Functional effects of Cx50 mutations associated with congenital cataracts
Functional effects of Cx50 mutations associated with congenital cataracts
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DOI：
10.1152/ajpcell.00098.2013
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发表时间：
2014-02-01
影响因子：
5.5
通讯作者：
Srinivas, Miduturu
中科院分区：
文献类型：
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作者：
Rubinos, Clio;Villone, Krista;Srinivas, Miduturu
在人类和小鼠中，连接蛋白50 （Cx50）的突变导致显性白内障。突变导致这些可变表型的确切机制尚不清楚。我们研究了在哺乳动物细胞系中表达的三种Cx50突变V44E、D47N和V79L所产生的间隙连接的功能特性。V44E被正常输送到质膜上，形成间隙连接斑块。然而，该突变体单独表达或与野生型（WT） Cx46和Cx50一起表达时均未形成功能性间隙连接，这表明V44E是WT连接蛋白功能的显性阴性抑制剂。相反，D47N亚基不定位于连接斑块或形成功能性的同型间隙连接；然而，Cx50或Cx46的D47N和WT亚基的混合表达导致功能性的细胞间通道，并具有高水平的偶联。单通道研究表明，D47N与WT Cx46形成了具有独特性质的异质通道。与V44E和D47N不同，V79L形成了功能性同型细胞间通道。然而，突变引起电压门控的改变和单通道打开概率的显著降低，导致单独表达V79L或与WT连接蛋白亚基一起表达的细胞的电导水平低得多。因此，每个突变在连接偶联的特性上产生了明显的变化。V44E在任何构型下都不能形成细胞间通道，D47N只形成带有WT连接蛋白的异质通道，V79L形成性质改变的同型和异质通道。这些结果表明，突变型和野生型晶状体连接蛋白之间的独特相互作用可能是人类各种白内障表型发展的基础。
Mutations in connexin50 (Cx50) cause dominant cataracts in both humans and mice. The exact mechanisms by which mutations cause these variable phenotypes are poorly understood. We have examined the functional properties of gap junctions made by three Cx50 mutations, V44E, D47N, and V79L, expressed in mammalian cell lines. V44E trafficked to the plasma membrane properly and formed gap junctional plaques. However, the mutant did not form functional gap junctions when expressed alone, or with wild-type (WT) Cx46 and Cx50, indicating that V44E is a dominant negative inhibitor of WT connexin function. In contrast, D47N subunits did not localize to junctional plaques or form functional homotypic gap junctions; however, mixed expression of D47N and WT subunits of either Cx50 or Cx46 resulted in functional intercellular channels, with high levels of coupling. Single-channel studies indicated that D47N formed heteromeric channels with WT Cx46 with unique properties. Unlike either V44E or D47N, V79L formed functional homotypic intercellular channels. However, the mutation caused an alteration in voltage gating and a dramatic reduction in the single-channel open probability, resulting in much lower levels of conductance in cells expressing V79L alone, or together with WT connexin subunits. Thus, each mutation produced distinct changes in the properties of junctional coupling. V44E failed to form intercellular channels in any configuration, D47N formed only heteromeric channels with WT connexins, and V79L formed homotypic and heteromeric channels with altered properties. These results suggest that unique interactions between mutant and wild-type lens connexins might underlie the development of various cataract phenotypes in humans.