Interaction of P-Glycoprotein with Defined Phospholipid Bilayers:  A Differential Scanning Calorimetric Study

Abstract

One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as a drug efflux pump. P-Glycoprotein is a member of the ABC superfamily of membrane proteins, and is composed of 12 hydrophobic membrane-spanning segments and 2 cytoplasmic nucleotide binding domains. Membrane lipids are known to play an important role in the function of P-glycoprotein. In the present study, purified P-glycoprotein of high specific ATPase activity was reconstituted into defined bilayers of dimyristoylphosphatidylcholine (DMPC), and its effects on lipid thermodynamic properties were then investigated using differential scanning calorimetry. P-Glycoprotein had a large perturbing effect on DMPC bilayers, even at relatively high lipid:protein ratios. The gel to liquid-crystalline phase transition temperature, Tm, was lowered on inclusion of P-glycoprotein in the bilayer, and the cooperativity of the transition was markedly reduced. The phase transition enthalpy, DeltaH, declined in a linear fashion with increasing P-glycoprotein content for lipid:protein ratios between 63:1 and 16:1 (w/w). Evaluation of these data using two different analytical methods indicated that P-glycoprotein perturbed either 375 or 485 phospholipids, withdrawing them from the phase transition. The DeltaH value for those lipids undergoing melting was similar to that of pure DMPC, which implies that their thermodynamic properties are essentially unchanged in the presence of P-glycoprotein. At lipid:protein ratios below 16:1 (w/w), transition enthalpy increased with higher P-glycoprotein content, until the DeltaH value reached that of pure DMPC. However, the lipid remained highly perturbed, as indicated by a very broad phase transition peak. This behavior may arise from either aggregation/oligomerization of P-glycoprotein within the bilayer or changes in the interaction of the transporter with the membrane at high density.

The ATPase and ATP‐binding functions of P‐glycoprotein

“…In the case of Pgp, it appears that a rate-limiting step is, in fact, enhanced when the host lipid is in the gel phase. Along the same lines, we recently reported [35] that Pgp was reconstituted into Myr 2GroPCho with higher efficiency and specific ATPase activity at temperatures below t m , suggesting that the protein may have a preference for gel-phase lipid. Clearly, the nature of the chemical barrier for catalysis by Pgp is affected by the lipid-phase state quite differently than for other membrane-bound enzymes.…”

“…SDS/PAGE was carried out as described previously [22]. Differential scanning calorimetry (DSC) measurements were used to determine the gel/ liquid-crystalline phase transition temperature, t m , of liposomes of PamMyrGroPCho and Myr 2 GroPCho alone, and proteoliposomes of PamMyrGroPCho and Myr 2 GroPCho containing 13:1 (by mass) and 10:1 (by mass) Pgp, respectively, as described previously [35].…”

“…Pgp purified to Ͼ 90% purity was fluorescently labeled with 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) as described previously [30]. MIANS-labeled Pgp was then reconstituted into PamMyrGroPCho or Myr 2 GroPCho proteoliposomes as outlined above [35], except that dithioerythritol was omitted from the lipid-solubilization buffer and the Sephadex G-50 column buffer. The proteoliposomes, which had a final lipid/protein ratio of Ϸ10:1 (by mass) were resuspended in 50 mM Tris/HCl, 0.15 M NH 4 Cl, 5 mM MgCl 2 , 0.02% (mass/ vol.)…”

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

“…Detergents and additional adjuvants from the purification and reconstitution procedures can affect the behavior of the transporter, as we have shown for CHAPS. The most frequently used detergents for the purification of P-gp beside CHAPS (26,28,29,32,39,42) are n-octyl b-D-glucopyranoside (octyl glucoside) (27,30,(49)(50)(51)(52)(53) and n-dodecyl or n-decyl b-Dmaltoside (14,22,25,33,51,(54)(55)(56). Octyl glucoside had activating as well as inhibiting effects on the ATPase activity of CHAPS-solubilized P-gp, depending on the glucoside concentration (39).…”

Transition State Analysis of the Coupling of Drug Transport to ATP Hydrolysis by P-glycoprotein

“…The linearity of the plots indicated that single overall rate-limiting steps were being observed. In contrast, some plasma membrane preparations and Pgp reconstituted in synthetic lipid mixtures did not produce linear Arrhenius relationships due to lipid phase transitions and other effects (43)(44)(45). Arrhenius relationships were measured for the drugs listed above as a function of concentration such that all phases of drug-dependent ATPase activities were investigated.…”