Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3
Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3
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DOI：
10.1021/bi002841o
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发表时间：
2001-05-08
期刊：
影响因子：
2.9
通讯作者：
Giacomini, KM
中科院分区：
文献类型：
--
作者：
Feng, B;Dresser, MJ;Giacomini, KM
有机阴离子转运蛋白（OAT）和有机阳离子转运蛋白（OCT）介导外源性物质穿过上皮细胞质膜的通量。OAT的底物通常携带负电荷，而OCT的底物是阳离子。本研究的目的是确定大鼠有机阴离子转运蛋白rOAT 3识别和转运有机阴离子所必需的结构域和氨基酸残基。含有rOAT 3的跨膜结构域1-5和rOCT 1的跨膜结构域6-12的rOAT 3/rOCT 1嵌合体保留了rOCT 1的特异性，表明参与底物识别的残基位于这些转运蛋白的羧基末端一半内。一个保守的碱性氨基酸残基，精氨酸454天冬氨酸（R454 D）的突变，揭示了这个氨基酸是必需的有机阴离子转运。在表达rOAT 3的卵母细胞中，对氨基马尿酸（PAH）、硫酸雌酮和赭曲霉毒素A的摄取分别增加了10倍、48倍和32倍，而R454 D仅增加了2倍、6倍和5倍。类似地，保守的赖氨酸370突变为丙氨酸（K370 A）表明K370对于有机阴离子转运是重要的。有趣的是，与rOAT 3相比，双突变体R454 DK 370 A的电荷特异性被逆转-与PAH相比，R454 DK 370 A优先转运有机阳离子MPP+（双突变体的MPP+摄取/PAH摄取= 3.21，rOAT 3为0.037）。这些数据表明，精氨酸454和赖氨酸370对rOAT 3的阴离子特异性至关重要。这些研究提供了第一次深入了解的分子决定因素，是至关重要的有机阴离子的识别和转运的有机阴离子转运蛋白家族的成员。
Organic anion transporters (OATs) and organic cation transporters (OCTs) mediate the flux of xenobiotics across the plasma membranes of epithelia. Substrates of OATs generally carry negative charge(s) whereas substrates of OCTs are cations. The goal of this study was to determine the domains and amino acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera containing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT 1 retained the specificity of rOCT1, suggesting that residues involved in substrate recognition reside within the carboxyl-terminal half of these transporters. Mutagenesis of a conserved basic amino acid residue, arginine 454 to aspartic acid (R454D), revealed that this amino acid is required for organic anion transport. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were similar to 10-, similar to 48-, and similar to 32-fold enhanced in oocytes expressing rOAT3 and were only similar to2-, similar to6-, and similar to5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lysine 370 to alanine (K370A) suggested that K370 is important for organic anion transport. Interestingly, the charge specificity of the double mutant, R454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially transported the organic cation, MPP+, in comparison to PAH (MPP+ uptake/PAH uptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity of rOAT3. The studies provide the first insights into the molecular determinants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.