Homo- and heterodimerization is a common feature of the solute carrier family SLC10 members

2019 ◽  
Vol 400 (10) ◽  
pp. 1371-1384 ◽  
Author(s):  
Saskia Noppes ◽  
Simon Franz Müller ◽  
Josefine Bennien ◽  
Matthias Holtemeyer ◽  
Massimo Palatini ◽  
...  
Keyword(s):  
Bile Acid ◽  
Transport Function ◽  
Solute Carrier Family ◽  
Bile Acid Transporters ◽  
Bile Acid Transporter ◽  
Solute Carrier ◽  
Steroid Sulfate ◽  
Functional Relevance ◽  
Two Hybrid

AbstractThe solute carrier family SLC10 consists of seven members, including the bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT), the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7). Previously, homodimerization of NTCP, ASBT and SOAT was described and there is increasing evidence that carrier oligomerization is an important regulatory factor for protein sorting and transport function. In the present study, homo- and heterodimerization were systematically analyzed among all SLC10 carriers (except for SLC10A3) using the yeast-two-hybrid membrane protein system. Strong homodimerization occurred for NTCP/NTCP, ASBT/ASBT and SLC10A7/SLC10A7. Heterodimerization was observed for most of the SLC10 carrier combinations. Heterodimerization of NTCP was additionally investigated by co-localization of NTCP-GFP and NTCP-mScarlet with respective SLC10 carrier constructs. NTCP co-localized with SLC10A4, SLC10A5, SOAT and SLC10A7. This co-localization was most pronounced for SLC10A4 and was additionally confirmed by co-immunoprecipitation. Interestingly, SLC10 carrier co-expression decreased the taurocholate transport function of NTCP for most of the analyzed constructs, indicating that SLC10 carrier heterodimerization is of functional relevance. In conclusion, homo- and heterodimerization is a common feature of the SLC10 carriers. The relevance of this finding for regulation and transport function of the SLC10 carriersin vivoneeds further investigation.

scholarly journals Substrate Specificities and Inhibition Pattern of the Solute Carrier Family 10 Members NTCP, ASBT and SOAT

2021 ◽  
Vol 8 ◽  
Author(s):  
Gary Grosser ◽  
Simon Franz Müller ◽  
Michael Kirstgen ◽  
Barbara Döring ◽  
Joachim Geyer
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Organic Anion ◽  
Hek293 Cells ◽  
Intestinal Lumen ◽  
Solute Carrier Family ◽  
Sodium Dependent ◽  
Bile Acid Transporter ◽  
Solute Carrier ◽  
Acid Transporter

Three carriers of the solute carrier family SLC10 have been functionally characterized so far. Na+/taurocholate cotransporting polypeptide NTCP is a hepatic bile acid transporter and the cellular entry receptor for the hepatitis B and D viruses. Its intestinal counterpart, apical sodium-dependent bile acid transporter ASBT, is responsible for the reabsorption of bile acids from the intestinal lumen. In addition, sodium-dependent organic anion transporter SOAT specifically transports sulfated steroid hormones, but not bile acids. All three carriers show high sequence homology, but significant differences in substrate recognition that makes a systematic structure-activity comparison attractive in order to define the protein domains involved in substrate binding and transport. By using stably transfected NTCP-, ASBT-, and SOAT-HEK293 cells, systematic comparative transport and inhibition experiments were performed with more than 20 bile acid and steroid substrates as well as different inhibitors. Taurolithocholic acid (TLC) was identified as the first common substrate of NTCP, ASBT and SOAT with Km values of 18.4, 5.9, and 19.3 µM, respectively. In contrast, lithocholic acid was the only bile acid that was not transported by any of these carriers. Troglitazone, BSP and erythrosine B were identified as pan-SLC10 inhibitors, whereas cyclosporine A, irbesartan, ginkgolic acid 17:1, and betulinic acid only inhibited NTCP and SOAT, but not ASBT. The HBV/HDV-derived myr-preS1 peptide showed equipotent inhibition of the NTCP-mediated substrate transport of taurocholic acid (TC), dehydroepiandrosterone sulfate (DHEAS), and TLC with IC50 values of 182 nM, 167 nM, and 316 nM, respectively. In contrast, TLC was more potent to inhibit myr-preS1 peptide binding to NTCP with IC50 of 4.3 µM compared to TC (IC50 = 70.4 µM) and DHEAS (IC50 = 52.0 µM). Based on the data of the present study, we propose several overlapping, but differently active binding sites for substrates and inhibitors in the carriers NTCP, ASBT, SOAT.

scholarly journals The solute carrier family 10 (SLC10): Beyond bile acid transport

2013 ◽  
Vol 34 (2-3) ◽  
pp. 252-269 ◽  
Author(s):  
Tatiana Claro da Silva ◽  
James E. Polli ◽  
Peter W. Swaan
Keyword(s):  
Bile Acid ◽  
Solute Carrier Family ◽  
Acid Transport ◽  
Bile Acid Transport ◽  
Solute Carrier

scholarly journals Evaluating Hepatobiliary Transport with 18F-Labeled Bile Acids: The Effect of Radiolabel Position and Bile Acid Structure on Radiosynthesis and In Vitro and In Vivo Performance

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Stef De Lombaerde ◽  
Ken Kersemans ◽  
Sara Neyt ◽  
Jeroen Verhoeven ◽  
Christian Vanhove ◽  
...  
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Membrane Vesicles ◽  
Hepatic Uptake ◽  
Hepatobiliary Transport ◽  
Bile Acid Transporters ◽  
Significant Difference ◽  
The Impact

Introduction. An in vivo determination of bile acid hepatobiliary transport efficiency can be of use in liver disease and preclinical drug development. Given the increased interest in bile acid Positron Emission Tomography- (PET-) imaging, a further understanding of the impact of 18-fluorine substitution on bile acid handling in vitro and in vivo can be of significance. Methods. A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3-, 7-, or 12-OH function is substituted with a fluorine atom (3α-[18F]FCA; 7β-[18F]FCA; 12β-[18F]FCA); a glycocholic and chenodeoxycholic acid analogue, substituted on the 3-position (3β-[18F]FGCA and 3β-[18F]FCDCA, resp.). Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines and efflux by BSEP in membrane vesicles. PET-scans with the tracers were performed in wild-type mice (n=3 per group): hepatobiliary transport was monitored and compared to a reference tracer, namely, 3β-[18F]FCA. Results. Compounds 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA were synthesized in moderate radiochemical yields (4–10% n.d.c.) and high radiochemical purity (>99%); 7β-[18F]FCA and 12β-[18F]FCA could not be synthesized and included further in this study. In vitro evaluation showed that 3α-FCA, 3β-FGCA, and 3β-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1, and BSEP. In vivo, 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3β-[18F]FCA epimers. Conjugation of 3β-[18F]FCA with glycine had no significant effect in vivo. Compound 3β-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines. Conclusion. A set of 18F labeled bile acids was synthesized that are substrates of the bile acid transporters in vitro and in vivo and can serve as PET-biomarkers for hepatobiliary transport of bile acids.

scholarly journals SLC36A1-mTORC1 signaling drives acquired resistance to CDK4/6 inhibitors

2019 ◽  
Vol 5 (9) ◽  
pp. eaax6352 ◽  
Author(s):  
Akihiro Yoshida ◽  
Yiwen Bu ◽  
Shuo Qie ◽  
John Wrangle ◽  
E. Ramsay Camp ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fragile X ◽  
Acquired Resistance ◽  
Mammalian Target Of Rapamycin ◽  
Cyclin Dependent Kinase ◽  
Solute Carrier Family ◽  
Potential Therapeutic Target ◽  
Mtorc1 Signaling ◽  
Solute Carrier

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome–associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

scholarly journals A novel bioluminescence-based method to investigate uptake of bile acids in living cells

2018 ◽  
Vol 315 (4) ◽  
pp. G529-G537 ◽  
Author(s):  
Alexander L. Ticho ◽  
Hyunjin Lee ◽  
Ravinder K. Gill ◽  
Pradeep K. Dudeja ◽  
Seema Saksena ◽  
...  
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Real Time ◽  
Sodium Taurocholate ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Bile Acid Transporters ◽  
Sodium Dependent ◽  
Bile Acid Transporter ◽  
Acid Transporter

Bile acid transporters, including the ileal apical sodium-dependent bile acid transporter (ASBT) and the hepatic sodium-taurocholate cotransporting polypeptide (NTCP), are crucial for the enterohepatic circulation of bile acids. Our objective was to develop a method for measuring bile acid transporter activity in real time to precisely evaluate rapid changes in their function. We designed a reporter system relying on a novel probe: cholic acid attached to luciferin via a disulfide-containing, self-immolating linker (CA-SS-Luc). Incubation of human embryonic kidney-293 cells coexpressing luciferase and ASBT with different concentrations of CA-SS-Luc (0.01–1 μM) resulted in bioluminescence with an intensity that was concentration- and time-dependent. The bioluminescence measured during incubation with 1 μM CA-SS-Luc was dependent on the levels of ASBT or NTCP expressed in the cells. Coincubation of CA-SS-Luc with natural bile acids enhanced the bioluminescence in a concentration-dependent manner with kinetic parameters for ASBT similar to those previously reported using conventional methods. These findings suggest that this method faithfully assesses ASBT function. Further, incubation with tyrosine phosphatase inhibitor III (PTPIII) led to significantly increased bioluminescence in cells expressing ASBT, consistent with previous studies showing an increase in ASBT function by PTPIII. We then investigated CA-SS-Luc in isolated mouse intestinal epithelial cells. Ileal enterocytes displayed significantly higher luminescence compared with jejunal enterocytes, indicating a transport process mediated by ileal ASBT. In conclusion, we have developed a novel method to monitor the activity of bile acid transporters in real time that has potential applications both for in vitro and in vivo studies.NEW & NOTEWORTHY This article reports the development of a real-time method for measuring the uptake of bile acids using a bioluminescent bile acid-based probe. This method has been validated for measuring uptake via the apical sodium-dependent bile acid transporter and the sodium-taurocholate cotransporting polypeptide in cell culture and ex vivo intestinal models.

scholarly journals Active Enterohepatic Cycling is Not Required for the Choleretic Actions of 24-norUrsodeoxycholic Acid in Mice

2021 ◽  
Author(s):  
Jianing Li ◽  
Jennifer K. Truong ◽  
Kimberly Pachura ◽  
Anuradha Rao ◽  
Sanjeev Gambeer ◽  
...  
Keyword(s):  
Liver Disease ◽  
Bile Acid ◽  
Bile Flow ◽  
Organic Anion ◽  
Cholestatic Liver Disease ◽  
Hepatic Expression ◽  
Bile Acid Sequestrants ◽  
Bile Acid Transporters ◽  
Liver Transcriptome ◽  
Bile Acid Transporter

AbstractThe superior ability of norursodeoxycholic acid (norUDCA) to induce a bicarbonate-rich hypercholeresis has been attributed to its ability to undergo cholehepatic shunting and norUDCA is currently being evaluated as a therapeutic for forms of liver disease. The goal of this study was to use mouse models to investigate contributions of bile acid transporters to the choleretic actions of norUDCA. Here, we show that the apical sodium-dependent bile acid transporter (ASBT) and Organic solute transporter-alpha (OSTα) are dispensable for norUDCA-stimulation of bile flow and biliary bicarbonate secretion in mice. Analysis of the liver transcriptome revealed that norUDCA induced hepatic expression of a limited number of transporter genes, particularly organic anion transporting polypeptide 1a4 (Oatp1a4). However, norUDCA potently stimulated a bicarbonate-rich hypercholeresis in Oatp1a/1b-deficient mice. Blocking intestinal bile acid reabsorption by co-administration of an ASBT inhibitor or bile acid sequestrant did not impact the ability of norUDCA to induce bile flow in wildtype mice. The results support the concept that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.

scholarly journals Ileal bile acid transporter inhibition, CYP7A1 induction, and antilipemic action of 264W94

2002 ◽  
Vol 43 (8) ◽  
pp. 1320-1330 ◽  
Author(s):  
Carolyn Root ◽  
Chari D. Smith ◽  
Scott S. Sundseth ◽  
Heather M. Pink ◽  
Joan G. Wilson ◽  
...  
Keyword(s):  
Bile Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Vesicles ◽  
Human Model ◽  
Diurnal Fluctuation ◽  
Bile Acid Transporter ◽  
Acid Transporter

264W94 was designed to inhibit the ileal bile acid transporter (IBAT). Evaluated in vitro, 264W94 dose-dependently inhibited sodium-dependent uptake of 10 μM [3H]taurocholic acid (TC) by rat and monkey brush border membrane vesicles with IC50s of 0.24 μM and 0.41 μM, and had a competitive profile with Ki of 0.2 μM against TC in Chinese hamster ovary cells expressing human IBAT. In distal ileum in situ, 1–10 μM of 264W94 rapidly decreased uptake of 3mM TC by 24–39%, with corresponding decreases in biliary recovery. In rats and mice in vivo, oral 264W94 decreased absorption of TC analog, 23,25-75Se-homocholic acid taurine (75SeHCAT; quantitated in feces), with ED30 of 0.02 mg/kg bid. 75SeHCAT traced through the GI-tract revealed that peak (97%) inhibition of 75SeHCAT absorption by the distal quarter of small intestine occurred at 4 h after single dose of 264W94 (0.1 mg/kg). Inhibition of IBAT by 264W94 in rats was associated with compensatory, same-day, 4-fold induction of hepatic cholesterol 7α-hydroxylase (CYP7A1) activity, exhibiting normal diurnal fluctuation for 3 days of dosing. In diet induced hypercholesterolemic rats, 264W94 (0.03–1.0 mg/kg bid) dose-dependently reduced serum LDL+VLDL cholesterol up to 61%.In conclusion, 264W94 is a potent new cholesterol lowering agent that acts through inhibition of IBAT and exhibits activity in a human model.

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