Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014, Helsinki, Finland.
School of Pharmacy, University of Eastern Finland, Kuopio, Finland.
Pharm Res. 2021 Oct;38(10):1663-1675. doi: 10.1007/s11095-021-03107-8. Epub 2021 Oct 13.
Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance.
Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC-MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions.
All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants.
Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.
有机阴离子转运多肽 1B1（OATP1B1）介导许多药物的肝内摄取和清除，包括他汀类药物。SLCO1B1 基因高度多态性，其功能受损的变体可使患者易发生不良反应。SLCO1B1 的罕见遗传变体的影响主要尚未被探索。我们研究了八种天然存在的罕见变体和众所周知的 SLCO1B1 c.521C > T（V174A）变体对体外转运活性、细胞定位和丰度的影响。
通过测量 OATP1B1 表达的 HEK293 细胞中瑞舒伐他汀和 2,7-二氯荧光素（DCF）的转运，评估变体活性的变化。免疫荧光和共聚焦显微镜确定 OATP1B1 的细胞定位，基于 LC-MS/MS 的定量靶向绝对蛋白质组学分析定量粗膜部分的 OATP1B1 量。
除 P336R 外，所有研究的变体均不同程度地降低了蛋白丰度。V174A 与野生型相比，降低了超过 90%的蛋白丰度。G76E、V174A、L193R 和 R580Q 变体丧失了转运功能。R181C 显著降低了活性，而 T345M 和 L543W 保留了野生型 OATP1B1 的大部分活性。P336R 显示出增加的活性，H575L 显著降低了 DCF 的转运，但不降低瑞舒伐他汀的转运。活性降低与研究变体中绝对蛋白丰度降低有关。
跨膜螺旋 2、4 和 11 似乎对 OATP1B1 的适当膜定位和功能至关重要。研究的四个变体被鉴定为失活变体，因此携带这些变体的个体可能易发生药代动力学改变和底物药物的不良反应。
