Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase--further experimental evidence for a multisite model

Eur J Biochem. 1997 Mar 1;244(2):664-73. doi: 10.1111/j.1432-1033.1997.00664.x.


P-glycoprotein, a plasma membrane protein overexpressed in multidrug-resistant (MDR) cells, exhibits in vitro an ATPase activity and is responsible for the energy-dependent efflux of structurally unrelated cytotoxic drugs (like vinblastine) and various MDR-reversing agents (like verapamil and progesterone) from these MDR cells. To investigate the mechanism of P-glycoprotein interaction with various compounds, we measured the P-glycoprotein ATPase activity on membrane vesicles prepared from the MDR cell line DC-3F/ADX, and we studied the effects of vinblastine, verapamil and progesterone on this ATPase activity. The basal P-glycoprotein ATPase activity is increased by verapamil and progesterone, with respective half-maximal activating concentrations of approximately 1.5 microM and approximately 25 microM, and activation factors of approximately 1.7 and approximately 2.2. Vinblastine inhibits the activation of P-glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant approximately 0.5 microM in both cases. This demonstrates that vinblastine has a specific modulating site on P-glycoprotein. The combined modulation of P-glycoprotein ATPase by vinblastine and verapamil reveals that these two drugs are mutually exclusive. Since these two molecules have different effects both on the basal P-glycoprotein ATPase activity and on the MgATP concentration dependence of P-glycoprotein ATPase activity, they could bind P-glycoprotein either on different and overlapping sites, or on distant but interacting sites. In contrast, the combined modulation of P-glycoprotein ATPase by vinblastine and progesterone reveals a non-competitive relationship between these two drugs, and hence shows that they can independently and simultaneously bind P-glycoprotein on distinct sites. Since verapamil and progesterone are mutual inhibitors of P-glycoprotein ATPase stimulation in a non-competitive manner, these two molecules can also bind independently P-glycoprotein on separated sites. This is confirmed here by the observation of a synergistic effect when mixtures of verapamil and progesterone are tested for the modulation of P-glycoprotein ATPase. Three MDR-related molecules, taken as models for interaction with P-glycoprotein, appear thus to bind on at least two different separated specific sites. These results favor a multisite model rather than a universal site model to describe the broad substrate specificity characterizing P-glycoprotein function.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / antagonists & inhibitors*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / chemistry
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Adenosine Triphosphatases / antagonists & inhibitors*
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • Animals
  • Binding Sites
  • Binding, Competitive
  • Cell Line
  • Cricetinae
  • Drug Resistance, Multiple
  • Drug Synergism
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • In Vitro Techniques
  • Kinetics
  • Models, Biological
  • Progesterone / metabolism
  • Progesterone / pharmacology
  • Verapamil / metabolism
  • Verapamil / pharmacology
  • Vinblastine / metabolism
  • Vinblastine / pharmacology


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Enzyme Inhibitors
  • Progesterone
  • Vinblastine
  • Verapamil
  • Adenosine Triphosphatases