A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ABC Transporter MDR3 [Enzymology]

December 22nd, 2014 by Kluth, M., Stindt, J., Droege, C., Linnemann, D., Kubitz, R., Schmitt, L.

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops phospholipids of the phosphatidylcholine family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem-affinity chromatography and determined MDR3 specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated twofold by liver PC or DOPC lipids. Furthermore, the crosslinking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similar, phosphatidylethanolamine (PE), phosphatidylserin (PS) and sphingomyelin (SM) lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues BeFx and AlFx led to complete inhibition of ATPase activity, while ortho-vanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain (NBD) in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the crosstalk of the NBD and the transmembrane domain.