Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC)

Abstract

Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.

Keywords: ABCB4; Functional characterization; LPAC; MDR3; Mutation.

Fig. 1

Effect of ABCB4 mutants on the transport activity. Inside-out membrane vesicles were prepared after transfection of ABCB4 reference (REF) or mutant-bearing plasmids into HEK-293T cells and ATP-dependent transport of paclitaxel was measured using various concentrations of paclitaxel. Values for transport activity were obtained by subtracting the uptake in empty vector (EV, pcDNA3.1)-transfected cells from that in cells transfected with ABCB4 reference or mutant-bearing vectors, at each corresponding paclitaxel concentration. The X-axis represents paclitaxel concentration. The Y-axis represents the amount of inorganic phosphate which was produced by the ATPase activity of MDR3 during the transport of paclitaxel. The data shown represent mean±SD from 5 separate experiments, with each experiment performed in triplicate wells. ^*p<0.05 vs. reference.

Fig. 2

Effect of ABCB4 mutants on the MDR3 expression. (A) HEK-293T cells expressing reference or mutant ABCB4 genes were lysed and proteins were immunoblotted with antibodies against MDR3, neomycin phosphotransferase II (NPII), and β-actin. (B) HEK-293T cells expressing reference or mutant ABCB4 genes were surface biotinylated and proteins were immunoblotted with antibodies against MDR3, Na⁺/K⁺ ATPase α-1, and aldolase. Immunoblotting with the anti-aldolase antibody confirmed that the samples were not contaminated with the cytosolic fraction of MDR3 protein. The intensity of each band was normalized by neomycin phosphotransferase II and β-actin (A) or Na⁺/K⁺ ATPase α-1 (B). Data (mean±SD) are from 3 separate experiments. ^*p<0.05, ^**p<0.01 vs. reference.

Fig. 3

Subcellular localization of the ABCB4 mutants. Immunofluorescence was performed after transfection of ABCB4 reference or mutant-bearing plasmids into HEK-293T cells. MDR3 protein was detected using the P3II26 antibody and Alexa Fluor® 488 rabbit anti-mouse IgG (green). Plasma membrane was stained by CellMask Plasma Membrane stain solution (red).