Charged amino acids in the sixth transmembrane helix of multidrug resistance protein 1 (MRP1/ABCC1) are critical determinants of transport activity

Abstract

The multidrug resistance protein, MRP1 (ABCC1), is an ATP-binding cassette transporter that confers resistance to chemotherapeutic agents. MRP1 also mediates transport of organic anions such as leukotriene C4 (LTC4), 17β-estradiol 17-(β-D-glucuronide) (E217βG), estrone 3-sulfate, methotrexate (MTX), and GSH. We replaced three charged amino acids, Lys332, His335, and Asp336, predicted to be in the sixth transmembrane (TM6) helix of MRP1 with neutral and oppositely charged amino acids and determined the effect on substrate specificity and transport activity. All mutants were expressed in transfected human embryonic kidney cells at levels comparable with wild-type MRP1, and confocal microscopy showed that they were correctly routed to the plasma membrane. Vesicular transport studies revealed that the MRP1-Lys332 mutants had lost the ability to transport LTC4, and GSH transport was reduced; whereas E217βG, estrone 3-sulfate, and MTX transport were unaffected. E217βG transport was not inhibited by LTC4 and could not be photolabeled with [3H]LTC4, indicating that the MRP1-Lys332 mutants no longer bound this substrate. Substitutions of MRP1-His335 also selectively diminished LTC4 transport and photolabeling but to a lesser extent. Kinetic analyses showed that Vmax (LTC4) of these mutants was decreased but Km was unchanged. In contrast to the selective loss of LTC4 transport in the Lys332 and His335 mutants, the MRP1-Asp336 mutants no longer transported LTC4, E217βG, estrone 3-sulfate, or GSH, and transport of MTX was reduced by >50%. Lys332, His335, and Asp336 of TM6 are predicted to be in the outer leaflet of the membrane and are all capable of forming intrahelical and interhelical ion pairs and hydrogen bonds. The importance of Lys332 and His335 in determining substrate specificity and of Asp336 in overall transport activity suggests that such interactions are critical for the binding and transport of LTC4 and other substrates of MRP1.