ABCB4 disease-causing variants S242R, S346I, T437I and T1077M significantly impair its function and display differential sensitivity to potentiators

The hepato-canalicular adenosine triphosphate (ATP)-binding cassette transporter ABCB4/MDR3 is responsible of the secretion of phosphatidylcholine (PC) into bile. Variations in ABCB4 gene induce a spectrum of cholestatic liver diseases, the most severe form is progressive familial intrahepatic cholestasis type 3 (PFIC3). The purpose of our study was to investigate the impact and potential rescue of four ABCB4 missense variants identified in patients, two of which (T437I and T1077M) affect homologous amino acids in the Walker A motifs in the two nucleotide-binding sites NBS2 and NBS1, respectively and the other two (S242R and S346I) affect residues of the transmembrane helices 4 and 6, respectively. The functional role of the four amino acids was assessed by analysis of three-dimensional (3D) structures and molecular dynamics (MD) simulations in a lipid bilayer. For functional validation, the mutants were reproduced in a plasmid encoding the human ABCB4 protein. The localization, the processing and the PC secretion activity of the mutants were studied after transfection in cell models. As the wild-type ABCB4, all four mutants expressed and trafficked efficiently to the canalicular membrane of HepG2 cells, but were dramatically impaired in PC secretion activity. This behavior can be explained by the critical positions of the amino acids in the NBSs and in a region participating in the substrate transport. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiators ivacaftor and Small Binder of CFTR 219 (SBC219) significantly rescued the function defect of the mutants with differential sensitivity. Our results demonstrate the importance of the four mutated residues for ABCB4 function, which may explain the pathogenic phenotype. They provide an experimental evidence that targeted pharmacotherapy for genetic diseases caused by ABCB4 deficiency is likely to be mutation-specific.