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

Abstract

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.

Keywords: ATP binding cassette subfamily B member 4; adenosine 5′-triphosphatase activity; adenosine 5′-triphosphate binding cassette transporter; bile; fatty acids.

Fig. 1.

A: Human ABCB4 was purified by tandem affinity purification in FC-16 micelles. Panel “CBB” displays 2 μg of the elution fraction after TAP stained with Coomassie brilliant blue. Panel “C219” shows the same purification on a Western blot with immunostaining using the monoclonal antibody C219. B: The same purification was loaded onto a different SDS gel without (w/o B) or after covalent modification with the thiol reactive maleimide-Bodipy fluorophore (w/ B) and exposed to UV light. The arrow indicates the position of ABCB4.

Fig. 2.

Relative ATPase activity measurements of ABCB4 in the presence of the substrate 16:0-16:0 PC. One hundred percent is defined as ATPase activity of ABCB4 in the absence of any lipid (basal activity). Data represent the average of three independent experiments with the errors reported as SD.

Fig. 3.

A: Change in the physical state of lipids with an increasing lipid to detergent molar ratio. E. coli polar lipids treated with Triton X-100 (in gray) were taken from (28) and correspond to a typical titration curve for destabilization of liposomes with detergent. All PC-lipid variants were measured at 492 nm. B: Slopes of the graph in A where a change in the physical state of lipids can be visualized at 492 nm with an increased lipid to detergent molar ratio.

Fig. 4.

Relative ATPase activities of detergent-solubilized and purified ABCB4 in the presence of different PC lipids with increasing concentrations. A: Activity in the presence of LTLE. The composition of LTLE of bovine liver is shown as a pie chart in the inset. B: Activity in the presence of a synthetic PC composition that resembles the natural PC composition of the bile canalicular membrane (shown as pie chart in the inset). C: The influence of the main bile PC lipids 16:0-18:1 PC (POPC, turquoise) and 16:0-18:2 PC (mocha) on the ATPase activity as well as 16:0-18:2 PC in brown and 16:0-20:4 PC in pink. D: ATPase activity in the presence of PC lipids with increasing chain length: 12:0-12:0 PC (orange), 14:0-14:0 PC (green), and 16:0-16:0 PC (red). E: ATPase activity in the presence of PC lipids with an increasing degree of unsaturation: 16:1-16:1 PC (blue) and 20:4-20:4 PC (purple). F: ATPase activity in the presence of symmetric PC lipids: 18:3-18:3 PC (black) and 18:1-18:1 PC (gray). All data points were analyzed using equation 1 and represent the average of three independent experiments with the errors reported as SD (18:1-18:1 PC; n = 6). Each kinetic is the result of approximately one independent protein purification.

Fig. 5.

Relative ATPase activity of detergent-solubilized and purified ABCB4 in the presence of different building blocks ranging from choline to a PC lipid, POPC (16:0-18:1 PC). 16:0-18:1 PC (black) and 16:0-18:1 PE (POPE, moss green) (A); lyso-16:0 PC (B); Lαgpc (blue), ppc (red), and choline (green) (C). D: Chemical structures of the above-mentioned compounds. Choline moieties are shown in green, phosphate in red, the glycerin backbone in blue, fatty acids in orange and black, and alkyl chains in gray. E: The zwitterionic detergent FC-16 is depicted in gray. Data points represent the average of two independent experiments with the errors reported as SD (16:0-18:1 PC; n = 3). Each kinetic is the result of approximately one independent protein purification.

Fig. 6.

A titration experiment with the concentrations of POPC (16:0-18:1 PC) ascending from 0 to 1,000 μM with four different but constant FC-16 concentrations and an exclusive kinetic without detergent. Each kinetic is plotted as Michaelis-Menten kinetics. The MM fit for orange kinetic of 100 μM FC-16 had to ignore the first three data points in order to be fitted accordingly. Data points that are not used for the MM fit of the 100 μM FC-16 are connected via a dashed line. Each kinetic is the result of approximately one independent protein purification.

Fig. 7.

Two-step enrichment of PC-lipid variants into the mixed micelles. A schematic view on mixed micelles (A) with a ratio of 14.2% other PC-lipid variants, 49.7% 16:0-18:2 PC, 26.4% 16:0-18:1 PC, 4.4% PE, 1.2% PS, 1.4% PI, and 2.7% SM (37) (B). C: Schematic view on the floppase ABCB4 [structure taken from (43)] actively generating a high abundance of PC lipids in the outer leaflet of the canalicular membrane (I). The second step of PC enrichment (II) may be performed by headgroup-independent bile salt selection. D: The canalicular membrane consists of 29.6% other PC-lipid variants, 4.1% 16:0-18:2 PC, 2.9% 16:0-18:1 PC, 24.5% PE, 11.5% PS, 4.5% PI, and 22.8% SM (37).