Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis
Structure-Function Studies of Claudin Extracellular Domains by Cysteine-scanning Mutagenesis
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DOI：
10.1074/jbc.m109.043752
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发表时间：
2009-10-16
影响因子：
4.8
通讯作者：
Yu, Alan S. L.
中科院分区：
文献类型：
--
作者：
Angelow, Susanne;Yu, Alan S. L.
紧密连接蛋白在紧密连接中形成尺寸和电荷选择性孔，控制无机离子和小分子的细胞旁通量。然而，人们对细胞旁孔离子选择性的结构基础知之甚少。在这里，我们应用半胱氨酸扫描来绘制claudin-2转染的Madin-Darby犬肾I型细胞的离子渗透的细胞旁途径。将第一个细胞外环中的四个潜在的孔衬氨基酸残基突变为半胱氨酸，并筛选它们对硫醇反应试剂的可及性。除 D65C 外，所有突变体均具有功能，D65C 通过分子间二硫键形成二聚体，导致电荷和尺寸选择性丧失。这表明claudin-2 孔是多聚体，并且Asp(65) 靠近蛋白质-蛋白质界面。不同大小和电荷的甲硫代磺酸盐试剂以及有机汞衍生物对（氯汞）苯磺酸通过一种表明孔的空间阻塞的机制显着降低了跨 I66C 转染细胞的细胞旁离子渗透。野生型claudin-2和其他半胱氨酸突变体的电导仅受到微弱影响。随着试剂尺寸的增加，与 I66C 的反应速率急剧下降，这表明 Ile(66) 深埋在孔的狭窄部分内，其侧基面向管腔。此外，用N-生物素氨乙基甲硫磺酸盐标记表明I66C具有弱反应性，而Y35C具有强反应性，表明Tyr(35)位于孔外的蛋白质表面。
Claudins form size- and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp(65) lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri) benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile(66) is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr(35) is located at the protein surface outside of the pore.