Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein)

Ronald P. J. Oude Elferink . Coen C. Paulusma - drug transporters ABCB1 (MDR1 P-glycoprotein), BCRP (ABCG2), and MRP2 (ABCC2) fulfill the important hepatic task of drug elimination into bile (for an overview of the relevant transporters, see Fig. 1). Functional characteristics of ABCB4 The physiological function of ABCB4 is clear: mice with a disrupted Abcb4 gene do not excrete any phosphatidylcholine into bile, while wild-type mice of the same genetic background excrete considerable amounts of PC [7, 8]. From these observations, it was suggested that Abcb4 flops PC within the membrane. Abcb4/ mice that were made transgenic for the human ABCB4 gene were completely rescued, demonstrating that these two orthologous genes have the same physiological function [9]. Despite the consensus on the physiological function of ABCB4, its molecular characteristics have not been extensively studied. The reason for this is the fact that it is quite difficult to develop reliable assays for the molecular mechanism of lipid translocators. In most studies, phospholipid analogs that have a fluorescent (NBD) group and a shortened fatty acid chain are used to increase their extractability from membranes. Polarized cells transduced with ABCB4 have increased rates of NBD-PC translocation to the outer leaflet [10]. In contrast, NBD-labeled phosphatidylethanolamine and phosphatidylsphingomyelin are not translocated, suggesting that ABCB4 has preference for phosphatidylcholine. However, the significance of these findings is limited, as the same study demonstrated that ABCB1 was able to flop all these NBD-labeled phospholipid analogs, including that of PC. Although ABCB1 has been shown to influence the asymmetric distribution of endogenous phosphatidylserine in plasma membranes of cells that overexpress this transporter, it is unlikely that ABCB1 is also capable of flopping natural phospholipids at high rates. This is due to the fact that Abcb4/ mice do not excrete any Fig. 1 Schematic representation of the most important transport processes in the canalicular membrane of hepatocytes mediated by ABC transporters. AQP aquaporin, NTCP Na+-dependent taurocholate transporter phospholipid into bile, irrespective of high expression of Abcb1 in hepatocytes of these animals. Similarly, ABCC1 (MRP1) was found to mediate the translocation of NBDlabeled phospholipids [1113], and possibly even natural PC [13]. In this case too, the question rises whether these translocation rates are physiologically important. ABCC2 has an almost identical substrate specificity as ABCC1 and is abundantly present in the canalicular membrane of the hepatocyte but is not able to rescue the PC translocation function in the absence of ABCB4. It may be assumed that the artificial NBD-labeled phospholipids are handled by these ABC transporters as amphiphilic drugs (that insert in the membrane) rather than as natural phospholipids [14, 15]. To date, the translocation of natural PC has been analyzed in only one study [16]. In these experiments, PC translocation was studied in fibroblasts overexpressing ABCB4 by metabolic labeling with radioactive choline. By addition of phosphatidylcholine-transfer protein to the medium of these cells, radioactive PC could be extracted from the membrane; the amount of radioactive PC in the extract was taken as a measure of floppase activity. Indeed, these studies demonstrated enhanced translocation of endogenous PC in the presence of ABCB4 expression. Excretion of phosphatidylcholine involves not only translocation but also extraction from the membrane As described above, it is generally accepted that ABCB4 flops phosphatidylcholine (PC) from the inner to the outer leaflet, but the mechanism of the subsequent step, i.e., extraction from the membrane, is largely unknown. With the discovery of the function of the two half-transporters ABCG5 and ABCG8, which translocate sterols, it became clear that translocation of the lipid molecule per se may not be the only important event in the excretion process. Although controversy exists on the spontaneous flip-flop rates of cholesterol in membranes, it is generally considered to be fast (half-times less than 1 s have been reported [17]). Nevertheless, in the absence of Abcg5/8, biliary cholesterol excretion in mice is reduced by about 75% [18, 19]. Small [20] suggested that not only translocation but also subsequent exposition of the cholesterol molecule out of the membrane bilayer by the transporter (termed lifting) is an essential step in the excretion (see also [21]). The exposed cholesterol molecule may then be transferred to mixed micelles of bile salts and phospholipids. Such a lifting or exposure mechanism may in fact also play a role in ABCB4-mediated phospholipid excretion. It has been known for many years that phospholipid and cholesterol excretion are driven by micelle forming bile salts. In the past, we have proposed that this involves vesiculation from the outer leaflet of the membrane, induced by local translocation of PC to the outer leaflet, combined with destabilization of the membrane by luminal bile salts [3]. Indeed, membrane-adherent vesicles have been detected in careful electron microscopic studies [22, 23]. Mechanistically it remains difficult, however, to envision a vesiculation process from a single (outer) leaflet of the membrane, as this would invoke highly unstable structures in the membrane. In the presence of high concentrations of bile salts, this might present a rather unfavorable situation. The discovery of ABCG5/8 as a cholesterol translocator demonstrated that PC and cholesterol excretion involve largely independent mechanisms; in the absence of either Abcg5 or Abcg8 (or both), cholesterol excretion in mice is strongly reduced, while PC excretion is much less affected [18, 19]. Cholesterol excretion is completely impaired in the Abcb4/ mouse, but this is caused by the absence of PC in bile, which renders the bile salt micelles as very poor cholesterol solubilizers [24]. Indeed, if more hydrophobic bile salts, such as taurodeoxycholate (which are better cholesterol solubilizers in the absence of PC), are infused in Abcb4/ mice, cholesterol excretion can be restored [24]. Hence, the excretion machineries of PC and cholesterol are more or less independent. It is difficult to envision a general lipid vesiculation mechanism from the outer leaflet driven by ABCB4mediated PC-flopping that does not involve simultaneous excretion of PC and cholesterol. It may therefore be suggested that PC excretion does not take place via vesiculation, but rather by translocation followed by exposition of the PC molecule by ABCB4, which subsequently allows extraction by bile salt micelles. This generates mixed micelles of bile salt and PC, which can subsequently act as acceptor for cholesterol that is exposed by ABCG5/8 (for an overview of our current model of biliary lipid excretion, see Fig. 2). Regulation of ABCB4 expression Expressi (...truncated)