Canalicular Membrane Of Hepatocytes Research Articles

Abcb4-defect cholangitis mouse model with hydrophobic bile acid composition by in vivo liver-specific gene deletion

Progressive familial intrahepatic cholestasis (PFIC) is a liver disease that occurs during childhood and requires liver transplantation. ABCB4 is localized along the canalicular membranes of hepatocytes, transports phosphatidylcholine into bile, and its mutation causes PFIC3. Abcb4 gene-deficient mice established as animal models of PFIC3 exhibit cholestasis-induced liver injury. However, their phenotypes are often milder than those of human PFIC3, partly because of the existence of large amounts of less toxic hydrophilic bile acids synthesized by the rodent-specific enzymes Cyp2c70 and Cyp2a12. Mice with double deletions of Cyp2c70/Cyp2a12 (CYPDKO mice) have a human-like hydrophobic bile acid composition. PFIC-related gene mutations were induced in CYPDKO mice to determine whether these triple-gene-deficient mice are a better model for PFIC. To establish a PFIC3 mouse model using CYPDKO mice, we induced abcb4 gene deletion in vivo using adeno-associated viruses expressing SaCas9 under the control of a liver-specific promoter and abcb4-target gRNAs. Compared to Abcb4-deficient wild-type mice, Abcb4-deficient CYPDKO mice showed more pronounced liver injury along with an elevation of inflammatory and fibrotic markers. The proliferation of intrahepatic bile ductal cells and hematopoietic cell infiltration were also observed. CYPDKO/abcb4-deficient mice show a predominance of taurine-conjugated chenodeoxycholic acid and lithocholic acid in the liver. In addition, phospholipid levels in the gallbladder bile were barely detectable. Mice with both human-like bile acid composition and Abcb4-defect exhibit severe cholestatic liver injury and are useful for studying human cholestatic diseases and developing new treatments.

Identification of new correctors for traffic-defective ABCB4 variants by a high-content screening approach

ABCB4 is located at the canalicular membrane of hepatocytes and is responsible for the secretion of phosphatidylcholine into bile. Genetic variations of this transporter are correlated with rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 patients most often require liver transplantation. In this context of unmet medical need, we developed a high-content screening approach to identify small molecules able to correct ABCB4 molecular defects. Intracellularly-retained variants of ABCB4 were expressed in cell models and their maturation, cellular localization and function were analyzed after treatment with the molecules identified by high-content screening. In total, six hits were identified by high-content screening. Three of them were able to correct the maturation and canalicular localization of two distinct intracellularly-retained ABCB4 variants; one molecule was able to significantly restore the function of two ABCB4 variants. In addition, in silico molecular docking calculations suggest that the identified hits may interact with wild type ABCB4 residues involved in ATP binding/hydrolysis. Our results pave the way for their optimization in order to provide new drug candidates as potential alternative to liver transplantation for patients with severe forms of ABCB4-related diseases, including PFIC3.

Assessing the degree of hepatic ischemia-reperfusion injury using physiologically based pharmacokinetic modeling of sodium fluorescein disposition in ex vivo machine-perfused livers.

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multidrug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a six-parameter version of this model to control time-course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only three unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT, found the mean estimated value for each parameter, and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at the 30-min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP.NEW & NOTEWORTHY We developed a computational model of sodium fluorescein (SF) biliary excretion in ex vivo machine perfusion and used this model to assess changes in model parameters associated with the activity of MRP2, a hepatocyte membrane transporter, in response to increasing warm ischemia time. We found a significant decrease in the parameter value describing MRP2 activity, consistent with a role of decreased MRP2 function in ischemia-reperfusion injury leading to decreased secretion of SF into bile.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

Natural Product Baohuoside I Impairs the Stability and Membrane Location of MRP2 Reciprocally Regulated by SUMOylation and Ubiquitination in Hepatocytes.

Epimedii Folium (EF) is a botanical dietary supplement to benefit immunity. Baohuoside I (BI), a prenylated flavonoid derived from EF, has exhibited the cholestatic risk before. Here, the mechanism of BI on the stability and membrane localization of liver MRP2, a bile acid exporter in the canalicular membrane of hepatocytes, was investigated. The fluorescent substrate of MRP2, CMFDA was accumulated in sandwich-cultured primary mouse hepatocytes (SCH) under BI stimulation, followed by reduced membrane MRP2 expression. BI triggered MRP2 endocytosis associated with oxidative stress via inhibition of the NRF2 signaling pathway. Meanwhile, BI promoted the degradation of MRP2 by reducing its SUMOylation and enhancing its ubiquitination level. Co-IP and fluorescence colocalization experiments all proved that MRP2 was a substrate protein for SUMOylation for SUMO proteins. CHX assays showed that SUMO1 prolonged the half-life of MRP2 and further increased its membrane expression, which could be reversed by UBC9 knockdown. Correspondingly, MRP2 accumulated in the cytoplasm by GP78 knockdown or under MG132 treatment. Additionally, the SUMOylation sites of MRP2 were predicted by the algorithm, and a conversion of lysines to arginines at positions 940 and 953 of human MRP2 caused its decreased stability and membrane location. K940 was further identified as the essential ubiquitination site for MRP2 by an in vitro ubiquitination assay. Moreover, the decreased ubiquitination of MRP2 enhanced the SUMOylation MRP2 and vice versa, and the crosstalk of these two modifiers could be disrupted by BI. Collectively, our findings indicated the process of MRP2 turnover from the membrane to cytoplasm at the post-translational level and further elucidated the novel toxicological mechanism of BI.

Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation.

The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation. Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells. Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased. Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin–Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

Genetic Analysis of ABCB4 Mutations and Variants Related to the Pathogenesis and Pathophysiology of Low Phospholipid-Associated Cholelithiasis.

Clinical studies have revealed that the ABCB4 gene encodes the phospholipid transporter on the canalicular membrane of hepatocytes, and its mutations and variants are the genetic basis of low phospholipid-associated cholelithiasis (LPAC), a rare type of gallstone disease caused by a single-gene mutation or variation. The main features of LPAC include a reduction or deficiency of phospholipids in bile, symptomatic cholelithiasis at <40 years of age, intrahepatic sludge and microlithiasis, mild chronic cholestasis, a high cholesterol/phospholipid ratio in bile, and recurrence of biliary symptoms after cholecystectomy. Needle-like cholesterol crystals, putatively “anhydrous” cholesterol crystallization at low phospholipid concentrations in model and native bile, are characterized in ABCB4 knockout mice, a unique animal model for LPAC. Gallbladder bile with only trace amounts of phospholipids in these mice is supersaturated with cholesterol, with lipid composition plotting in the left two-phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. In this review, we summarize the molecular biology and physiological functions of ABCB4 and comprehensively discuss the latest advances in the genetic analysis of ABCB4 mutations and variations and their roles in the pathogenesis and pathophysiology of LPAC in humans, based on the results from clinical studies and mouse experiments. To date, approximately 158 distinct LPAC-causing ABCB4 mutations and variants in humans have been reported in the literature, indicating that it is a monogenic risk factor for LPAC. The elucidation of the ABCB4 function in the liver, the identification of ABCB4 mutations and variants in LPAC patients, and the exploration of gene therapy for ABCB4 deficiency in animal models can help us to better understand the cellular, molecular, and genetic mechanisms underlying the onset of the disease, and will pave the way for early diagnosis and prevention of susceptible subjects and effective intervention for LPAC in patients.

MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Digital Imaging Analysis for Quantitative Subcellular Localization of MRP2 during Hepatic Ischemia–Reperfusion Injury in a Rat Model: Can You Capture Time?

Introduction: The mechanism of cholestasis in hepatic ischemia–reperfusion injury (IRI) remains to be elucidated. Short-term changes in bile formation are regulated by translocating transporter proteins between the cell membrane and the cytosolic compartment. We sought to develop a method that quantifies the degree of translocation of multidrug resistance-associated protein 2 (MRP2) along the hepatocyte canalicular membrane. Method: Fifteen male Sprague Dawley rats were divided into three groups based on warm ischemia time (WIT): 0 min, 10 min, and 20 min. IRI was induced by temporarily clamping pedicles to the median and lateral lobes, followed by 60 min of reperfusion. Liver tissue was analyzed for subcellular protein fractionation and immunofluorescence staining. CD13 served as a reference protein for the canalicular membrane. Digital imaging analyses measured the area of MRP2 and CD13 staining. The size of the MRP2 area without overlap with CD13 (i.e., MRP2 in the cytosolic compartment) in proportion to the total MRP2 area was defined as the MRP2 translocation index (MTI). Results: The proportion of CD13 along the canalicular membrane was larger than that of MRP2 (median 93.3% versus 43.4%, respectively; P<0.0001, Figure A). The subcellular distribution of CD13 was consistent among the three WIT groups. Immunofluorescence colocalization analysis for CD13 and MRP2 demonstrated a negative linear correlation between MTI and WIT (P=0.0183, Y=-1.052X+25.00, Figure B). Conclusions: The degree of MRP2 translocation relative to CD13 was quantified, which correlated with WIT. This novel approach can be used to determine the pathophysiology of cholestasis in hepatic IRI.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic.

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants.

ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. We observed that the correctors C10, C13, and C17, as well as the combinations of C3+C18 and C4+C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

Molecular Regulation of Canalicular ABC Transporters.

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters’ folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

Monomeric bile acids modulate the ATPase activity of detergent-solubilized ABCB4/MDR3

ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4. Using in vitro experiments, we investigated the modulation of ATP hydrolysis of ABC by different bile acids commonly present in humans. We demonstrated that all tested bile acids stimulated ATPase activity except for taurolithocholic acid, which inhibited ATPase activity due to its hydrophobic nature. Additionally, we observed a nearly linear correlation between the critical micelle concentration and maximal stimulation by each bile acid, and that this modulation was maintained in the presence of PC lipids. This study revealed a large effect of 24-nor-ursodeoxycholic acid, suggesting a distinct mode of regulation of ATPase activity compared with other bile acids. In addition, it sheds light on the molecular cross talk of canalicular ABC transporters of the human liver.

Stimulation of ABCB4/MDR3 ATPase activity requires an intact phosphatidylcholine lipid

ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.

Histological demonstration of BSEP/ABCB11 inhibition in transient neonatal cholestasis: a case report

BackgroundIdiopathic or transient neonatal cholestasis (TNC) represents a group of cholestatic disorders with unidentified origin and remains a diagnosis of exclusion. Dysfunction of hepatobiliary transporters mediating excretion of biliary constituents from hepatocytes may play a central role in the pathogenesis of cholestasis. Despite variants of bile salt (BS) export pump (BSEP/ABCB11) have already been described in TNC, the pathogenic role of BSEP dysfunction in TNC remained so far elusive.Case presentationWe report on a newly-identified heterozygous ABCB11 missense variant (c.1345G > A, p.Glu449Lys) which was associated with prolonged cholestasis in a term infant after a complicated neonatal period. Moreover, we show for the first time almost completely abolished BSEP expression on the hepatocellular membrane in TNC.ConclusionThis report demonstrates for the first time a close association between the prolonged cholestasis in infancy and impaired BSEP expression on the hepatocyte canalicular membrane in a heterozygous carrier of newly-identified ABCB11 variant.

Measurement of Hepatic ABCB1 and ABCG2 Transport Activity with [11C]Tariquidar and PET in Humans and Mice.

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in the canalicular membrane of hepatocytes mediate the biliary excretion of drugs and drug metabolites. To measure hepatic ABCB1 and ABCG2 activity, we performed positron emission tomography (PET) scans with the ABCB1/ABCG2 substrate [11C]tariquidar in healthy volunteers and wild-type, Abcb1a/b(-/-), Abcg2(-/-), and Abcb1a/b(-/-)Abcg2(-/-) mice without and with coadministration of unlabeled tariquidar. PET data were analyzed with a three-compartment pharmacokinetic model. [11C]Tariquidar underwent hepatobiliary excretion in both humans and mice, and tariquidar coadministration caused a significant reduction in the rate constant for the transfer of radioactivity from the liver into bile (by -74% in humans and by -62% in wild-type mice), suggesting inhibition of canalicular efflux transporter activity. Radio-thin-layer chromatography analysis revealed that the majority of radioactivity (>87%) in the mouse liver and bile was composed of unmetabolized [11C]tariquidar. PET data in transporter knockout mice revealed that both ABCB1 and ABCG2 mediated biliary excretion of [11C]tariquidar. In vitro experiments indicated that tariquidar is not a substrate of major hepatic basolateral uptake transporters (SLCO1B1, SLCO1B3, SLCO2B1, SLC22A1, and SLC22A3). Our data suggest that [11C]tariquidar can be used to measure hepatic canalicular ABCB1/ABCG2 transport activity without a confounding effect of uptake transporters.

Manganese transporter Slc30a10 controls physiological manganese excretion and toxicity.

Manganese (Mn), an essential metal and nutrient, is toxic in excess. Toxicity classically results from inhalational exposures in individuals who work in industrial settings. The first known disease of inherited Mn excess, identified in 2012, is caused by mutations in the metal exporter SLC30A10 and is characterized by Mn excess, dystonia, cirrhosis, and polycythemia. To investigate the role of SLC30A10 in Mn homeostasis, we first generated whole-body Slc30a10-deficient mice, which developed severe Mn excess and impaired systemic and biliary Mn excretion. Slc30a10 localized to canalicular membranes of hepatocytes, but mice with liver Slc30a10 deficiency developed minimal Mn excess despite impaired biliary Mn excretion. Slc30a10 also localized to the apical membrane of enterocytes, but mice with Slc30a10 deficiency in small intestines developed minimal Mn excess despite impaired Mn export into the lumen of the small intestines. Finally, mice with Slc30a10 deficiency in liver and small intestines developed Mn excess that was less severe than that observed in mice with whole-body Slc30a10 deficiency, suggesting that additional sites of Slc30a10 expression contribute to Mn homeostasis. Overall, these results indicated that Slc30a10 is essential for Mn excretion by hepatocytes and enterocytes and could be an effective target for pharmacological intervention to treat Mn toxicity.

Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models

Adenosine triphosphate binding cassette transporter, subfamily B member 4 (ABCB4) is the transporter of phosphatidylcholine at the canalicular membrane of hepatocytes. ABCB4 deficiency, due to genetic variations, is responsible for progressive familial intrahepatic cholestasis type 3 (PFIC3) and other rare biliary diseases. Roscovitine is a molecule in clinical trial that was shown to correct the F508del variant of cystic fibrosis transmembrane conductance regulator (CFTR), another ABC transporter. In the present study, we hypothesized that roscovitine could act as a corrector of ABCB4 traffic-defective variants. Using HEK and HepG2 cells, we showed that roscovitine corrected the traffic and localisation at the plasma membrane of ABCB4-I541F, a prototypical intracellularly retained variant. However, roscovitine caused cytotoxicity, which urged us to synthesize non-toxic structural analogues. Roscovitine analogues were able to correct the intracellular traffic of ABCB4-I541F in HepG2 cells. Importantly, the phospholipid secretion activity of this variant was substantially rescued by three analogues (MRT2-235, MRT2-237 and MRT2-243) in HEK cells. We showed that these analogues also triggered the rescue of intracellular traffic and function of two other intracellularly retained ABCB4 variants, i.e. I490T and L556R. Our results indicate that structural analogues of roscovitine can rescue genetic variations altering the intracellular traffic of ABCB4 and should be considered as therapeutic means for severe biliary diseases caused by this class of variations.

ABCB4/MDR3 in health and disease - at the crossroads of biochemistry and medicine.

Several ABC transporters of the human liver are responsible for the secretion of bile salts, lipids and cholesterol. Their interplay protects the biliary tree from the harsh detergent activity of bile salts. Among these transporters, ABCB4 is essential for the translocation of phosphatidylcholine (PC) lipids from the inner to the outer leaflet of the canalicular membrane of hepatocytes. ABCB4 deficiency can result in altered PC to bile salt ratios, which led to intrahepatic cholestasis of pregnancy, low phospholipid associated cholelithiasis, drug induced liver injury or even progressive familial intrahepatic cholestasis type 3. Although PC lipids only account for 30-40% of the lipids in the canalicular membrane, 95% of all phospholipids in bile are PC lipids. We discuss this discrepancy in the light of PC synthesis and bile salts favoring certain lipids. Nevertheless, the in vivo extraction of PC lipids from the outer leaflet of the canalicular membrane by bile salts should be considered as a separate step in bile formation. Therefore, methods to characterize disease causing ABCB4 mutations should be considered carefully, but such an analysis represents a crucial point in understanding the currently unknown transport mechanism of this ABC transporter.