Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4).
Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4).
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人有机阴离子转运蛋白 4 (hOAT4) 中组氨酸残基的突变分析。
DOI：
10.1042/bj20040751
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
You,Guofeng
中科院分区：
文献类型：
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作者：
Zhou,Fanfan;Pan,Zui;Ma,Jianjie;You,Guofeng
人有机阴离子转运蛋白4(HOAT4)属于一个有机阴离子转运蛋白家族，在抗HIV治疗药物、抗肿瘤药物、抗生素、抗高血压药物和抗炎药物等临床重要药物的体内转运中发挥重要作用。组氨酸修饰剂DEPC(焦碳酸二乙酯)可抑制hOAT4介导的有机阴离子雌酮在COS-7细胞中的转运。因此，我们用定点突变的方法研究了组氨酸残基在hOAT4功能中的作用。HOAT4的5个组氨酸残基被单独或组合转化为丙氨酸。单独更换HIS-47或同时更换HIS-47/52/83或HIS-47/52/83/305/469(H-less)导致运输活动减少50%-80%。尽管这些突变体的细胞总表达与野生型hOAT4相似，但这些突变体的转运活性降低与细胞表面表达的减少有关。这些结果表明，47、47/52/83和47/52/83/305/469位的突变损害了膜的表达，而不是功能。我们还发现，虽然大多数hOAT4的组氨酸突变体对DEPC的抑制敏感，但H469A(His-469→Ala)对该试剂的抑制完全不敏感。因此，His-469的修饰与DEPC对hOAT4的抑制有关。
Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. hOAT4-mediated transport of the organic anion oestrone sulphate in COS-7 cells was inhibited by the histidine-modifying reagent DEPC (diethyl pyrocarbonate). Therefore the role of histidine residues in the function of hOAT4 was examined by site-directed mutagenesis. All five histidine residues of hOAT4 were converted into alanine, singly or in combination. Single replacement of His-47, or simultaneous replacement of His-47/52/83 or His-47/52/83/305/469 (H-less) led to a 50–80% decrease in transport activity. The decreased transport activity of these mutants was correlated with a decreased amount of cell-surface expression, although the total cell expression of these mutants was similar to that of wild-type hOAT4. These results suggest that mutation at positions 47, 47/52/83 and 47/52/83/305/469 impaired membrane expression rather than function. We also showed that, although most of the histidine mutants of hOAT4 were sensitive to inhibition by DEPC, H469A (His-469→Ala) was completely insensitive to inhibition by this reagent. Therefore modification of His-469 is responsible for the inhibition of hOAT4 by DEPC.