Electrophysiological characterization of human Na+/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes
Electrophysiological characterization of human Na+/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes
复制标题
非洲爪蟾卵母细胞异源表达的人钠/牛磺胆酸共转运多肽（hNTCP）的电生理学特征
DOI：
10.1016/j.abb.2014.08.010
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发表时间：
2014
影响因子：
3.9
通讯作者：
Miyauchi S
中科院分区：
文献类型：
--
作者：
Masuda M;Ichikawa Y;Shimono K;Shimizu M;Tanaka Y;Nara T;Miyauchi S
Na+/牛磺胆酸盐共转运多肽（NTCP）在Na+依赖性胆汁酸摄取到肝细胞中起主要作用。本研究的目的是建立人NTCP（hNTCP）在非洲爪蟾细胞中的异源表达，并阐明胆汁酸通过hNTCP的运输是否是用电生理技术。首先，我们利用[3 H]-标记的牛磺胆酸盐（TCA）评估了异种卵母细胞中异源表达的hNTCP对TCA的摄取。与注射H2O的卵母细胞相比，注射cRNA的卵母细胞对1.2 μM TCA的摄取增加了100倍以上，表明hNTCP在卵母细胞中稳健表达。hNTCP介导的TCA转运是饱和的，米氏常数为10.5 ± 2.9 μM。描述Na+浓度与hNTCP的TCA摄取速率大小之间关系的Na+活化动力学呈S形，希尔系数为2.3 ± 0.4，表明转运过程中涉及一种以上的Na+。在大鼠原代培养肝细胞中，NTCP的Na+激活动力学与TCA摄取速率相似，Hill系数为1.9 ± 0.1，表明hNTCP能在卵母细胞中正常表达，并具有Na+偶联TCA转运的产电特性。随后通过在电压（−50 mV）下TCA摄取诱导的内向电流在卵母细胞中确定TCA通过hNTCP的转运。200微摩尔TCA诱导显著的内向电流，完全取消的取代Na+与N-甲基-D-葡糖胺（NMDG）在灌流液中，表明TCA诱导的电流是强制性依赖于Na+的存在。TCA诱导的电流是饱和的，并且电流的半最大诱导所需的底物浓度与米氏常数一致。可转运底物，如瑞舒伐他汀和氟伐他汀，也诱导电流。hNTCP在异种卵母细胞中异源表达的结果直接证明hNTCP是一种产电Na+依赖性转运蛋白。
The Na+/taurocholate cotransporting polypeptide (NTCP) plays a major role in Na+-dependent bile acid uptake into hepatocytes. The purpose of the present study was to establish the heterologous expression of human NTCP (hNTCP) inXenopus laevisoocytes and to elucidate whether the transport of bile acid via hNTCP is electrogenic using electrophysiological techniques. First, we evaluated the uptake of taurocholate (TCA) by hNTCP heterologously expressed inXenopusoocytes utilizing [3H]-labeled TCA. The uptake of 1.2 μM TCA by cRNA-injected oocytes increased more than 100-fold compared to H2O-injected oocytes, indicating that hNTCP is robustly expressed in the oocytes. hNTCP-mediated transport of TCA is saturable with a Michaelis constant of 10.5 ± 2.9 μM. The Na+-activation kinetics describing the relationship between the concentration of Na+and the magnitude of the TCA uptake rate by hNTCP were sigmoidal with a Hill coefficient of 2.3 ± 0.4, indicating the involvement of more than one Na+in the transport process. Ntcp in primary cultured hepatocytes from rats exhibited similar Na+-activation kinetics of TCA uptake rate with a Hill coefficient of 1.9 ± 0.1, suggesting that hNTCP could be expressed properly in the oocytes and exhibit the electrogenic property of Na+-coupled TCA transport. The transport of TCA via hNTCP was subsequently determined in the oocytes by the inward currents induced via TCA uptake under voltage (−50 mV). Two hundred micromolar TCA induced significant inward currents that were entirely abolished by the substitution of Na+with N-methyl-d-glucamine (NMDG) in the perfusate, indicating that the TCA-induced currents were obligatorily dependent on the presence of Na+. The TCA-induced currents were saturable, and the substrate concentration needed for half-maximal induction of the current was consistent with the Michaelis constant. Transportable substrates, such as rosuvastatin and fluvastatin, also induced currents. These results in the hNTCP heterologously expressed inXenopusoocytes directly demonstrated that hNTCP is an electrogenic Na+-dependent transporter.