IntroductionHuman bile, which is formed at the canalicular membrane of hepatocytes, consists mainly of mixed micelles formed by predominately PC lipids, bile acids and cholesterol as well as to a smaller extent bilirubin, glucuronides and organic anions. All these molecules are transported into the bile canaliculi by different ABC transporters. Bile salts are secreted by ABCB11 [bile salt export pump (BSEP)] (1Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. et al.The Sister of P-glycoprotein Represents the Canalicular Bile Salt Export Pump of Mammalian Liver.), PC lipids are translocated by ABCB4 (2Smit J.J.M. Schinkel A.H. Elferink R.P.J.O. Groen A.K. Wagenaar E. van Deemter L. et al.Homozygous disruption of the murine MDR2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.) and cholesterol is the substrate of the heterodimeric ABC transporter ABCG5/G8 (3Graf G.A. Yu L. Li W.P. Gerard R. Tuma P.L. Cohen J.C. et al.ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion.). Furthermore ABCC2 (MRP2) excretes bilirubin (4Kamisako T. Kobayashi Y. Takeuchi K. Ishihara T. Higuchi K. Tanaka Y. et al.Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance.) and glucuronidated metabolites (5Buchler M. Konig J. Brom M. Kartenbeck J. Spring H. Horie T. et al.cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats., 6The apical conjugate efflux pump ABCC2 (MRP2).). Further information concerning human hepatobiliary ABC transporter are summarized in (7Kroll T. Prescher M. Smits S.H.J. Schmitt L. Structure and Function of Hepatobiliary ATP Binding Cassette Transporters.). In general, the major component of bile are bile acids, which make up approximately 70% of human gallbladder bile (8Sex differences in the bile acid composition of human bile: studies in patients with and without gallstones.). In humans, four different bile acids are present (Figure1 A-D), which can be divided into primary and secondary bile acids. Primary bile acids are derived from cholesterol and are synthesised in hepatocytes by a twostep pathway resulting either in the trihydroxy cholic acid (CA) (Figure 1A,) or the two hydroxy chenodeoxycholic acid (CDCA) (Figure 1B) (9Chemistry and enterohepatic circulation of bile acids., 10Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem., 11Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics., 12Monte M.J. Marin J.J. Antelo A. Vazquez-Tato J. Bile acids: chemistry, physiology, and pathophysiology., 13Bile formation and secretion.). For higher solubility both are conjugated either with glycine or taurine. In humans, the major conjugation is glycine (8Sex differences in the bile acid composition of human bile: studies in patients with and without gallstones., 11Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics., 12Monte M.J. Marin J.J. Antelo A. Vazquez-Tato J. Bile acids: chemistry, physiology, and pathophysiology.). In rodents (e.g. mouse or rats), however, taurine is the main conjugate. These conjugated primary bile acids are then secreted via ABCB11 into bile, stored in mixed micelles, until bile is secreted into the intestine. There, bile acids are required to solubilize hydrophobic compounds, e.g. vitamins or fatty acids. However, the bile acids themselves remain not unmodified in the intestine. Intestinal bacteria modify the conjugated primary bile acids by deconjugation and dehydroxylation specifically at position 7 (14Ridlon J.M. Kang D.J. Hylemon P.B. Bile salt biotransformations by human intestinal bacteria.). This results in so-called secondary bile aids. The two hydroxy deoxycholic acid (DCA) (Figure 1C) is derived from CA, while the monohydroxy lithocholic acid (LCA) (Figure 1D) is derived from CDCA. Through these modifications, the bile pool becomes more hydrophobic. Next re-absorption (active or passive) results in uptake of nearly 95% of the bile acids from the ileal segment into blood (15Dietschy J.M. Turley S.D. Control of cholesterol turnover in the mouse.), where it is transported back in to the liver. Here, the sodium taurocholate transporting peptide (NTCP) takes up primary and secondary bile acids (16The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation.). This circulation of bile acids is called ‘enterohepatic circulation’. For detailed information we recommend the review of Martinez-Augustin (17Martinez-Augustin O. Sanchez de Medina F. Intestinal bile acid physiology and pathophysiology.). Furthermore, next to the four bile acids described above, two non-human bile acids are relevant in medical treatments. Ursodeoxycholic acid (UDCA) (Figure 1E) is part of the Chinese black bear bile pool and possesses the highest similarity to CDCA. The only but important difference is the stereochemistry of the hydroxy group at position 7. While the hydroxy group at position 7 (if present) in all human bile acids is in the alpha position, the hydroxy group of UDCA is in the beta position (18Pharmacology of ursodeoxycholic acid, an enterohepatic drug.). UDCA is a common drug in the treatment of cholestatic liver diseases (19Mechanisms of action and therapeutic efficacy of ursodeoxycholic acid in cholestatic liver disease.), such as primary biliary cirrhosis (20Poupon R.E. Balkau B. Eschwege E. Poupon R. A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. UDCA-PBC Study Group., 21Heathcote E.J. Cauch-Dudek K. Walker V. Bailey R.J. Blendis L.M. Ghent C.N. et al.The Canadian Multicenter Double-blind Randomized Controlled Trial of ursodeoxycholic acid in primary biliary cirrhosis., 22Lindor K.D. Dickson E.R. Baldus W.P. Jorgensen R.A. Ludwig J. Murtaugh P.A. et al.Ursodeoxycholic acid in the treatment of primary biliary cirrhosis.), intrahepatic cholestasis of pregnancy (23Palma J. Reyes H. Ribalta J. Hernandez I. Sandoval L. Almuna R. et al.Ursodeoxycholic acid in the treatment of cholestasis of pregnancy: a randomized, double-blind study controlled with placebo.) or progressive familial intrahepatic cholestasis (24Jacquemin E. Hermans D. Myara A. Habes D. Debray D. Hadchouel M. et al.Ursodeoxycholic acid therapy in pediatric patients with progressive familial intrahepatic cholestasis., 25Gordo-Gilart R. Andueza S. Hierro L. Martinez-Fernandez P. D'Agostino D. Jara P. et al.Functional analysis of ABCB4 mutations relates clinical outcomes of progressive familial intrahepatic cholestasis type 3 to the degree of MDR3 floppase activity., 26van der Woerd W.L. Houwen R.H. van de Graaf S.F. Current and future therapies for inherited cholestatic liver diseases.). Since UDCA is such a powerful and widely distributed drug, it was also the target of synthetic modifications. These investigations resulted in a sidechain shortened derivate, 24-nor-ursodeoxycholic acid (Nor-UDCA). In comparison to UDCA, it lacks a methylene unit of its side chain (Figure 1F). This minimal chemical modification results in resistance to amidation with taurine or glycine compared with UDCA (27Yoon Y.B. Hagey L.R. Hofmann A.F. Gurantz D. Michelotti E.L. Steinbach J.H. Effect of side-chain shortening on the physiologic properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents.). Additionally, Nor-UDCA does not undergo the entire enterohepatic circulation, instead it undergoes cholehepatic shunting, which represents the reabsorption by cholangiocytes (28Hofmann A.F. Zakko S.F. Lira M. Clerici C. Hagey L.R. Lambert K.K. et al.Novel biotransformation and physiological properties of norursodeoxycholic acid in humans.). Nor-UDCA is seen as a novel approach in cholestatic and metabolic liver diseases (29Trauner M. Halilbasic E. Claudel T. Steinacher D. Fuchs C. Moustafa T. et al.Potential of nor-Ursodeoxycholic Acid in Cholestatic and Metabolic Disorders., 30Halilbasic E. Steinacher D. Trauner M. Nor-Ursodeoxycholic Acid as a Novel Therapeutic Approach for Cholestatic and Metabolic Liver Diseases.). In the case of ABCB4 knock-out mice, Nor-UDCA was superior to UDCA in the treatment of sclerosing cholangitis (31Fickert P. Wagner M. Marschall H.U. Fuchsbichler A. Zollner G. Tsybrovskyy O. et al.24-norUrsodeoxycholic acid is superior to ursodeoxycholic acid in the treatment of sclerosing cholangitis in Mdr2 (Abcb4) knockout mice., 32Fickert P. Hirschfield G.M. Denk G. Marschall H.U. Altorjay I. Farkkila M. et al.norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholangitis.).

Figure 1Chemical structure of relevant bile acids. Bile acids are unconjugated (R equals a hydroxyl group) after synthesis, but get conjugated with either glycine or taurine (R equals glycine or taurine) prior to transport. The primary bile acids cholic (A) and chenodeoxycholic (B) acid are synthesized by two different pathways resulting in either a hydroxylation at position twelve for cholic acid or no hydroxylation at position twelve for chenodeoxycholic acid. Secondary bile acids are derived from the primary ones by dehydroxylation at position seven by bacteria in the ilium. While deoxycholic acid (C) derives from cholic acid (A) lithocholic acid (D) is the result of dehydroxylation of chenodexycholic acid (B). Ursodeoxycholic acid (E) can be found in small amounts in human, but is one of the major bile acids in Chinese black bear (Ursus thibetanus). Nor-Ursodeoxycholic acid (F) in comparison to UDCA lacks a methylene group in its side chain (highlighted in violet). Therefore, it is not conjugated like the other bile acids.

ABCB4 belongs to the superfamily of ATP binding cassette (ABC) transporters. The membrane proteins within this superfamily are present in all three kingdoms of life and share a common blueprint (33Davidson A.L. Dassa E. Orelle C. Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems., 34Multifaceted structures and mechanisms of ABC transport systems in health and disease.). In the human genome, 48 genes coding for ABC transporter have been identified, which are divided into seven subfamilies (35Dean M. Hamon Y. Chimini G. The human ATP-binding cassette (ABC) transporter superfamily.). ABCB4 is part of the subfamily B and consists of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) encoded on one single gene. Thus, ABCB4 represents a so-called full-size transporter (36van der Bliek A.M. Kooiman P.M. Schneider C. Borst P. Sequence of mdr3 cDNA encoding a human P-glycoprotein.), which structure was determined in 2019 (37Prescher M. Kroll T. Schmitt L. ABCB4/MDR3 in health and disease - at the crossroads of biochemistry and medicine.). Due to the high identity (76%) and similarity (86%) of ABCB4 to the P-glycoprotein (P-gp, ABCB1), also termed multidrug resistance protein 1 (MDR1), ABCB4 was termed MDR3. In contrast to the ubiquitous expressed P-gp, ABCB4 is only expressed in hepatocytes (38Tissue-specific mRNA expression profiles of human ATP-binding cassette and solute carrier transporter superfamilies., 39Fagerberg L. Hallstrom B.M. Oksvold P. Kampf C. Djureinovic D. Odeberg J. et al.Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.) and specifically targeted to the apical (canalicular) membrane. Additionally, ABCB4 possesses a different function as P-gp. First indications were derived from the murine homologue of ABCB4, Mdr2. Homozygous mdr2-/- knock-out mice lacked cholesterol and phosphatidylcholine (PC) lipids in their bile (2Smit J.J.M. Schinkel A.H. Elferink R.P.J.O. Groen A.K. Wagenaar E. van Deemter L. et al.Homozygous disruption of the murine MDR2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.). Complementation of mice mdr2-/- by human ABCB4 demonstrated that human ABCB4 carried out the same function as Mdr2 (40Smith A.J. de Vree J.M. Ottenhoff R. Oude Elferink R.P. Schinkel A.H. Borst P. Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (-/-) mice.). Finally, it was shown that ABCB4 specifically recognizes PC lipids (41Smith A.J. Timmermans-Hereijgers J.L. Roelofsen B. Wirtz K.W. van Blitterswijk W.J. Smit J.J. et al.The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice., 42van Helvoort A. Smith A.J. Sprong H. Fritzsche I. Schinkel A.H. Borst P. et al.MDR1 P-Glycoprotein Is a Lipid Translocase of Broad Specificity, While MDR3 P-Glycoprotein Specifically Translocates Phosphatidylcholine., 43Prescher M. Smits S.H.J. Schmitt L. Stimulation of the ATPase activity of MDR3/ABCB4 requires an intact phosphatidylcholine lipid.). Nevertheless, data demonstrated that ABCB4 recognized certain P-gp substrates and inhibitors (44Smith A.J. van Helvoort A. van Meer G. Szabo K. Welker E. Szakacs G. et al.MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping., 45Kino K. Taguchi Y. Yamada K. Komano T. Ueda K. Aureobasidin A, an antifungal cyclic depsipeptide antibiotic, is a substrate for both human MDR1 and MDR2/P-glycoproteins.). Based on these findings, it is now general accepted that in vivo ABCB4 specifically translocates lipids of the PC family from the inner to the outer leaflet of the canalicular membrane of hepatocytes and therefore is part of the bile triumvirate. This bile triumvirate is composed of ABCB4, the bile acid export pump ABCB11 (or BSEP) and the cholesterol heterodimeric transporter ABCG5/G8. Since the substrates of this bile triumvirate are the major compounds of primary bile and form mixed micelles, one might speculate that these three ABC exporters act in concert to ensure proper formation of bile and balance of their compounds (46Role of ABC transporters in secretion of cholesterol from liver into bile.). For ABCB11 it was demonstrated that the amount of membrane cholesterol has an effect on its activity (47Kis E. Ioja E. Nagy T. Szente L. Heredi-Szabo K. Krajcsi P. Effect of membrane cholesterol on BSEP/Bsep activity: species specificity studies for substrates and inhibitors.). Furthermore, it was shown for detergent purified ABCG5/G8 that bile acids stimulate ATPase activity in a concentration depending manner (48Johnson B.J.H. Lee J.-Y. Pickert A. Urbatsch I.L. Bile Acids Stimulate ATP Hydrolysis in the Purified Cholesterol Transporter ABCG5/G8.). For ABCB4 it is known that the presence of a bile acid such as taurocholic acid (TCA) can increase the PC-lipid and cholesterol content in the extracellular medium (49Morita Sy Kobayashi A. Takanezawa Y. Kioka N. Handa T. Arai H. et al.Bile salt–dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4.). For example, studies with murine ABCB4 demonstrated an explicit higher PC lipid content in mouse bile in presence of TCA compared to the situation in the absence of TCA (50Enhancement of Mdr2-mediated phosphatidylcholine translocation by the bile salt taurocholate. Implications for hepatic bile formation.). In line with these findings is a preferential release of PC lipids from rat liver canalicular vesicles in the presence of TCA (51Gerloff T. Meier P.J. Stieger B. Taurocholate induces preferential release of phosphatidylcholine from rat liver canalicular vesicles.). For human ABCB4 expressed in HEK cells, it was demonstrated that adding TCA to the extracellular medium results in an increased amount of PC-lipid and cholesterol in the extracellular medium after 24 hours (49Morita Sy Kobayashi A. Takanezawa Y. Kioka N. Handa T. Arai H. et al.Bile salt–dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4.). Furthermore, in a model cell line (LLC-PK1) expressing all three ABC transporters involved in bile formation, higher NBD-labelled PC lipid concentrations in the medium were observed, if cells were treated with albumin or TCA (52Mahdi Z.M. Synal-Hermanns U. Yoker A. Locher K.P. Stieger B. Role of Multidrug Resistance Protein 3 in Antifungal-Induced Cholestasis.). These assays however cannot distinguish between a direct effect of the bile acid on ABCB4 or whether only PC lipid extraction is improved (37Prescher M. Kroll T. Schmitt L. ABCB4/MDR3 in health and disease - at the crossroads of biochemistry and medicine.).In this study we aimed to address this question by measuring the ATPase activity of ABCB4 under defined conditions. The rationale behind this is the coupling between ATP hydrolysis and PC translocation, i.e. higher ATPase activity is the prerequisite for more efficient PC transport. Therefore, we used the previously established heterologous expression system in Picheria pastoris, which allows the purification of human ABCB4 (53Ellinger P. Kluth M. Stindt J. Smits S.H. Schmitt L. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris.), which enabled the determination of the kinetic parameters of the basal ATPase activity of ABCB4 wild type (54Kluth M. Stindt J. Droge C. Linnemann D. Kubitz R. Schmitt L. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.). In the same study, we were able to demonstrate that lipids of the PC family such as 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) stimulated ATPase activity of detergent purified ABCB4, while such a stimulation did not occur for non-PC lipids (54Kluth M. Stindt J. Droge C. Linnemann D. Kubitz R. Schmitt L. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.). This clearly reflects the in vivo situation (40Smith A.J. de Vree J.M. Ottenhoff R. Oude Elferink R.P. Schinkel A.H. Borst P. Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (-/-) mice., 41Smith A.J. Timmermans-Hereijgers J.L. Roelofsen B. Wirtz K.W. van Blitterswijk W.J. Smit J.J. et al.The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice., 42van Helvoort A. Smith A.J.