中的功能影响。方法采用聚合酶链反应（PCR）诱变法构建D47A基因。将编码野生型小鼠Cx50 （Cx50wt）、野生型大鼠Cx46 （Cx46wt）、D47A或野生型和突变型连接蛋白的组合的体外转录的cRNA注入爪蟾卵母细胞。然后将卵母细胞去卵化并配对。采用双微电极电压钳技术测量间隙结电导（Gj）。结果表达野生型Cx50或Cx46的同型卵母细胞偶联良好。相比之下，注射D47A cRNA的卵母细胞在同型配对时不形成间隙连接通道。为了测试D47A突变是否能以显性负性方式与野生型连接蛋白相互作用，我们模拟杂合条件，向卵母细胞注射等量的突变型和野生型连接蛋白cRNA。D47A的表达并未抑制野生型Cx50或Cx46诱导的配对卵母细胞结导的发展。结论这些结果表明，D47A突变是一种功能丧失突变，没有很强的显性抑制作用。在No2小鼠中，预计该突变会导致细胞间通讯减少，从而导致白内障的发生。它还可能引起其他质的变化，如小分子的渗透率的变化。
PURPOSE Recently, a missense mutation in the mouse connexin 50 (Cx50) gene has been associated with the nuclear opacity 2 (No2) mouse cataract. This missense mutation (D47A) resulted in an aspartate-to-alanine substitution at amino acid position 47 in the first extracellular domain of Cx50. To better understand the role of Cx50 in the pathogenesis of congenital cataract, the functional consequences of the D47A mutation in the Xenopus oocyte expression system were studied. METHODS D47A was constructed using polymerase chain reaction (PCR) mutagenesis. Xenopus oocytes were injected with in vitro transcribed cRNA encoding wild-type mouse Cx50 (Cx50wt), wild-type rat Cx46 (Cx46wt), D47A, or combinations of wild-type and mutant connexins. The oocytes were then devitellinized and paired. Gap junctional conductance (Gj) was measured using a dual two-microelectrode voltage-clamp technique. RESULTS Homotypic oocyte pairs expressing wild-type Cx50 or Cx46 were well coupled. In contrast, oocytes injected with D47A cRNA did not form gap junctional channels when paired homotypically. To test whether the D47A mutation could interact with wild-type connexins in a dominant negative manner, oocytes were injected with equal amounts of mutant and wild-type connexin cRNA, mimicking the heterozygous condition. Expression of D47A did not inhibit the development of junctional conductance in paired oocytes induced by wild-type Cx50 or Cx46. CONCLUSIONS These results indicate that the D47A mutation acts as a loss-of-function mutation without strong dominant inhibition. In No2 mice, the mutation would be predicted to result in a reduction in intercellular communication, leading to cataractogenesis. It may also cause other qualitative changes such as a change in permeability for small molecules.