Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity.
Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity.
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DOI：
10.1097/fpc.0b013e328342f5b1
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发表时间：
2011-03
影响因子：
2.6
通讯作者：
Kim RB
中科院分区：
文献类型：
--
作者：
Schwarz UI;Meyer zu Schwabedissen HE;Tirona RG;Suzuki A;Leake BF;Mokrab Y;Mizuguchi K;Ho RH;Kim RB
摄取载体有机阴离子转运多肽1B3（OATP1B3，基因SLCO1B3）参与异生素的肝脏清除，包括他汀类药物、紫杉烷类药物和霉酚酸。我们考虑评估 SLCO1B3 编码区中尚未鉴定的多态性，并分析它们的功能相关性。我们使用不同种族受试者的 DNA 进行聚合酶链式反应 (PCR)，并通过测序或温度依赖性毛细管电泳确定多态性。然后，我们通过定点诱变创建了变体 OATP1B3 表达质粒，使用放射性标记底物在 HeLa 细胞中瞬时表达并进行功能表征。我们鉴定了 6 个非同义多态性，包括新变体 439A>G (Thr147Ala)、767G>C (Gly256Ala)、1559A>C (His520Pro) 和 1679T>C (Val560Ala)。等位基因频率的发生与种族有关，后者仅在非裔美国人中观察到（3.6%）。在 HeLa 细胞中表达后，与野生型相比，变体 His520Pro、Val560Ala 和 Met233Ile 或 Met233Ile_Ser112Ala 单倍型表现出胆囊收缩素-8 (CCK8) 和瑞舒伐他汀的摄取活性降低，但阿托伐他汀则不然。动力学 CCK8 分析显示 Vmax 降低但 Km 没有改变。 His520Pro 和 Val560Ala 表现出总蛋白和质膜蛋白表达降低。 Val560 映射到 OATP1B3 的结构模型表明这是底物-转运蛋白相互作用的关键区域。 His520 位于预测的细胞外区域，该区域被认为对 OATP1B3 活性的 pH 依赖性成分至关重要。 Pro520 变体在 pH 7.4 和 8.0 时的活性损失明显大于在 pH 6.5 时的活性损失。 OATP1B3 多态性导致表达、底物特异性和 pH 依赖性活性改变，可能与体内底物药物的肝脏清除具有潜在相关性。
The uptake carrier Organic Anion-transporting Polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms, and to analyze their functional relevance. We used DNA of ethnically diverse subjects for polymerase chain reaction (PCR), and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in HeLa cells using radiolabeled substrates. We identified six non-synonymous polymorphisms including novel variants 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro) and 1679T>C (Val560Ala). Allelic frequencies occurred ethnicity-dependent, with the latter observed only in African Americans (3.6%). After expression in HeLa cells, variants His520Pro, Val560Ala, and Met233Ile or Met233Ile_Ser112Ala haplotype demonstrated decreased uptake activity compared to wildtype for cholecystokinin-8 (CCK8) and rosuvastatin, but not atorvastatin. Kinetic CCK8 analysis revealed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expression. Val560 mapped onto a structural model of OATP1B3 revealed this is a key region for substrate–transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant. OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.