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Transport of Cisplatin by the Copper Efflux Transporter ATP7B

Moores University of California San Diego Cancer Center, University of California, San Diego, La Jolla, California (R.S., M.L.R., S.B.H.); Department of Surgery, Hino Hospital, Hino-cho, Tottori Prefecture, Japan (S.O.); and Stanford University, School of Medicine, Stanford, California (B.J.L.)

ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that expressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-dependent ATPase activity, copper-induced acyl-phosphate formation, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP-dependent accumulation of DDP with an apparent K_m of 1.2 ± 0.5 (S.E.M.) µM and V_max of 0.03 ± 0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper.

Address correspondence to: Dr. Roohangiz Safaei, Department of Medicine, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093-0819; E-mail: rsafaei{at}ucsd.edu