Received for publication December 28, 2006.
Revised May 22, 2007.
Accepted for publication May 24, 2007.

ABCG2 (BCRP/MXR) ATPase assay - a useful tool to detect drug - transporter interactions

Abstract

The ATPase assay utilizing membrane preparations from recombinant baculovirus infected Sf9 cells is widely used to detect the interaction of compounds with different ABC transporters. However, Sf9 membrane preparations containing the wild type ABCG2 transporter show an elevated baseline vanadate sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty i) we purified membranes from a selected human cell line expressing wild type ABCG2, and ii) inhibited the baseline ATPase activity with different inhibitors. In our modified assay ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

Key words: ABC transporters, membrane transport, transporters