The remarkable transport mechanism of P-glycoprotein: a multidrug transporter

Abstract

Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that utilizes the energy of ATP hydrolysis to pump toxic xenobiotics out of cells. P-glycoprotein employs a most unusual molecular mechanism to perform this drug transport function. Here we review our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling, and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised to place our results within a proper structural context.

Fig. 1

Transport of spin-labeled verapamil by P-glycoprotein. Panel A. The reaction mixture contained 50 μM of SL-verapamil and 2 μM P-glycoprotein, in unilamellar proteoliposomes with an average hydrodynamic radius of 420 Å, at pH 7.5 and 23 °C. Transport was started in the EPR spectrometer by addition of ATP to the reaction mixture containing a potent ATP regenerating system (Omote and Al-Shawi, 2002). The concentration of SL-verapamil in the aqueous phase is plotted as a function of time. After maximum steady state transport was achieved (120 min), vesicles and supernatant were separated by centrifugation. Vesicles were resuspended in buffer and spectra recorded at the times indicated. Representative EPR spectra are shown for various time points along the transport assay. The post centrifugation vesicular spectrum is expanded to show fine details. The sum of the vesicular and supernatant spectra was identical to the transport assay spectrum at 120 min. Panel B. Schematic representation of the steady state concentrations of SL-verapamil at 120 min. Calculated values were obtained from the deconvoluted steady state spectrum at 120 min of panel A. These results were in absolute agreement with results obtained by analysis of the component spectra of the supernatant and vesicles obtained by centrifugation and SL-verapamil concentration analysis.

Fig. 2

Catalytic and drug transport cycles of P-glycoprotein. A partitioning model of P-glycoprotein catalytic and transport cycles is shown. E refers to P-gp species. Inner-leaflet, high-affinity, drug-loading “ON-sites” are shown in green (loading site, [unk]). Extracellular facing, low-affinity, drug-unloading “OFF-sites” are shown in orange (unloading site, [unk]). Red stars show the rate-limiting transition states for the two cycles. The centrally located species _ATPE [unk] [unk] indicates a molecule of P-glycoprotein that has one bound ATP. This species is thought to be a mobile carrier form of the protein in which the unloaded high-affinity and unloaded low-affinity drug-binding sites are in equilibrium. Upper right cycle shows the drug-activated, coupled activity; lower left cycle shows the uncoupled basal activity. If there is insufficient drug and two ATP molecules bind, P-gp partitions to the uncoupled cycle and hydrolyses ATP without any transport work. In this cycle the drug sites are in a low-affinity unloading conformation. However, if there is sufficient transport drug present, P-gp partitions to the coupled activity cycle. The coupled cycle is the alternating catalytic cycle previously described (Senior et al., 1995a). Transport drug binds first to a high-affinity loading site followed by ATP to form the ternary complex. After passing through the high-energy transition state, drug is released to the other side of the membrane (successful transport). Different transport drugs lead to different energy levels of the rate-limiting coupling transition state (Al-Shawi et al., 2003; Omote et al., 2004). Due to the instability of the higher-energy transition states, there is a greater probability of nonproductive transition state decay without drug transport for drugs with higher-energy transition states (failed transport). Failed transport is observed when wild-type P-glycoprotein is transporting colchicine or etoposide. The mutation G185V increases the strength of colchicine and etoposide interaction with the transition state and improves the transport of these drugs by reducing the level of failed transport (Omote et al., 2004).