The ATPase and ATP‐binding functions of P‐glycoprotein

Abstract

P-glycoprotein functions as an active efflux pump for lipophilic compounds and plays an important role in the resistance of human cancers to chemotherapeutic drugs. Drug transport is powered by ATP hydrolysis at two highly conserved nucleotide-binding domains, which are proposed to be located at the cytosolic face of the protein. The ATPase activity of P-glycoprotein depends on the presence of phospholipids, and various lipids affect both basal ATPase activity and its stimulation or inhibition by drug substrates. The modulating effects of the lipid-phase state and effects on the function of the nucleotidebinding domains of P-glycoprotein have been studied in reconstituted vesicles of the synthetic phospholipids 1-palmitoyl-2-myristoylphosphatidylcholine (PamMyrGroPCho) and dimyristoylphosphatidylcholine (Myr 2GroPCho). The kinetic parameters for P-glycoprotein ATPase activity were determined, and a fluorescence-quenching technique was used to measure the K d for ATP binding. The values of both the K m for ATP hydrolysis and K d for ATP binding were significantly different above and below the gel/liquidcrystalline phase transition temperature (t m) of PamMyrGroPCho and Myr2GroPCho, whereas they were similar at the same temperatures for P-glycoprotein in detergent solution. A discontinuity at 21Ϫ24°C was observed in the Arrhenius plots of P-glycoprotein ATPase activity in a membrane environment, but not in detergent solution. In addition, the activation energies for ATP hydrolysis in the gel and liquidcrystalline phases of the lipid bilayer were significantly different. P-glycoprotein in PamMyrGroPCho bilayers displayed an unusually low activation energy just below the melting transition. These results indicate that both ATP binding and ATP hydrolysis by P-glycoprotein are affected by the phase state of the host lipids in which it is reconstituted. Lipids may modulate the function of the nucleotide-binding domains of P-glycoprotein by interacting with the transmembrane regions of the protein, or the nucleotidebinding domains themselves may interact with the surface of the bilayer.

Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

“…Since the transporter is known to be sensitive to detergent and lipid composition [40,73,[75][76][77][78][79], these differences in ATP hydrolysis kinetics were attributed to our procedure for reconstituting the transporter into the liposomes and our efforts to minimize the DDM during the protein purification process.…”

ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells

“…It is possible that membrane composition may be a factor. The cholesterol content of insect cell membranes is only ϳ10% that of mammalian cell membranes and membrane composition is known to influence the transport characteristics of some ABC proteins including their ATPase activity (52,68). The trafficking of the wild-type and mutant proteins may also differ.…”

Drug resistance-associated changes in sphingolipids and ABC transporters occur in different regions of membrane domains

“…These tightly bound lipids have been identified as mainly PE and PS (15,16). Furthermore, it has been shown that PE stimulates the ATPase activity of both Pgp and MRP1, whereas PS additionally stimulates MRP1 ATPase activity (17)(18)(19)(20).…”

“…Pgp and MRP1 are known to depend on the lipid environment for optimal functioning (13,14). In membrane model systems, the ATPase activity of both proteins is dependent on the close proximity of specific phospholipids, especially phosphatidylethanolamine (PE) (15)(16)(17)(18)(19)(20). Moreover, Pgp has a higher affinity for its substrates when the surrounding lipids are in the gel phase rather than in the liquid-crystalline phase (21).…”