Two ABCB4 mutations involving two strategic NBD-motifs do not prevent the targeting to the plasma membrana but promote MDR3 dysfunction

MDR3 protein translocates phosphatidylcoline (PC) from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency, related to ABCB4 mutations, favours the formation of “toxic bile”. A continuum of hepatobiliary diseases have been associated with ABCB4 mutations but, for most of them, the detrimental effect on the protein is speculative only. The functional relevance of two strategic mutations within the N-terminal Nucleotide-Binding-Domain was examined with stably transfected HUH28-cell-lines expressing wild type and mutant MDR3 proteins by westernblotting, immunocytochemistry and chromatographic quantification of lipids, collected from culture medium after sodium-taurocholate (NaTC) stimulation. As suggested by our three-dimensional model of MDR3 (Degiorgio D et al., 2007), the p.Y403H mutation involves the A-loop while the p.L481R mutation is contained into the Q-loop. Our results show that both MDR3-mutant proteins were expressed in a comparable way to the MDR3-wild-type protein: a molecular mass of 160KDa associated with a green fluorescent signal, intenser and sharper in the plasma membranes, was constantly identified. However, compared to the stably transfected HUH28-cell-line expressing wild-type-MDR3 protein in the presence of NaTC 3mM, the lipid dosage into culture medium has shown (with five indipendent experiments) that i) the efflux of PC is reduced (p<0.01) from cell lines expressing p.Y403H and p.L481R mutant proteins; ii) the efflux of cholesterol is increased (p<0.01) from cell line expressing the p.Y403H mutant protein. In conclusion these mutations could promote in vivo formation of toxic bile with reduced amounts of PC (p.L481R) or with reduced amounts of PC and increased level of cholesterol (p.Y403H).