The Membrane Lipid Environment Modulates Drug Interactions with the P-Glycoprotein Multidrug Transporter

Abstract

The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be modulated by the properties of the lipid bilayer. The interactions with P-glycoprotein of two drugs (vinblastine and daunorubicin) and a chemosensitizer (verapamil) were characterized by quenching of purified fluorescently labeled protein in the presence of various phospholipids. Biphasic quench curves were observed for vinblastine and verapamil, suggesting that more than one molecule of these compounds may bind to the transporter simultaneously. All three drugs bound to P-glycoprotein with substantially higher affinity in egg phosphatidylcholine (PC), compared to brain phosphatidylserine (PS) and egg phosphatidylethanolamine (PE). The nature of the lipid acyl chains also modulated binding, with affinity decreasing in the order egg PC > dimyristoyl-PC (DMPC) > dipalmitoyl-PC (DPPC). Following reconstitution of the transporter into DMPC, all three compounds bound to P-glycoprotein with 2-4-fold higher affinity in gel phase lipid relative to liquid-crystalline phase lipid. The P-glycoprotein ATPase stimulation/inhibition profiles for the drugs were also altered in different lipids, in a manner consistent with the observed changes in binding affinity. The ability of the drugs to partition into bilayers of phosphatidylcholines was determined. All of the drugs partitioned much better into egg PC relative to DMPC and DPPC. The binding affinity increased (i.e., the value of Kd decreased) as the drug-lipid partition coefficient increased, supporting the proposal that the effective concentration of the drug substrate in the membrane is important for interaction with the transporter. These results provide support for the vacuum cleaner model of P-glycoprotein action.

Identification and Characterization of the Binding Sites of P-Glycoprotein for Multidrug Resistance-Related Drugs and Modulators

“…Moreover, while evidence suggests that these drug-binding sites are located in TM segments of P-glycoprotein within the lipid bilayer, it is possible that the hydrophobic region(s) of the drugs may bind to the TM segments and the hydrophilic region(s) may interact close to the cytosolic domains of P-glycoprotein near the TM segments [197]. In fact, it has been recently shown that the binding affinities of Pglycoprotein-related drugs increase as the drug-lipid partition coefficient increases [198], suggesting that effective concentrations of the drugs in the membrane are important for interacting with P-glycoprotein. Photoaffinity labeled drugs have been useful for identifying regions of P-glycoprotein that bind to cytotoxic drugs and chemosensitizers.…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…Several experimental results indicate that ABCB1 binds its drug substrates within the plasma membrane and most probably in the inner leaflet of the plasma membrane (Clay & Sharom, 2013;Higgins & Gottesman, 1992;Sharom, 1997). Drug partitioning into the lipid membrane and drug binding to ABC multidrug transporters show close correlation (Clay & Sharom, 2013;Romsicki & Sharom, 1999). FRET studies, applying a fluorescent substrate drug (doxorubicin) of ABCB1 and a lipophilic fluorescent compound (iodonaphtalen-1-azide) that can be directly attached to ABCB1 through photoaffinity labeling, indicated drug binding to the transporter within the lipid phase (Raviv, Pollard, Bruggemann, Pastan, & Gottesman, 1990).…”

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

“…Effects of Potential Substrates on the ATPase Activity of CTSThe ATPase activity of many ABC transporters is increased above basal rates upon substrate binding (45)(46)(47)(48)(49)(50). Exploiting this behavior, it is possible to screen a range of compounds to identify putative substrates for a given ATP transporter.…”