The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation
The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation
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DOI：
10.1167/iovs.18-26459
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发表时间：
2019-05-01
影响因子：
4.4
通讯作者：
Beyer, Eric C.
中科院分区：
文献类型：
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作者：
Berthoud, Viviana M.;Gao, Junyuan;Beyer, Eric C.
目的。connexin50 （Cx50）和connexin46 （Cx46）的突变导致白内障。由于Cx46fs380的表达导致间隙连接偶联减少和钙沉淀的形成，我们研究了Cx50D47A晶体，以检验Cx50突变体是否也因钙沉淀而导致白内障。免疫印迹法测定连接蛋白水平。从完整透镜的细胞内阻抗研究中计算间隙结耦合电导。使用微电极/压力计系统测量细胞内静水压力。使用Fura-2和荧光成像测量细胞内游离钙离子浓度（[Ca2+](i)）。采用茜素红染色评价钙沉淀，并与暗场图像中混浊物的分布进行比较。在Cx50D47A晶体中，与野生型相比，2.5个月时Cx50水平为11%（杂合子）和1.2%（纯合子），Cx46水平为52%（杂合子）和30%（纯合子）。与野生型相比，Cx50D47A晶状体分化纤维的间隙结偶率分别为49%（杂合子）和29%（纯合子），成熟纤维的间隙结偶率分别为24%（杂合子）和4%（纯合子）。Cx50D47A镜头的静水压力明显增加。[Ca2+](i)在Cx50D47A透镜中显著增加。纯合子Cx50D47A晶体中存在茜素红染色的钙沉淀，其分布与白内障相似。Cx50D47A表达通过降低连接蛋白水平和间隙连接耦合改变晶状体内循环。通过间隙连接的水和离子流出量减少，增加了细胞内静水压力梯度和游离钙离子浓度。在这些晶状体中，钙离子积聚、沉淀，形成白内障。这些结果表明晶状体纤维连接蛋白突变导致钙沉淀，这可能导致白内障。
PURPOSE. Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation.METHODS. Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca2+](i)) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images.RESULTS. In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca2+](i) was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts.CONCLUSIONS. Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calciumions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.