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J. Biol. Chem., Vol. 279, Issue 31, 32700-32708, July 30, 2004

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Arsenic Transport by the Human Multidrug Resistance Protein 1 (MRP1/ABCC1)

EVIDENCE THAT A TRI-GLUTATHIONE CONJUGATE IS REQUIRED^*

Elaine M. Leslie¶, Anass Haimeur||, and Michael P. Waalkes**

From the Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, NCI at NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ^||Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada

Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C₄) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (As^III) only in the presence of GSH but does not transport arsenate (As^V) (with or without GSH). The non-reducing GSH analogs L--glutamyl-L--aminobutyryl glycine and S-methyl GSH did not support As^III transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of As^III was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)₃). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport As^III even in the presence of GSH but did transport synthetic As(GS)₃. The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent As^III transport. The apparent K_m of As(GS)₃ for MRP1 was 0.32 µM, suggesting a remarkably high relative affinity. As(GS)₃ transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (K_i 2.8 µM). As(GS)₃ transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)₃ and leukotriene C₄. Finally, human MRP2 also transported As(GS)₃. In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process.

Received for publication, May 3, 2004 , and in revised form, May 21, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipients of Canadian Institutes of Health Research fellowships.

^¶ Recipient of the Davies Charitable Foundation Research Fellowship Award.

^** To whom correspondence should be addressed: Inorganic Carcinogenesis Section, NCI at NIEHS, P. O. Box 12233, Mail drop F0-09, 111 Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-2328; Fax: 919-541-3970; E-mail: waalkes{at}niehs.nih.gov.

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