title: Electrophysiological characterization of human Na⁺/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes.
abstract: 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) in Xenopus laevis oocytes 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 in Xenopus oocytes utilizing [(3)H]-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 in Xenopus oocytes directly demonstrated that hNTCP is an electrogenic Na(+)-dependent transporter.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Masuda
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.
authors.forename: Yukari
authors.initials: Y
authors.lastname: Ichikawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Shimono
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Shimizu
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Nara
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan. Electronic address: seiji.miyauchi@phar.toho-u.ac.jp.
authors.forename: Seiji
authors.initials: S
authors.lastname: Miyauchi
mesh_terms: D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D055724:Electrophysiological Phenomena; D015723:Gene Library; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D009865:Oocytes; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000737:chemistry*; D027981:Symporters / Q000378:metabolism*; D013656:Taurocholic Acid / Q000737:chemistry*; D014982:Xenopus laevis
chemical_list: D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter; D013656:Taurocholic Acid; D012964:Sodium
keywords: Bile acid; Electrogenicity; Hepatic uptake; NTCP; Na(+)-dependent transporter; SLC10A1; Xenopus laevis oocyte