P-Glycoprotein in Proteoliposomes with Low Residual Detergent: The Effects of Cholesterol

Abstract

Purpose. There is evidence that cholesterol affects the ATPase and transport functions of P-glycoprotein (P-gp). To study the influence of cholesterol on P-gp in a well defined lipid environment, we reconstituted P-gp in egg phosphatidylcholine (PhC) and PhC/cholesterol proteoliposomes with negligible residual amounts of detergents. Materials and methods. P-gp proteoliposomes were prepared by continuous dialysis from micelles consisting of P-gp, lipids, sodium dodecyl sulfate and cholate. Basal and modulator-induced ATPase activities were studied in an established enzyme assay. Modulator affinities to P-gp and to the lipid bilayers were determined by equilibrium dialysis. Results. In the absence of cholesterol the basal ATPase activity was six fold lower than in the presence of 20 or 40% cholesterol, and no P-gp binding and ATPase induction was detected for the tested modulators verapamil and progesterone. In proteoliposomes containing 20 and 40% cholesterol, respectively, the modulators showed significant P-gp binding and ATPase activation. The concentration of the modulators for half maximal activation of the ATPase was higher with 40% than with 20% cholesterol. Conclusions. Cholesterol influences P-gp in three ways: (a) it enhances its basal ATPase activity, (b) it renders P-gp sensitive towards the modulators verapamil and progesterone and (c) it affects the modulator concentration at half maximal ATPase activation.

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…ABCB1 ATPase activity was measured in most cases in native membranes prepared from ABCB1-overexpressing cells. While the basal ATPase activity of ABCB1 was increased by cholesterol, the substrate-stimulated activity was less modified (Belli, Elsener, Wunderli-Allenspach, & Kramer, 2009;Bucher, Belli, Wunderli-Allenspach, & Kramer, 2007). In addition, in the case of smaller size drug substrates, cholesterol had a much greater influence on the substrate-dependent ABCB1-ATPase activity than in the case of larger drug substrates (Kimura, Kioka, Kato, Matsuo, & Ueda, 2007).…”

“…The analysis of the effects of the length of lipid side chains revealed an optimum bilayer width, and the headgroups of the lipids were also found to be important (Sharom, 2014). The presence of cholesterol is not necessary for purified ABCB1 activity in liposomes (Bucher et al, 2007;Kimura, Kioka, et al, 2007), and not only other sterols but also α-tocopherol or even DPPC caused the same moderate effect as cholesterol on the ABCB1 function in PC liposomes (Belli et al, 2009). …”

“…The homodimeric ABCG2 protein was even more sensitive to detergents than ABCB1, and the addition of extra lipids at the solubilization step was indispensable for maintaining activity (Telbisz et al, 2014). Reconstitution studies showed that purified ABCB1 can be functional in several types of liposomes, composed of different phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures, and the addition of cholesterol is not required for full activity (Bucher et al, 2007;Doige et al, 1993;Kimura, Kioka, et al, 2007;Modok, Heyward, & Callaghan, 2004). ABCB1 was active even in pure DMPE or DPPE liposomes, but the application of PC:PE mixtures increased activity and unsaturated lipids also had an activating role .…”

Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs

“…P-gp is known to be sensitive to both the lipid environment (Orlowski et al, 2006) and the detergent used during the purification process (Bucher et al, 2007). Disruption of the lipid-protein interface has been shown to result in almost complete inactivation of the protein (Callaghan et al, 1997); in fact, a common practice in the purification of P-gp is to add external lipid to maintain this crucial interface (Ambudkar et al, 1998; Taylor et al, 2001).…”

Membrane protein reconstitution for functional and structural studies

“…Based on the fact that the averaged density of proteoliposomes (between 1.03 and 1.05 g cm 3 [44]) is heavier than that of liposomes, a discontinuous gradient (e.g., metrizamide, Nycodenz, and sucrose), is used to separate these two populations (e.g., 100,000200,000×g for 318 h). For example, protein-free liposomes were found in the 3% Nycodenz layer (10% sucrose layer), while proteoliposomes appeared at the boundary between 5% and 15% Nycodenz layers (or 40% and 50% sucrose layers) [8,28].…”