Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46.
Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46.
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DOI：
10.1152/ajpcell.00344.2012
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发表时间：
2012-01
期刊：
影响因子：
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通讯作者：
Jun-Jie Tong;Bonnie C. H. Sohn;A. Lam;D. Eric Walters;Barbara M. Vertel;L. Ebihara
中科院分区：
文献类型：
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作者：
Jun-Jie Tong;Bonnie C. H. Sohn;A. Lam;D. Eric Walters;Barbara M. Vertel;L. Ebihara
连接蛋白46的突变与先天性白内障有关。本研究的目的是研究Cx46D3Y和Cx46L11S这两个位于连接蛋白46 NH2末端的先天性白内障相关突变的细胞和功能特性，我们发现这两个突变定位于HeLa细胞中类似野生型Cx46的缝隙连接斑块。对注射野生型和突变型大鼠Cx46的非洲爪哇卵母细胞对的双微电极-电压钳研究表明，注射D3Y或L11S cRNA的卵母细胞对未能诱导缝隙连接偶联，而注射Cx46的卵母细胞对则表现出高度的偶联。D3Y，但不是L11S，与野生型Cx46在功能上配对。为了确定D3Y或L11S的共表达是否影响野生型晶状体连接蛋白产生的连接电导，我们研究了共注射等量突变型和野生型连接蛋白cRNA的卵母细胞对。D3Y或L11S的表达几乎完全消除了Cx46诱导的缝隙连接偶联。相反，D3Y或L11S的表达不能抑制CX50诱导的连接电导。为了检测D3Y和L11S突变对半通道活动的影响，对注射连接蛋白cRNA的卵母细胞进行了半通道电流的测量。表达D3Y的卵母细胞表现出半通道活性降低，电压门控和电荷选择性改变，而表达L11S的卵母细胞没有半通道活性。此外，突变体与野生型CX50或Cx46共表达产生了具有不同电生理特性的半通道，表明突变体连接蛋白与野生型连接蛋白形成了异构体通道。这些数据表明，D3Y和L11S通过相似但不相同的机制导致白内障。
Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling. D3Y, but not L11S, functionally paired with wild-type Cx46. To determine whether coexpression of D3Y or L11S affected the junctional conductance produced by wild-type lens connexins, we studied pairs of oocytes coinjected with equal amounts of mutant and wild-type connexin cRNA. Expression of D3Y or L11S almost completely abolished gap junctional coupling induced by Cx46. In contrast, expression of D3Y or L11S failed to inhibit junctional conductance induced by Cx50. To examine effects of the D3Y and L11S mutations on hemichannel activity, hemichannel currents were measured in connexin cRNA-injected oocytes. Oocytes expressing D3Y exhibited reduced hemichannel activity as well as alterations in voltage gating and charge selectivity while oocytes expressing L11S showed no hemichannel activity. Moreover, coexpression of mutant with wild-type Cx50 or Cx46 gave rise to hemichannels with distinct electrophysiological properties, suggesting that the mutant connexins were forming heteromeric channels with wild-type connexins. These data suggest D3Y and L11S cause cataracts by similar but not identical mechanisms.