P-Glycoprotein Kinetics Measured in Plasma Membrane Vesicles and Living Cells

Abstract

P-glycoprotein (MDR1, ABCB1) is an ATP-dependent efflux transporter of a large variety of compounds. To understand P-glycoprotein in more detail, it is important to elucidate its activity in the cellular ensemble as well as in plasma membrane vesicles (under conditions where other ATP dependent proteins are blocked). We measured P-glycoprotein activity in inside-out vesicles formed from plasma membranes of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) for comparison with previous measurements of P-glycoprotein activity in living NIH-MDR1-G185 cells. In plasma membrane vesicles activity was measured by monitoring phosphate release upon ATP hydrolysis and in living cells by monitoring the extracellular acidification rate upon ATP synthesis via glycolysis. P-glycoprotein was stimulated as a function of the concentration with 19 structurally different drugs, including local anesthetics, cyclic peptides, and cytotoxic drugs. The concentrations of half-maximum P-glycoprotein activation, K1, were identical in inside-out plasma membrane vesicles and in living cells and covered a broad range of concentrations (K1 approximately (10(-8)-10(-3)) M). The influence of the pH, drug association, and vesicle aggregation on the concentration of half-maximum P-glycoprotein activation was investigated. The turnover numbers in plasma membrane vesicles and in living cells were also approximately identical if the latter were measured in the presence of pyruvate. However, in the absence of pyruvate they were higher in living cells. The rate of ATP hydrolysis/ATP synthesis decreased exponentially with decreasing free energy of drug binding from water to the transporter, DeltaG0(tw)(1) (or increasing binding affinity). This suggests that drug release from the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.

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

“…These values are very similar to the average K m and K i values determined for mouse Pgp in Ehrlich membranes of 2.5 and 225 μM [74] and for human Pgp in NIH-MDR1-G185 cells of 1.0 and 843.6 μM respectively [20]. The V MAX was also similar to previous determinations with mouse Pgp and the half maximal ATPase activity of 4.2 μM with mouse Pgp was close to our estimates [39].…”

“…The effect of verapamil and digoxin on the Pgp-coupled ATPase activity Biphasic verapamil kinetics with the transporter has been observed previously with hamster [70][71][72][73], human [20] and mouse [74] Pgp. These values are very similar to the average K m and K i values determined for mouse Pgp in Ehrlich membranes of 2.5 and 225 μM [74] and for human Pgp in NIH-MDR1-G185 cells of 1.0 and 843.6 μM respectively [20].…”

“…To remove aggregated Pgp, the reconstituted liposomes were centrifuged for 5 min at 100 g in a Sorvall Legend Micro 21 centrifuge (ThermoScientific). To determine the orientation of mouse Pgp in the liposomes, the permeability of the reconstituted liposomes was tested with CHAPS detergent to expose nucleotide-binding domains (NBDs) oriented within the liposome [20,44,45]. Since there was no increase in the ATPase activity with increasing CHAPS concentrations, Pgp was assumed to be in an inside-out orientation.…”

“…From results of in vitro studies, the drug is known to activate Pgp-coupled ATP-hydrolysis [20]. This drug manifests a spectrum of characteristics, ranging from being a good substrate to a non-substrate for the transporter, which depends on the cell type being evaluated in in vitro cell studies [21][22][23][24][25][26] or host tissue type in in vivo studies [27][28][29].…”

“…Because verapamil and digoxin have been the focus of a number of in vitro [20] and in vivo studies [27], these drugs are ideal for studying DDIs with the transporter. Many molecular and mechanistic details of verapamil-digoxin DDIs with Pgp remain unresolved.…”