Download P-glycoprotein Activation Monitored via ATP Hydrolysis and ATP

P-glycoprotein Activation Monitored via ATP Hydrolysis and
ATP Synthesis
Päivi Äänismaa, Anna Seelig
Biozentrum der Universität Basel, Abteilung für Biophysikalische Chemie,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland
We investigated the relationship between the rate of ATP hydrolysis and ATP synthesis
upon P-glycoprotein activation for several structurally different drugs, including local
anaesthetics, cyclic peptides, and cytotoxic drugs. ATP hydrolysis was assessed by
spectroscopically monitoring the release of inorganic phosphate in inside-out cellular
vesicles of MDR1-transfected cells and ATP synthesis was assessed by measuring the
extracellular acidification rate, ECAR, which corresponds to the rate of lactate efflux in
living MDR1-transfected cells by means of a Cytosensor microphysiometer. Both
processes were investigated as a function of drug concentration. The kinetic data were
evaluated with a model taking into account activation with one, and inhibition with two
molecules bound to P-glycoprotein (1, 2). The experiments revealed that the
concentrations of half-maximum P-glycoprotein activation, K1, were identical in insideout plasma membrane vesicles and in living cells and covered a broad range of
concentrations (K1 approximately 10-8-10-3 M). The rate of ATP-hydrolysis and ATPsynthesis upon Pgp activation is also approximately identical if measured in the
presence of pyruvate. However, in the absence of pyruvate they were higher in living
cells. The rate of Pgp activation, V1, varies significantly with the substrate transported.
It decreases with increasing free energy of drug binding from water to the transporter
suggesting drug release from the transmembrane domains has to occur before ATP is
hydrolyzed for resetting the transporter (2).
(1) Gatlik-Landwojtowicz, E., Äänismaa, P., Seelig, A., (2006) Biochemistry 45, 3020-3032
(2) Äänismaa, P., and Seelig, A., (2007) Biochemistry 46, 3394-3404