Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation
Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation
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DOI：
10.1016/j.jmb.2010.07.060
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发表时间：
2010-10-01
影响因子：
5.6
通讯作者：
Fujiyoshi, Yoshinori
中科院分区：
文献类型：
--
作者：
Mitsuma, Tadanori;Tani, Kazutoshi;Fujiyoshi, Yoshinori
细胞质环D中Ser180的磷酸化已被证明会降低水通道蛋白（AQP） 4的水通透性，AQP是大脑中主要的水通道。然而，当利用电子衍射图对模拟磷酸化Ser180的S180D突变体（AQP4M23S180D）的结构进行2.8埃分辨率测定时，它与野生型通道的结构没有显着差异。高分辨率的密度图通常不能解析只有部分有序的蛋白质区域，但有时可以在电子显微镜计算的低分辨率密度图中看到这些区域。因此，我们使用二维晶体图像，在10埃分辨率下确定了AQP4M23S180D的结构。10埃密度图的特征与先前确定的原子模型的特征一致；特别是在细胞质孔入口附近没有任何阻塞的迹象。此外，体外和体内的水电导测量显示，野生型和突变型AQP4M23的水渗透性相同，这表明S180D突变既没有通过构象改变减少水传导，也没有通过与阻碍细胞质通道入口的蛋白质相互作用来减少水传导。最后，10埃图显示了四个相邻四聚体之间的细胞质密度，这很可能代表了四个N端的关联。这一发现支持了AQP4的N端在稳定正交阵列中的关键作用，以及它们通过脂质修饰长N端异构体中半胱氨酸残基的干扰。(C) 2010 Elsevier Ltd.版权所有。
Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 angstrom resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 angstrom resolution. The features of the 10-angstrom density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-angstrom map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform. (C) 2010 Elsevier Ltd. All rights reserved.