| 2022 |
Cryo-EM structures of human, bovine, and rat NTCPs in the apo state reveal a tunnel across the membrane and a possible transport route for the bile acid substrate. The structure of human NTCP in complex with the myristoylated preS1 domain of the HBV large surface protein, together with mutation and transport assays, shows that preS1 and the bile acid substrate compete for the extracellular opening of the tunnel, establishing the binding mode for viral entry. |
Cryo-electron microscopy, mutation assays, transport assays |
Nature |
High |
35580629
|
| 2022 |
Cryo-EM structure of NTCP bound to an antibody shows the transporter lacks the first transmembrane helix found in other SLC10 proteins and has an N-terminus exposed on the extracellular face. Comparison with related proteins indicates a common mechanism of bile acid transport, and an additional pocket formed by residues known to interact with preS1 was identified. |
Cryo-electron microscopy, structural comparison |
Nature |
High |
35580630
|
| 2024 |
Cryo-EM structure of bulevirtide (BLV/Myrcludex B)-bound human NTCP reveals BLV forms two domains: a plug lodged in the bile salt transport tunnel of NTCP and a string covering the extracellular surface, with the N-terminal myristoyl group interacting with the lipid-exposed surface of NTCP. This structure explains how BLV blocks bile salt transport and provides a structural basis for HBV/HDV host specificity. |
Cryo-electron microscopy |
Nature communications |
High |
38509088
|
| 2013 |
NTCP (SLC10A1) was confirmed as the functional HBV entry receptor: HepG2 cells engineered to overexpress human NTCP became susceptible to HBV infection, and knockdown of NTCP blocked infection. Compounds that inhibit NTCP transporter activity (including cyclosporin A and oxysterols) also blocked HBV infection, linking bile acid transport function to viral entry. |
NTCP overexpression in HepG2 cells, siRNA knockdown, HBV infection assay, inhibitor studies |
Biochemical and biophysical research communications |
High |
24342612
|
| 2013 |
Cyclosporin A inhibits HBV and HDV entry by directly interfering with NTCP receptor function in a cyclophilin-independent manner. Binding of the HBVpreS1 domain to NTCP was blocked by cyclosporin A, and an NTCP variant deficient in HBVpreS1 binding but competent for bile salt transport was resistant to cyclosporin A, mapping the interaction site to the preS1-binding domain on NTCP. |
HepaRG cells and NTCP-expressing hepatoma cell lines, taurocholate uptake assay, HBVpreS1 binding assay, cyclophilin siRNA, NTCP variant studies |
Journal of hepatology |
High |
24295872
|
| 2014 |
The myristoylated preS1 domain of HBV inhibits bile acid transport by NTCP, and inversely, bile acid conjugates (taurine/glycine conjugates of cholic acid and ursodeoxycholic acid) inhibit HBV infection in a concentration-dependent manner. NTCP expression, transport function, preS1 peptide binding, and HBV infection follow comparable kinetics, establishing direct functional overlap between the bile acid transport site and the HBV receptor site. |
Bile acid transport assays, HBV infection assays, myr-preS1 peptide binding in primary hepatocytes and NTCP-transfected cells |
Journal of hepatology |
High |
24845614
|
| 2005 |
Dephosphorylation of Ser-226 in the third cytoplasmic loop of NTCP facilitates plasma membrane retention. Mutation of Ser-226 to Ala decreased NTCP phosphorylation by 30% and increased taurocholate uptake and plasma membrane retention 2.5–3.2-fold; cAMP failed to further increase translocation of S226A-NTCP, establishing Ser-226 as the cAMP-regulated phosphorylation site controlling NTCP plasma membrane localization. |
Metabolic [32P] labeling, clostripain digestion, site-directed mutagenesis, taurocholate uptake assay, cell surface expression in transfected HuH-7 cells |
The Journal of biological chemistry |
High |
16027164
|
| 2002 |
PP2B (calcineurin), a Ca2+/calmodulin-dependent phosphatase, mediates cAMP-induced dephosphorylation and translocation of NTCP to the plasma membrane. The PP2B inhibitor cypermethrin reversed cAMP-mediated NTCP dephosphorylation and translocation, and PP2B directly dephosphorylated immunoprecipitated NTCP from control but not cAMP-treated hepatocytes. |
PP2B inhibitors (cypermethrin, FK-506), taurocholate uptake assay, NTCP immunoprecipitation, PP2B activity assay in isolated rat hepatocytes |
American journal of physiology. Gastrointestinal and liver physiology |
High |
12065290
|
| 2002 |
Protein kinase B (PKB/Akt) mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and NTCP translocation to the plasma membrane via the PI3K/PKB signaling pathway. Dominant-negative PKB blocked cAMP- and swelling-induced increases in TC uptake and NTCP translocation, while constitutively active PKB increased both. |
Dominant-negative and constitutively active PKB transfection, taurocholate uptake assay, NTCP translocation assay in HuH-Ntcp cells |
The Journal of biological chemistry |
High |
12034724
|
| 2006 |
NTCP-containing vesicles are present on intracellular recycling endosomes and move bidirectionally on microtubules using kinesin-1 and dynein motors. PKCζ is specifically required for microtubule-based motility of NTCP vesicles: PI(3,4,5)P3 activates PKCζ and enhances motility, while specific inhibition of PKCζ blocks motility of NTCP-containing vesicles but not late vesicles. |
In vitro and whole-cell immunofluorescence microscopy, live-cell imaging of NTCP-GFP, PKCζ inhibitor, colocalization with endosomal markers |
Traffic (Copenhagen, Denmark) |
High |
16734659
|
| 2005 |
Rat Ntcp/human NTCP is degraded by the ubiquitin-proteasome system via ER-associated degradation (ERAD). Proteasome inhibition led to accumulation of core-glycosylated (ER-resident) intracellular NTCP that co-localized with ubiquitin at the microtubule organization center as polyubiquitinated aggresomes. |
Proteasome inhibitors (MG-132, lactacystin), co-localization studies, polyubiquitination assay, glycosylation analysis in stably transfected HepG2 cells |
Biological chemistry |
High |
16218878
|
| 2002 |
Interleukin-1β suppresses Ntcp gene expression via a JNK-dependent mechanism: JNK phosphorylates RXR, reducing nuclear RXR:RAR binding activity and thereby decreasing RXR:RAR-mediated transactivation of the Ntcp promoter. JNK inhibition by curcumin or dominant-negative JNK completely blocked IL-1β-mediated suppression, while ERK and p38 inhibitors had no effect. |
Ntcp promoter luciferase assays, JNK expression plasmids (dominant-negative and wild-type), pharmacological inhibitors, EMSA, primary rat hepatocytes |
The Journal of biological chemistry |
High |
12105223
|
| 2003 |
HNF1α, HNF4α, and RXRα/RARα activate the rat Ntcp promoter but not the human or mouse NTCP/Ntcp promoters, whereas C/EBPβ activation is specific to human and mouse. HNF3β is the only transcription factor binding and active at a conserved motif in all three species, where it inhibits NTCP/Ntcp promoter activity. Small heterodimer partner (SHP) does not affect NTCP/Ntcp promoter activity from the conserved region. |
Transfection-based luciferase reporter assays, electrophoretic mobility shift assay (EMSA), cotransfection in Huh7 cells |
American journal of physiology. Gastrointestinal and liver physiology |
High |
14701722
|
| 2002 |
Short heterodimer partner 1 (SHP-1) induction by retained bile acids temporally precedes and likely mediates downregulation of Ntcp in obstructive cholestasis. In bile duct-ligated mice, SHP-1 mRNA peaked at 6 h after ligation, followed 6 h later by the nadir of Ntcp mRNA and protein. |
Northern and Western blotting of SHP-1 and Ntcp in CBDL mice, immunofluorescence |
American journal of physiology. Gastrointestinal and liver physiology |
Medium |
11751172
|
| 2005 |
In obstructive cholestasis (CBDL), Ntcp downregulation is bile acid–mediated and FXR-dependent, not cytokine-mediated. FXR knockout mice fail to repress Ntcp after bile acid feeding or CBDL; LPS-induced Ntcp repression is FXR-independent. The mechanism involves reduced nuclear levels and DNA binding of HNF-1α, HNF-4α, RXRα, and RARα. |
FXR knockout mice, bile duct ligation, cholic acid feeding, LPS treatment, EMSA, Western blotting, cytokine inactivation |
American journal of physiology. Gastrointestinal and liver physiology |
High |
16002565
|
| 2005 |
Kupffer cell depletion with liposomal clodronate prevents endotoxin-induced suppression of Ntcp expression. In endotoxin-exposed rats, KC depletion preserved Ntcp RNA and protein expression and maintained RXR:RAR and HNF1α transcription factor binding activity. |
Liposomal clodronate KC depletion, LPS exposure, Northern/Western blotting, EMSA in rats |
Journal of hepatology |
Medium |
15629514
|
| 2005 |
In obstructive cholestasis in rodents, accumulating bile acids—independent of cytokines—downregulate Ntcp through repression of HNF-1α and HNF-4α. Both TNF-α/IL-1β neutralization and Kupffer cell depletion failed to restore Ntcp expression, while SHP mRNA increased 3–5-fold. |
Cytokine neutralization (etanercept, anakinra), KC depletion by liposome clodronate, Western/Northern blotting, EMSA in CBDL rats and mice |
Hepatology (Baltimore, Md.) |
Medium |
15723437
|
| 2003 |
NTCP (Ntcp) facilitates hepatocellular uptake of the lethal mushroom toxin α-amanitin. HepG2 cells stably transfected with rat Ntcp showed markedly increased sensitivity to α-amanitin cytotoxicity compared to non-transfected cells, as measured by suppression of cytokine-induced IL-1Ra mRNA synthesis. |
Stable transfection of rat Ntcp in HepG2 cells, taurocholate uptake kinetics, functional toxicity assay (IL-1Ra mRNA induction) |
Archives of toxicology |
Medium |
14598021
|
| 2006 |
Human NTCP and BSEP reconstituted together in LLC-PK1 polarized cells mediate vectorial transcellular transport of bile acids from the basolateral to apical side, with substrate specificities showing taurine conjugates > glycine conjugates > unconjugated bile salts for NTCP. Single-transporter cells showed no vectorial transport. |
Double transfection of LLC-PK1 cells with NTCP and BSEP, transcellular bile acid flux assays, kinetic analysis |
American journal of physiology. Gastrointestinal and liver physiology |
High |
16474011
|
| 2017 |
N-linked glycosylation of NTCP at residues N5 and N11 is required for efficient trafficking to the plasma membrane and for HBV infection. NTCP lacking both glycosylation sites (N5,11Q) failed to support HBV infection, showed minimal cellular expression, and was degraded in lysosomes, while single-glycan variants retained normal bile acid transport function and HBV infectability. |
Site-directed mutagenesis of glycosylation sites, cell surface biotinylation, lysosomal degradation assay, HBV infection assay, bile acid uptake assay in HepG2 cells |
PloS one |
High |
28125599
|
| 2003 |
The C800T (Ser267Phe) polymorphism in NTCP causes near-complete loss of bile acid uptake function while preserving transport of the non-bile acid substrate estrone sulfate, identifying position 267 as part of a region critical and specific for bile acid substrate recognition. Cell surface biotinylation showed normal plasma membrane expression of this variant. |
Cell surface biotinylation, taurocholate and estrone sulfate transport assays, immunofluorescence confocal microscopy in HepG2 and transfected cells |
The Journal of biological chemistry |
High |
14660639
|
| 2013 |
A dileucine motif in the third intracellular loop of Ntcp is essential for both endocytosis and plasma membrane targeting, indicating a dual trafficking function. PKC-mediated endocytosis of Ntcp from the plasma membrane is clathrin-dependent and is followed by lysosomal degradation. Mutation of Thr225 and Ser226 inhibited PKC-mediated endocytosis. |
Flow cytometry, immunofluorescence, Western blotting, site-directed mutagenesis of dileucine motif and phosphorylation sites in HepG2 cells |
American journal of physiology. Gastrointestinal and liver physiology |
High |
24008362
|
| 2010 |
Nitric oxide (NO) inhibits taurocholate uptake by NTCP via S-nitrosylation of cysteine residues on NTCP, causing non-competitive inhibition (decreased Vmax, unchanged Km) and reduction of NTCP at the plasma membrane. Dithiothreitol reversed both S-nitrosylation and transport inhibition. |
Biotin switch assay for S-nitrosylation, taurocholate kinetic uptake assay, plasma membrane biotinylation, NO donor treatment in HuH-NTCP cells |
American journal of physiology. Gastrointestinal and liver physiology |
High |
21109590
|
| 2012 |
Plasma membrane localization of PKCδ, rather than its kinase activity, is necessary for cAMP-induced NTCP translocation and Rab4 activation. Kinase-dead dominant-negative PKCδ and PKCδ knockdown both still increased plasma membrane NTCP and Rab4 activity when PKCδ was at the membrane. In contrast, kinase activity of PKCδ is required for cAMP-induced MRP2 translocation. |
Wild-type, kinase-dead DN-PKCδ transfection, siRNA knockdown, Rab4 activity assay, rottlerin and LY294002 inhibitors in HuH-NTCP cells |
American journal of physiology. Gastrointestinal and liver physiology |
Medium |
22744337
|
| 2015 |
IL-6 inhibits HBV entry into hepatocytes by downregulating NTCP: IL-6 pretreatment reduced NTCP mRNA by 98%, reduced NTCP-mediated taurocholate uptake by 80%, and inhibited HBV entry by up to 90%. Restoration of NTCP expression suppressed the inhibitory effect of IL-6, confirming that NTCP downregulation mediates the effect. |
HBV infection assay (cccDNA and HBsAg readouts), taurocholate uptake assay, NTCP mRNA quantification, NTCP restoration experiment |
Virology |
High |
25765005
|
| 2014 |
Retinoic acid receptor (RAR) regulates human NTCP promoter activity via a binding site at nucleotides −112 to −96 of the NTCP gene. Pharmacological antagonism of RAR (Ro41-5253, CD2665) reduced NTCP mRNA and protein expression, blocked viral entry, and inhibited HBV spread across multiple genotypes. |
NTCP promoter luciferase assays, RAR ChIP, pharmacological RAR antagonists, HBV infection assay, NTCP mRNA/protein quantification in HepG2-hNTCP cells |
The Journal of biological chemistry |
Medium |
25550158
|
| 2016 |
NTCP augments HCV infection by mediating bile-acid-dependent repression of interferon-stimulated genes (ISGs), including IFITM3. Gain- and loss-of-function studies in hepatocytes showed NTCP regulates innate antiviral immunity, with NTCP-mediated bile acid transport suppressing ISG expression and thereby increasing permissiveness to HCV infection. |
NTCP overexpression and knockdown in hepatoma cells, ISG expression profiling, HCV infection assay, bile acid treatment |
Cell reports |
Medium |
27783949
|
| 2021 |
NTCP oligomerization occurs downstream of the NTCP-EGFR interaction and is required for HBV internalization. Troglitazone directly binds NTCP allosterically (non-competitive inhibition of bile acid uptake) and blocks NTCP oligomerization, inhibiting HBV internalization without affecting HBV attachment or the NTCP-EGFR interaction. The F274A mutation in NTCP disrupts oligomerization and HBV internalization without affecting viral surface binding. |
Surface plasmon resonance, transporter kinetics, alanine scanning mutagenesis, coimmunoprecipitation, NTCP oligomerization assay in transfected cells |
Journal of virology |
High |
34613794
|
| 2022 |
IFITM3 is a protein-protein interaction partner of NTCP identified by membrane yeast-two-hybrid and confirmed by co-immunoprecipitation. IFITM3 knockdown significantly reduced HBV and HDV infection of NTCP-expressing HuH7 cells and primary human hepatocytes, despite intact myr-preS1 peptide binding to NTCP, indicating IFITM3 facilitates a post-attachment step of HBV/HDV entry. |
Membrane yeast-two-hybrid, co-immunoprecipitation, IFITM3 siRNA knockdown, HBV/HDV infection assays, myr-preS1 binding assay |
Viruses |
Medium |
35458456
|
| 2019 |
OATP1B3 forms hetero-oligomers with NTCP in both HEK293 cells and frozen human liver sections. Coexpression of NTCP with OATP1B3 in HEK293 cells increased OATP1B3 plasma membrane expression but decreased its apparent turnover rate for the substrate cholecystokinin-8. |
Co-immunoprecipitation, proximity ligation assay, plasma membrane expression quantification, transport assay in HEK293T cells and human liver sections |
Drug metabolism and disposition |
Medium |
32482756
|
| 2014 |
SLC10A1 deficiency (NTCP deficiency) was identified as an inborn error of metabolism in a patient with a homozygous R252H mutation. Functional studies showed markedly reduced taurocholate uptake activity, and immunofluorescence/surface biotinylation showed the mutant protein is virtually absent from the plasma membrane, confirming NTCP is the main hepatic import system for conjugated bile salts. |
SLC10A1 sequencing, taurocholic acid uptake assay, immunofluorescence, surface biotinylation |
Hepatology (Baltimore, Md.) |
High |
24867799
|
| 2019 |
Cyclin D1 transcriptionally suppresses NTCP expression by inhibiting NTCP promoter activity during cell cycle progression. Ectopic expression of NTCP in HepG2 and Huh-7 cells suppressed hepatocyte growth by arresting cells in G0/G1 phase, identifying a functional link between NTCP expression and cell cycle regulation. |
NTCP promoter luciferase assay, cyclin D1 overexpression, cell cycle analysis (flow cytometry), ectopic NTCP expression in HCC cell lines |
Oncotarget |
Medium |
28915572
|
| 2020 |
HBV uptake into HepG2-NTCP cells is dependent on the actin cytoskeleton and occurs via clathrin-mediated endocytosis. HBV internalisation was inhibited by pitstop-2 and siRNA silencing of clathrin heavy chain, adaptor protein AP-2, and dynamin-2. Entry via caveolae or macropinocytosis was excluded. HBV entry in clathrin-coated pits was visualised by electron microscopy and cryo-EM with immunogold labelling. |
siRNA knockdown of clathrin, AP-2, dynamin-2; pharmacological inhibitors; electron microscopy; cryo-EM immunogold labelling in HepG2-NTCP cells |
Cellular microbiology |
High |
32216005
|
| 2019 |
NTCP genetic deletion in mice reduces diet-induced obesity, attenuates hepatic steatosis, and lowers plasma cholesterol by prolonging postprandial plasma bile acid elevations. The effect on obesity protection is TGR5-independent (NTCP/TGR5 double knockout mice showed equal protection). Mechanistically, NTCP knockout was associated with decreased intestinal fat absorption and increased uncoupled respiration in brown adipose tissue. |
NTCP knockout mice, NTCP/TGR5 double knockout mice, metabolic phenotyping, intestinal fat absorption assay, brown adipose tissue respiration measurement |
JCI insight |
High |
31237863
|
| 2019 |
Myrcludex B (bulevirtide) binds NTCP with very high affinity, and the NTCP:Myrcludex B interaction is extremely long-lived. Pre-bound Myrcludex B can transfer from one NTCP molecule to newly synthesized NTCP, partly escaping co-degradation. The normalization of plasma bile salt levels correlates with NTCP protein turnover rate. |
FITC-labeled Myrcludex B tracking, biotin-labeled NTCP turnover assay, FRET by fluorescence lifetime imaging microscopy in U2OS cells |
JHEP reports |
Medium |
32039379
|
| 2011 |
The A64T polymorphism of NTCP (SLC10A1) significantly decreased transport of both taurocholate and rosuvastatin compared to wild-type. The S267F polymorphism showed decreased taurocholate uptake but increased rosuvastatin uptake (substrate-dependent functional change), demonstrating position 267 as a substrate selectivity determinant. |
Stable transfection of NTCP variants, taurocholate and rosuvastatin uptake assays in transfected cells |
Xenobiotica |
Medium |
21341987
|
| 1999 |
Two alternatively spliced isoforms of mouse Ntcp (Ntcp1 encoding 362 aa and Ntcp2 encoding 317 aa with a shorter C-terminus from intron retention) both mediate saturable Na+-dependent transport of taurocholate when expressed in Xenopus oocytes. |
cDNA library screening, Xenopus oocyte expression, taurocholate uptake assay |
Biochimica et biophysica acta |
High |
10209268
|
| 2020 |
Position S267 in NTCP functions as an evolutionary 'rheostat': all 20 amino acid substitutions at this position produce progressive, varied effects on transport kinetics (Km and Vmax) and substrate specificity for taurocholic acid, estrone-3-sulfate, and rosuvastatin, rather than simple on/off effects. Stability modeling showed substitution tolerance correlates with surface expression but not transport activity. |
Systematic mutagenesis of all 20 amino acids at position 267, transport assays with three substrates, surface expression analysis, Rosetta stability modeling |
The Journal of biological chemistry |
High |
33168628
|