ABSTRACT

A single amino acid substitution, Gly Val, in the human P-glycoprotein (Pgp) was previously shown to cause an altered pattern of drug resistance in cell lines transfected with the MDR1 cDNA carrying this mutation. To further define the function of amino acid 185 in the Pgp, the wild-type and the mutant Val Pgps were expressed in Sf9 insect cells, and their biochemical properties were compared. Verapamil- and colchicine-stimulated ATPase activities were markedly increased with concomitant increase in affinity for these compounds with Gly Val substitution in the Pgp. However, the vinblastine-stimulated ATPase activities of the wild-type and Val Pgps were nearly identical. Because transport substrate-induced ATP hydrolysis is generally thought to reflect transport function, these data suggest that colchicine and verapamil are transported at an increased rate with Gly Val substitution in the Pgp. These results also indicate that amino acid 185 is involved in verapamil and colchicine, but not in vinblastine, binding/transport. Kinetic analyses indicate that cyclosporin A, an inhibitor of Pgp, binds to the verapamil and vinblastine binding/transport site(s) in the Pgp. Taken together, the results presented herein reveal that the verapamil and vinblastine binding/transport site(s) are in close proximity and that the cyclosporin A binding site spans the common region of these two drug binding/transport site(s) in the Pgp molecule.

FOOTNOTES

*

This work was supported by Grant IRG 15-34 from the American Cancer Society (to U. S. R.) and Grant CA58491 from the National Institutes of Health (to Gene A. Scarborough). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Pharmacology, CB 7020, Burnett-Womack Bldg., University of North Carolina, Chapel Hill, NC 27599-7020. Tel.: 919-966-1077; Fax: 919-966-7524.

()

The abbreviations used are: MDR, multidrug resistance; Pgp, P-glycoprotein; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis.

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