Abstract
P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.
Highlights
The ATPase activity was measured in native membranes, DDM micelles, and proteoliposomes as described under “Experimental procedures.”
As both hP-gp and mP-gp recognize and transport a variety of chemically dissimilar amphipathic or hydrophobic compounds including toxins and anticancer agents [2, 23, 24], and a number of detergents have been reported to interact as ligands or “allocrits” with this multidrug transporter when tested in native membranes [21, 25, 26], we investigated the interactions of selected modulators and substrates with purified mP-gp and hP-gp in the presence of DDM micelles
We found that substitution of two tyrosine residues and one glutamate residue with alanine disrupts the high-affinity interaction of these three modulators with P-gp, which is similar to our observations reported here with purified wild-type P-gp in a DDM micelle environment
Summary
The detergent DDM was used for crystallization of mouse and Caenorhabditis elegans P-gp, and the ATPase activity of P-gp was measured in DDM micelles in the presence of added lipid [16, 17]. To determine the effect of changing the environment from membranes to detergent micelles, ATP hydrolysis by mP-gp in native membranes and after its purification in DDM micelles in the presence of three known and well characterized inhibitors (tariquidar, elacridar, and zosuquidar) was measured as detailed under “Experimental procedures.”. A and B, when the detergent was removed by Bio-Beads SM2 in mP-gp proteoliposomes, both tariquidar and elacridar inhibited ATP hydrolysis in a concentration-dependent manner similar to their observed effect in native membranes (Fig. 1). Summary of IC50 and EC50 values of tariquidar, elacridar, zosuquidar, verapamil, and QZ59-SSS for ATP hydrolysis of mouse P-gp in native membranes, DDM micelles, and proteoliposomes. The ATPase activity was measured in native membranes, DDM micelles, and proteoliposomes as described under “Experimental procedures.” M EC50 ϭ 0.51 Ϯ 0.07 EC50 ϭ 2.18 Ϯ 0.40 EC50 ϭ 11.82 Ϯ 2.34 EC50 ϭ 421.8 Ϯ 93.30 EC50 ϭ 2.08 Ϯ 0.48
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