RT Journal Article
SR Electronic
T1 Functional Role of Arginine 375 in Transmembrane Helix 6 of Multidrug Resistance Protein 4 (MRP4/ABCC4)
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 964
OP 971
DO 10.1124/mol.107.043661
VO 74
IS 4
A1 Azza A. K. El-Sheikh
A1 Jeroen J. M. W. van den Heuvel
A1 Elmar Krieger
A1 Frans G. M. Russel
A1 Jan B. Koenderink
YR 2008
UL http://molpharm.aspetjournals.org/content/74/4/964.abstract
AB Multidrug resistance protein (MRP) 4 transports a variety of endogenous and xenobiotic organic anions. MRP4 is widely expressed in the body and specifically localized to the renal apical proximal tubule cell membrane, where it mediates the excretion of these compounds into urine. To characterize the MRP4 substrate-binding site, the amino acids Phe368, Phe369, Glu374, Arg375, and Glu378 of transmembrane helix 6, and Arg998 of helix 12, localized in the intracellular half of the central pore, were mutated into the corresponding amino acids of MRP1 and MRP2. Membrane vesicles isolated from human embryonic kidney 293 cells overexpressing these mutants showed significantly reduced methotrexate (MTX) and cGMP transport activity compared with vesicles that expressed wild-type MRP4. The only exception was substitution of Arg375 with serine, which had no effect on cGMP transport but significantly decreased the affinity of MTX. Substitution of the same amino acid with a positively charged lysine returned the MTX affinity to that of the wild type. Furthermore, MTX inhibition of MRP4-mediated cGMP transport was noncompetitive, and the inhibition constant was increased by introduction of the R375S mutation. A homology model of MRP4 showed that Arg375 and Arg998 face right into the central aqueous pore of MRP4. We conclude that positively charged amino acids in transmembrane helices 6 and 12 contribute to the MRP4 substrate-binding pocket. The American Society for Pharmacology and Experimental Therapeutics