Uncouplers of Mitochondrial Oxidative Phosphorylation Are Not Substrates of the Erythrocyte Glutathione-S-Conjugate Pump

Uncouplers of mitochondrial oxidative phosphorylation, dinitrophenol (DNP) and carbonyl cyanideptrifluoromethoxyphenylhydrazone (FCCP), were found to stimulate Mg2+-ATPase activity of human erythrocyte membranes in a manner competitive with respect to 2,4-dinitrophenyl-S-glutathione (DNP-SG) which suggested that these compounds may also be substrates of the glutathione-S-conjugate pump. We confirm that the stimulation of erythrocyte membrane ATPase activity by DNP and by another uncoupler, carbonyl cyanidem-chlorophenylhydrazone (CCCP), is competitive with respect to DNP-SG. However, we found no evidence for active transport of DNP and CCCP out of erythrocytes and demonstrate that they inhibit the low-affinity component of DNP-SG transport noncompetitively while stimulating the high-affinity DNP-SG transport (mediated by multidrug resistance-associated protein, MRP1). Implications of these findings may indicate the electrogenic nature of MRP1-mediated transport of glutathione-Sconjugates and stimulation of aminophospholipid translocase (flippase) rather than the glutathione-S-conjugate pump by the uncouplers.