Real-time monitoring of membrane-protein reconstitution by isothermal titration calorimetry.

Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K(+) channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-ß-d-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

Lipid composition determines interaction of liposome membranes with Pluronic L61.

Triblock copolymers of ethylene oxide (EO) and propylene oxide (PO) of EO(n/2)PO(m)EO(n/2) type (Pluronics) demonstrate a variety of biological effects that are mainly due to their interaction with cell membranes. Previously, we have shown that Pluronics can bind to artificial lipid membranes and enhance accumulation of the anti-tumor drug doxorubicin (DOX) inside the pH-gradient liposomes and transmembrane migration (flip-flop) of NBD-labeled phosphatidylethanolamine in the liposomes composed from one component-lecithin. Here, we describe the effects caused by insertion of other natural lipids in lecithin liposomes and the significance of the lipid composition for interaction of Pluronic L61 with the membrane. We used binary liposomes consisting of lecithin and one of the following lipids: cholesterol, phosphatidylethanolamine, ganglioside GM1, sphingomyelin, cardiolipin or phosphatidic acid. The influence of the additives on (1) membrane microviscosity; (2) binding of Pluronic L61; (3) the copolymer effect on lipid flip-flop and membrane permeability towards DOX was studied. The results showed that insertion of sphingomyelin and cardiolipin did not influence membrane microviscosity and effects of Pluronic on the membrane permeability. Addition of phosphatidic acid led to a decrease in microviscosity of the bilayer and provoked its destabilization by the copolymer. On the contrary, cholesterol increased microviscosity of the membrane and decreased binding of Pluronic and its capacity to enhance flip-flop and DOX accumulation. Analogous tendencies were revealed upon incorporation of egg phosphatidylethanolamine or bovine brain ganglioside GM1. Thus, a reverse dependence between the microviscosity of membranes and their sensitivity to Pluronic effects was demonstrated. The described data may be relevant to mechanisms of Pluronic L61 interaction with normal and tumor cells.

Membrane solubilisation and reconstitution by octylglucoside: comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry.

We have studied the solubilisation and reconstitution of lipid membranes composed of either synthetic phosphatidylcholine or Escherichia. coli polar lipid extract by the non-ionic detergent octylglucoside. For both lipid systems, composition-dependent transformations of unilamellar vesicles into micelles or vice versa were followed by high-sensitivity isothermal titration calorimetry. Data obtained over a range of detergent and lipid concentrations could be rationalised in terms of a three-stage phase separation model involving bilayer, bilayer/micelle coexistence, and micellar ranges, yielding the detergent/lipid phase diagrams and the bilayer-to-micelle partition coefficients of both detergent and lipid. The most notable difference between the lipids investigated was a substantial widening of the bilayer/micelle coexistence range for E. coli lipid, which was due to an increased preference of the detergent and a decreased affinity of the lipid for the micellar phase as compared with the bilayer phase. These effects on the bilayer-to-micelle partition coefficients could be explained by the high proportion in E. coli membranes of lipids possessing negative spontaneous curvature, which hampers both their transfer into strongly curved micellar structures as well as the insertion of detergent into condensed bilayers.

Influence of amphiphilic block copolymer induced changes in membrane ion conductance on the reversal of multidrug resistance.

Block copolymers are able to reverse multidrug resistance (MDR) of tumor cells by a yet unknown mechanism. The drug efflux system's direct and indirect inhibition mediated by polymer P-glycoprotein (Pgp) interactions or adenosine triphosphate (ATP) depletion, respectively, may be involved in MDR reversal as well as damage to the membrane barrier caused by polymer insertion into the membrane. To test the latter hypothesis, cellular drug accumulation was monitored in the presence of both overexpressed fluorescently labeled Pgp and different block copolymers. Therefore, a new triblock copolymer (poly(ethylene oxide)- block-poly(hexafluoropropylene oxide)- block-poly(ethylene oxide)) was designed and synthesized by combined polymerization and polymer analogous reaction. Its administration induced drug uptake, whereas control cells with high Pgp expression levels showed no drug accumulation. Drug uptake was even more pronounced in the presence of another triblock copolymer: (poly(perfluorohexylethyl methacrylate)- block-poly(ethylene oxide)- block-poly(perfluorohexylethyl methacrylate). The latter polymer's lack of ionophoric activity suggests that ion transport facilitation by polymers is not a determinative factor for MDR reversal.

Ionophoric activity of pluronic block copolymers.

Pluronic block copolymers (triblock copolymers of poly(ethylene oxide) and poly(propylene oxide)) exhibit a chemosensitizing effect on multidrug resistant cell lines. Changes in membrane permeability are hypothesized to be responsible because inhibition of drug transport mediated by both the multidrug-resistance-associated protein and the P-glycoprotein drug efflux system has been observed. To test this hypothesis, we now have studied the ion conductivity mediated by Pluronic L61. Besides a detergent-like action, the copolymer was able to form regular channels and to exhibit carrier activity. Long living ion channels were formed by polymer oligomerization. Aggregate equilibrium was shifted toward L61 monomers and dimers, which operated as mobile carriers. Copolymer-induced membrane permeability for potassium ions (1 M KCl) was less than 10(-8) cm s(-1), whereas the permeability for uncharged doxorubicin molecules (1 mM) was equal to 5 x 10(-4) cm s(-1). The results are consistent with reports about an increased doxorubicin accumulation in cells (Venne, Li, S., Mandeville, R., Kabanov, A., and Alakhov, V. Y. (1996) Cancer Res. 56, 3626-3629). However, the increased permeability contrasts with the polymer-mediated decrease of drug efflux from cells. Preferential polymer binding to membrane proteins may mask the unspecific effect of L61 observed on lipid bilayers.

Membrane destabilization by ricin.

Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.

Pluronic L61 accelerates flip-flop and transbilayer doxorubicin permeation.

It has recently been found that Pluronics (block copolymers of ethylene oxide, EO, and propylene oxide, PO) favor the permeability and accumulation of anthracycline antibiotics, for example doxorubicin (Dox), in tumor cells. In an effort to understand these results, the interaction of EO(2)/PO(32)/EO(2) (Pluronic L61) with unilamellar egg yolk vesicles (80-100 nm in diameter) was examined. A partition coefficient K(p)=[Pl](membrane)/[Pl](water)=45 was determined. This corresponds to adsorption of about 20 polymer molecules to the surface of each vesicle in a 20 microM polymer solution. Despite this rather weak adsorption, Pluronic has a substantial effect upon the transmembrane permeation rate of Dox and upon the phospholipid flip-flop rate within the bilayers. Thus, the Dox permeation rate increases threefold and the flip-flop rate increases sixfold in 20 microM Pluronic. The two rates increase linearly with the amount of adsorbed polymer. The obvious ability of Pluronics to increase the mobility of membrane components may have important biomedical consequences.