ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2.
BACKGROUND
Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney.


METHODS
Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1.


RESULTS
Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2.


CONCLUSIONS
PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.
Leier
I
I
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany. i.leier@dkfz-heidelberg.de
Hummel-Eisenbeiss
J
J
Cui
Y
Y
Keppler
D
D
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009793:Ochratoxins / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D010130:p-Aminohippuric Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009793:Ochratoxins; D011994:Recombinant Proteins; D010130:p-Aminohippuric Acid; C000715087:ABCC2 protein, human; C025589:ochratoxin A; C421109:multidrug resistance-associated protein 1