Recovery of human erythrocytes from the echinocyte shape induced by added choline-phospholipids is dependent on the acyl chain length

Addition of an amphiphilic lipid, such as phosphatidylcholine (PC) species with two identical saturated chains or lysophosphatidylcholine (lysoPC) species with one saturated acyl chain of various lengths, into a suspension of intact human erythrocytes resulted in lipid incorporation into the erythrocytes membrane to produce echinocytes (crenated cells). The altered shape gradually reverted on incubation at 37 degrees C until the cells reassumed their normal disc shape. The rate of such recovery of shape increased with decreasing acyl chain length for both PC with C8-C12 acyl chains and lysoPC with a C14-C18 acyl chain, and was strongly influenced by incubation temperature. The identical rate of recovery of shape was observed for cells with normal, decreased or increased ATP content, implying that the metabolic state of the cell had no influence on the recovery process. Recovery of shape is therefore considered to be caused by translocation of the incorporated lipid molecules from the outer to the inner leaflet of the membrane lipid bilayer and the rate of recovery increases with decreasing hydrophobicity of the lipid.     

Shape change of human erythrocytes induced by phosphatidylcholine and lysophosphatidylcholine species with various acyl chain lengths

Intact human erythrocytes were treated, under non-haemolytic conditions at 37 degrees C, with synthetic phosphatidylcholine which has homologous, saturated acyl chains of 8-18 even-numbered carbon atoms (C8-C18-PC) or with lysophosphatidylcholine which has a saturated acyl chain of 8-18 carbon atoms (C8-C18-lysoPC). The C8-C14-PC and C12-C18-lysoPC species were rapidly incorporated into the erythrocytes and induced a shape change of the crenation (echinocyte formation) type. The site of the incorporation was found to be most probably on the outer leaflet of the membrane lipid bilayer. The extent of the shape change was dependent on the amount of each lipid incorporated. When the same amount of a PC or lysoPC species was incorporated into the membrane, about the same extent of crenation was induced, independent of acyl chain length. However, C16-PC, C18-PC, C8-lysoPC and C10-lysoPC, which were not incorporated into the erythrocytes, did not induce any shape change. It is therefore suggested that the hydrophobic moiety of these amphiphilic lipids may greatly contribute to their transfer from the outer medium into the erythrocyte membrane, but do not influence so much the perturbation of the membrane lipid bilayer which may be responsible for induction of the shape change.     

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.     

Effect of fatty acyl chain length of phosphatidylcholine on their transfer from liposomes to erythrocytes and transverse diffusion in the membranes inferred by TEMPO-phosphatidylcholine spin probes

TEMPO-phosphatidylcholine (PC) spin probes which have homologous saturated acyl chains of 10, 12, 14 and 16 carbon atoms, were synthesized as analogues of PC. Transfer of TEMPO-PCs from liposomal membrane to the ghost membrane of human erythrocyte and transverse diffusion of TEMPO-PCs within the membrane of intact erythrocytes were determined by measurement of spontaneous increase and decrease in signal amplitude of an anisotropic triplet spectrum, due to dilution of the label by natural phospholipid of the membrane and reduction of the label by the cytoplasmic content of the erythrocyte, respectively. TEMPO-PC molecules in TEMPO-PC liposomes, except dipalmitoyl TEMPO-PC, were rapidly incorporated into the ghost membrane by incubation at 37 degrees C; the PC having shorter acyl chains was transferred faster. The cytoplasmic content of the erythrocyte rapidly reduced the nitroxide radical of the spin probe. The central peak height of ESR signal was once increased by incorporation of TEMPO-PC into the erythrocyte membrane and then was spontaneously decreased during further incubation at 37 degrees C. This decrease indicates that PC molecules traverse from the outer to the inner layer of the membrane lipid bilayer. The decrease of signal amplitude was faster with PC of shorter acyl chain. These findings suggest that both transfer between membranes and transverse diffusion in the membrane may be favored to the PC species with shorter acyl chains.     

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non-specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6-times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP-depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Quantitative studies on translocation and metabolic conversion of lysophosphatidylcholine incorporated into the membrane of intact human erythrocytes from the medium

The fate of palmitoyl-lysophosphatidylcholine (lysoPC) incorporated into the membrane of intact human erythrocytes from a medium was investigated under nonhemolytic conditions at 37 degrees C by means of 14C-labeled tracers. The lysoPC was first incorporated into the outer half of the membrane lipid bilayer and then gradually translocated into the inner half during the incubation. At the same time it was metabolically converted into phosphatidylcholine (PC) and free fatty acid (FFA) plus glycerophosphorylcholine by the actions of acyltransferase and lysophospholipase, respectively. The half times of the conversion were about 14 h, while the value of 0.5 h was obtained when the half time was measured with the hemolysate of the lysoPC-loaded erythrocytes. Chymotrypsin treatment of unsealed ghosts caused a definite decrease in lysophospholipase activity, while similar treatment of resealed ghosts did not. This together with other evidence already reported in the literature suggests that both lysophospholipase and acyltransferase may be located in the inner surface of the membrane. The above findings strongly suggest that the most of the lysoPC loaded to the membrane is gradually translocated from the outer to the inner half of the bilayer and soon converted to either PC or FFA.     

Flip-flop rates of individual molecular species of phosphatidylcholine in the human red cell membrane

Trace amounts of four different, well-defined species of phosphatidyl[N-methyl-14C]choline ([14C]PC), differing in their fatty acyl constituents, were introduced exclusively into the outer membrane leaflet of the intact erythrocyte by using a PC-specific phospholipid transfer protein. The rate of transbilayer equilibration of these probe molecules was calculated from the time-dependent decay in specific radioactivity of the PC pool in the outer monolayer, which was discriminated from that in the inner leaflet by treating the intact cells with phospholipase A2 in the presence of sphingomyelinase C. At 37 degrees C, 1,2-dipalmitoyl-, 1,2-dioleoyl-, 1-palmitoyl-2-linoleoyl- and 1-palmitoyl-2-arachidonoyl-PC revealed halftime values for the rate of their transbilayer equilibration of 26.3 +/- 4.4, 14.4 +/- 3.5, 2.9 +/- 1.7 and 9.7 +/- 1.6 h, respectively.     

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.     

A model for the extracellular release of PAF: the influence of plasma membrane phospholipid asymmetry.

Recent studies suggesting that cellular activation leads to enhanced transbilayer movement of phospholipids and loss of plasma membrane phospholipid asymmetry lead us to hypothesize that such events may govern the release of PAF, a potent, but variably release, lipid mediator synthesized by numerous inflammatory cells. To model these membrane events, we studied the transbilayer movement of PAF across the human erythrocyte and erythrocyte ghost plasma membrane, membranes with documented phospholipid asymmetry which can be deliberately manipulated. Utilizing albumin to extract outer leaflet PAF, transbilayer movement of PAF was shown to be significantly enhanced in erythrocytes and ghosts altered to lose membrane asymmetry when compared to movement in those with native membrane asymmetry. Verification of membrane changes was demonstrated using merocyanine 540 (MC540), a dye which preferentially stains loosely packed or hydrophobic membranes, and acceleration of the modified Russell's viper venom clotting assay by externalized anionic phospholipids. Utilizing the erythrocyte ghost loaded with PAF in either the outer or the inner leaflet, enhanced transbilayer movement to the opposite leaflet was seen to accompany loss of membrane asymmetry. Studies utilizing ghosts loaded with albumin intracellularly demonstrated that 'acceptor' molecules binding PAF further influence the disposition of PAF across the plasma membrane. Taken together, these findings suggest that the net release of PAF from activated inflammatory cells will depend on localization of PAF to the plasma membrane, transbilayer movement, which is facilitated by alteration of membrane phospholipid asymmetry, and removal from the membrane by extracellular and intracellular 'acceptor' molecules.     

Accelerated transbilayer movement of phosphatidylcholine in sickled erythrocytes. A reversible process

The transbilayer mobility of phosphatidylcholine (PC) molecules in the membrane of homozygous reversible sickle cells (RSCs) was studied using a PC-specific exchange protein from beef liver. In deoxygenated RSCs, all of the PC present in the membrane of the intact cell is rapidly available for exchange, mediated by this protein. Since a substantial amount of the PC is present in the inner membrane leaflet of these cells, this observation implies that the PC molecules in their membranes do experience rapid transbilayer movements. To determine the actual rate of transbilayer movement of the PC, radioactive PC was introduced into the outer monolayer of oxygenated RSCs using the PC-specific exchange protein. Subsequently, the cells were incubated at 37 degrees C under oxy- and deoxygenating conditions to enable the PC to equilibrate within the bilayer. At various time intervals, samples were taken and treated with phospholipase A2, which selectively degrades the PC in the outer monolayer. Analysis of the specific radioactivities of the lyso-PC thus produced, as well as of the residual PC, enabled us to follow the fate of the radioactive PC previously introduced into the outer membrane layer. The half-time value for transbilayer equilibration of the PC in deoxygenated RSCs was determined to be 3.5 h, which is about four times lower than that for oxygenated RSCs. This increased transbilayer mobility of PC, observed in deoxygenated RSCs, is immediately restored to the normal low rate upon reoxygenation of the cells, indicating a complete reversibility of this phenomenon.     

Rapid transbilayer movement of spin-labeled steroids in human erythrocytes and in liposomes

The transbilayer movement and distribution of spin-labeled analogs of the steroids androstane (SLA) and cholestane (SLC) were investigated in the human erythrocyte and in liposomes. Membranes were labeled with SLA or SLC, and the analogs in the outer leaflet were selectively reduced at 4C using 6-O-phenylascorbic acid. As shown previously, 6-O-phenylascorbic acid reduces rapidly nitroxides exposed on the outer leaflet, but its permeation of membranes is comparatively slow and thus does not interfere with the assay. From the reduction kinetics, we infer that transbilayer movement of SLA in erythrocytes is rapid at 4C with a half-time of approximately 4.3 min and that the probe distributes almost symmetrically between both halves of the plasma membrane. We have no indication that a protein-mediated transport is involved in the rapid transbilayer movement of SLA because 1) pretreatment of erythrocytes with N-ethyl maleimide affected neither flip-flop nor transbilayer distribution of SLA and 2) flip-flop of SLA was also rapid in pure lipid membranes. The transbilayer dynamics of SLC in erythrocyte membranes could not be resolved by our assay. Thus, the rate of SLC flip-flop must be on the order of, or even faster than, that of probe reduction rate on the exoplasmic leaflet (half-time approximately 0.5 min). The results are discussed with regard to the transbilayer dynamics of cholesterol.     

Acyl Chain Length and Saturation Modulate Interleaflet Coupling in Asymmetric Bilayers: Effects on Dynamics and Structural Order

A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.     

Rapid transbilayer movement of ceramides in phospholipid vesicles and in human erythrocytes

The transbilayer diffusion of unlabeled ceramides with different acyl chains (C6-Cer, C10-Cer, and C16-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (approximately 0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 degrees C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after approximately 15 min of incubation at 20 degrees C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.     

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.     

The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains.     

Topological Biosynthesis of Phosphatidylcholine in Brain Microsomes

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.     

31P- and 1H-NMR investigations of the effect of n-alcohols on the hydrolysis by phospholipase A2 of phospholipid vesicular membranes

31P- and 1H-NMR spectroscopy of small, unilamellar egg yolk phosphatidylcholine (PC) vesicles in the presence of the lanthanide ion Dy3+ have been used to study the effect of various n-alcohols on the permeability induced by the action of the enzyme phospholipase A2 (PLA2). The method allows the monitoring of the number of PC and lysoPC molecules in the outer and inner monolayers. The results indicate that the initial rate of hydrolysis of PC by PLA2 is increased by all the n-alcohols but in a chain-length dependent manner and that the maximum rate occurs at n = 8 (octan-1-ol). The subsequent rate is dependent upon the rate of transbilayer lipid exchange (flip-flop) of PC molecules from the inner to the outer monolayer. The vesicles only become permeable to the Dy3+ ions when lysoPC is mobilised in the flip-flop process of exchange of lipid molecules between the two monolayers. The n-alcohols affect both the time taken to initiate flip-flop of inner monolayer PC and the subsequent rate of permeability to Dy3+. The n-alcohols are seen to affect all the above rates in an identical chain-length dependent manner, indicating a common cause for all observations which we identify as the degree of clustering of the n-alcohol molecules in the bilayer. The results are discussed in terms of the chain-length dependent mechanism of n-alcohol interactions with the membrane and the mechanism by which the vesicles become permeable to Dy3+ ions.     

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach.

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.     

Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter

The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degrees C was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.