Expression Cloning and Characterization of ROAT1
Expression Cloning and Characterization of ROAT1
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DOI：
10.1074/jbc.272.48.30088
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发表时间：
1997-11
期刊：
影响因子：
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通讯作者：
D. H. Sweet;N. Wolff;J. B. Pritchard
中科院分区：
文献类型：
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作者：
D. H. Sweet;N. Wolff;J. B. Pritchard
利用爪蟾卵母细胞的表达克隆技术，从大鼠肾脏中分离出一种有机阴离子转运蛋白。利用poly(A)+ RNA构建cDNA文库，筛选对氨基马嘌呤（PAH）的对乙酰氨基马嘌呤敏感转运。分离得到一个2227个碱基对的cDNA克隆，内含1656个碱基对的开放阅读框，编码551个氨基酸。roat1介导的50 μm [3H]多环芳烃的转运与膜电位的施加变化无关。在4°C或与其他有机阴离子孵育时，转运明显受到抑制，但有机阳离子四乙基铵、多药耐atp酶抑制剂环孢素A或尿酸盐不受抑制。外戊二酸和α-酮戊二酸（1 mm）都是基底外侧多环芳烃交换的反离子，也抑制了运输，这表明ROAT1在功能上与基底外侧多环芳烃载体相似。与这一结论一致的是，预负荷戊二酸盐的卵母细胞对多环芳烃的摄取受到反式刺激，而不被基底外侧交换器接受的二羧酸盐甲基琥珀酸则没有反式刺激。最后，roat1介导的多环芳烃转运是饱和的，估计K m为70 μm。这些性质与先前描述的分离膜囊泡或完整肾小管中基底外侧α-酮戊二酸/多环芳烃交换剂的性质相同。
Expression cloning in Xenopus laevisoocytes was used to isolate an organic anion transport protein from rat kidney. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for probenecid-sensitive transport of p-aminohippurate (PAH). A 2,227-base pair cDNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1. ROAT1-mediated transport of 50 μm [3H]PAH was independent of imposed changes in membrane potential. Transport was significantly inhibited at 4 °C, or upon incubation with other organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate. External glutarate and α-ketoglutarate (1 mm), both counterions for basolateral PAH exchange, also inhibited transport, suggesting that ROAT1 is functionally similar to the basolateral PAH carrier. Consistent with this conclusion, PAH uptake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basolateral exchanger, did nottrans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K m of 70 μm. Each of these properties is identical to those previously described for the basolateral α-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.