Communication between multiple drug binding sites on P-glycoprotein

P-glycoprotein, a member of the ATP-binding cassette transporter family, is able to confer resistance on tumors against a large number of functionally and chemically distinct cytotoxic compounds. Several recent investigations suggest that P-glycoprotein contains multiple drug binding sites rather th an a single site of broad substrate specificity. In the present study, radi oligand-binding techniques were used to directly characterize drug interact ion sites on P-glycoprotein and how these multiple sites interact. The drug s used were classified as either 1) substrates, which are known to be trans ported by P-glycoprotein (e.g., vinblastine) or 2) modulators, which alter P-glycoprotein function but are not themselves transported by the protein ( e.g., XR9576). Drug interactions with P-glycoprotein were either competitiv e, at a common site, or noncompetitive, and therefore at distinct sites. Ba sed on these data, we can assign a minimum of four drug binding sites on P- glycoprotein. These sites fall into two categories: transport, at which tra nslocation of drug across the membrane can occur, and regulatory sites, whi ch modify P-glycoprotein function. Intriguingly, however, some modulators i nteract with P-glycoprotein at a transport site rather than a regulatory si te. The pharmacological data also demonstrate that both transport and regul atory sites are able to switch between high- and low-affinity conformations . The multiple sites on P-glycoprotein display complex allosteric interacti ons through which interaction of drug at one site switches other sites betw een high- or low-affinity conformations. The data are discussed in terms of a model for the mechanism of transport by P-glycoprotein.