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Drug Metabolism and Disposition Fast Forward
First published on March 2, 2005; DOI: 10.1124/dmd.105.003657

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Received for publication January 11, 2005.
Revised February 18, 2005.
Accepted for publication February 25, 2005.

Functional Analysis of the Human Variants of BCRP: I206L, N590Y, and D620N

R. Robert Vethanayagam 1, Honggang Wang 1, Anshul Gupta 1, Yi Zhang 1, Fred Lewis 1, Jashvant D. Unadkat 1, Qingcheng Mao 1*

1 University of Washington

* Address correspondence to: E-mail: qmao{at}u.washington.edu

Abstract

Previous studies have shown that the V12M and Q141K variants of BCRP can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y and D620N variants on protein expression, plasma membrane localization and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in HEK cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, while the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All the three variants transported mitoxantrone, pheophorbide a and BODIPY-prazosin. After normalization for differences in BCRP expression, I206L, N590Y and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. While D620N conferred a similar topotecan resistance as the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, while N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP.


Key words: ABC transporters, membrane transport, pharmacogenetics, transporters


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