Inhibition and stimulation of phospholipid scrambling activity. Consequences for lipid asymmetry, echinocytosis, and microvesiculation of erythrocytes.

An increase of the intracellular Ca(2+) concentration in erythrocytes is known to activate rapid nonspecific bidirectional translocation of membrane-inserted phospholipid probes and to decrease the asymmetric distribution of endogenous membrane phospholipids. These scrambling effects are now shown to be suppressed by pretreatment of cells with the essentially impermeable reagents 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 2,4,6-trinitrobenzenesulfonate. The inhibitory effects are no longer observed during renewed activation of scrambling following a first transient activation by Ca(2+). Assuming the involvement of the human scramblase, this suggests a conformational alteration of this protein during activation by Ca(2+). Marked suppression of scrambling activity is also observed in cells pretreated with the disulfide reducing agent dithioerythritol which can be reverted by the SH oxidizing agent diamide. This indicates the importance of intramolecular and/or intersubunit disulfide bonds for the function of the scramblase. On the other hand, treatment of cells with the SH reagents N-ethylmaleimide and phenylarsine oxide enhances Ca(2+)-activated scrambling and diminution of asymmetry of membrane phospholipids. This suggests an allosteric connection of several protein SH groups to the translocation mechanism. The inhibitors retain their strong suppressive effects. Besides covalent modification, addition of oligomycin highly stimulates and addition of clotrimazole suppresses the Ca(2+)-activated translocation. No evidence for a role of the ATP-binding cassette transporter ABCA1 in the Ca(2+)-activated outward translocation is obtained. Suppression of phospholipid scrambling by dithioerythritol inhibits Ca(2+)-induced spheroechinocytosis and reduces the extent of subsequent microvesiculation. Scrambling of endogenous phospholipids is proposed to induce echinocytosis and to have only a stimulatory effect on microvesiculation.

Complex effects of papain on function and inhibitor sensitivity of the red cell anion exchanger AE1 suggest the presence of different transport subsites.

Band 3 (AE1), the anion exchanger of the human erythrocyte membrane, mediates not only fluxes of small hydrophilic anions (e.g., chloride, oxalate), but also the flip-flop of long-chain amphiphilic anions (e.g., dodecylsulfate). Treatment of erythrocytes with papain, long known to inhibit the transport of the former type of anions, accelerates the transport of the latter type. In an attempt to elucidate the basis of these opposite responses to papain, several small amphiphilic arylalkyl sulfonates and -sulfates were tested for the response of their transport, via AE1, to papain. Although all these probes are most likely transported by a flux and not by flip-flop, their transport was inhibited by papain only in some cases, but accelerated in others. Different responses to papain therefore most likely do not reflect differences between transport by flux or by flip. The transports of different species of anions also differed considerably in the changes of their sensitivity, to noncovalent and some covalent inhibitors, brought about by papain treatment. While oxalate transport remained as sensitive as in native cells, transports of small amphiphilic anions lost their sensitivity to a major extent, regardless of the inhibition or acceleration of their transport by papain. The results are discussed in the light of present concepts of the structural organisation of AE1, and interpreted in terms of a model of different transport subsites for different species of anions in this transporter.

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length.

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.

Extensive electroporation abolishes experimentally induced shape transformations of erythrocytes: a consequence of phospholipid symmetrization?

As shown in earlier work (M.M. Henszen et al., Mol. Membr. Biol. 14 (1997) 195-204), exposure of erythrocytes to single brief electric field pulses (5-7 kV cm(-1)) enhances the transbilayer mobility of phospholipids and produces echinocytes which can subsequently be transformed into stomatocytes in an ATP-dependent process. These shape transformations arise from partly reversible changes of the transbilayer disposition of phospholipids, in agreement with the bilayer couple concept. Extensive membrane modification by repetitive (

Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells.

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degree C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipids.

Band 3-mediated flip-flop and phosphatase-catalyzed cleavage of a long-chain alkyl phosphate anion in the human erythrocyte membrane.

In pursuit of the characterization of the recently discovered flippase mode of operation of the anion transporter (band 3, AE1) of the human erythrocyte membrane, the transbilayer translocation (flip) of a fluorescently labeled, membrane-intercalated long-chain alkyl phosphate, 10-(alpha-napthyl)-1-decyl-phosphate (NDP) was investigated. In contrast to the alkyl sulfonates and esters of phosphatidic acid studied as yet, NDP moves exclusively via band 3. NDP is, however, dephosphorylated at the inner membrane surface by a cytoplasmic phosphatase likely to interact specifically with endofacial membrane structures of the erythrocyte. This phosphatase shares characteristic inhibitor sensitivities with protein tyrosine phosphatases present in the erythrocyte interior. Vanadate as an inhibitor of NDP dephosphorylation provided a means to study the kinetic properties and patterns of inhibition (by inhibitors of anion exchange) and stimulation (by proteolysis of band 3 and aliphatic alcohols) of the flip of NDP. NDP is also an inhibitor of the exchange of hydrophilic anions via band 3, while hydrophilic anions interfere with the flip of NDP. The results are compared with the characteristics of the flip, via Band 3, of other amphiphilic anions and of the exchange of hydrophilic anions. Attempts are presented to understand the low flip rate of long-chain amphiphilic anions on the basis of their molecular properties and the thermodynamics of the "transition state" of the flip process.

Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane.

Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1, 3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner membrane leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner membrane leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.

Nonmediated flip-flop of anionic phospholipids and long-chain amphiphiles in the erythrocyte membrane depends on membrane potential.

The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer membrane leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier.

Transbilayer reorientation of phospholipid probes in the human erythrocyte membrane. Lessons from studies on electroporated and resealed cells.

In order to characterize in more detail the previously observed (Dressler et al. (1983) Biochim. Biophys. Acta 732, 304-307) increases in transbilayer mobility of phospholipids in the erythrocyte membrane following electroporation at 0 degrees C and subsequent resealing at 37 degrees C of the cells, we have studied rates of flip and flop as well as steady state distributions of the fluorescent N-(NBD)-aminohexanoyl-analogues of the four major membrane phospholipids. Measurements comprised the passive non-mediated components as well as those mediated by specific translocases (flippase and floppase). The major new findings and insights can be summarized as follows. (1) The enhancement of passive transbilayer mobility which increases with the strength, duration, and number of field pulses at 0 degrees C, cannot be fully reversed by subsequent resealing at 37 degrees C. Flip-flop remains considerably elevated relative to the original values.(2) Enhanced mobilities induced by electroporation differ for the probes studied in the sequence SM <<< PS << PC < PE. Other membrane perturbations going along with enhanced flip-flop share only in part this pattern. (3) Mediated, ATP-dependent components of flip and flop of the probes are suppressed in electroporated/resealed cells, partly due to loss of cellular Mg2+, partly - in case of flippase - due to competition by externalized endogenous PS. (4) Electroporated/resealed cells provide an elegant means to demonstrate the contribution of various components of flip and flop to the steady state transbilayer distribution of phospholipids, in particular the role of passive mobility. The new, detailed information on the displacements of phospholipid between the two leaflets of the membrane bilayer in porated/resealed cells will help to understand erythrocyte shape changes following poration and during resealing (Henszen et al. (1993) Biol. Chem. Hoppe-Seyler 374, 114).

Electric field pulses induce reversible shape transformation of human erythrocytes.

Electric field pulses > 2-3 kV cm-1, long known to induce membrane poration and fusion of erythrocytes as well as to enhance the transbilayer mobility of phospholipids and to perturb aminophospholipid asymmetry, are shown to induce, at 0 degree C, transformation of the discocytic cells into echinocytes and spheroechinocytes. The extent of transformation increases with strength, duration and number of pulses. Its time course is biphasic, a major rapid phase (t/2 approximately 5 s) being followed by a minor one, lasting for 2-3 h. Shape transformation goes along with the exofacial exposure of phosphatidylserine (PS), detected by FITC-annexin V binding and quantified by a calibration curve established via externally inserted dilauroylphosphatidylserine. Incubation of these echinocytes at 37 degrees C leads to a rapid recovery of the discocytic shape followed by slower formation of stomatocytes. Shape recovery is temperature dependent (Ea approximately 100 kJ/mol), and can be impaired by depletion of ATP or Mg++ and by addition of vanadate or fluoride. Shape recovery and stomatocyte formation go along with a rapid loss of annexin binding in about 45% of the cells while the rest maintains its binding capacity. In the presence of vanadate, annexin binding increases in all cells. The results are discussed in the light of the bilayer couple concept of erythrocyte shape and the enhanced transverse mobility of phospholipids. Echinocyte formation is most likely caused by the reorientation of endofacial aminophospholipids to the outer leaflet of the bilayer. Shape recovery and stomatocyte formation probably result from a continuous reinternalization of PS via the ATP dependent aminophospholipid translocase, but may also be supported by downhill movement of PC to the inner leaflet and by other yet unidentified processes.

Pathways for flip-flop of mono- and di-anionic phospholipids in the erythrocyte membrane.

The inward translocations (flip), from the outer to the inner membrane leaflet of human erythrocytes, of di-anionic NBD-labeled phospholipids containing as a head group phosphate esters of glycolate, butyrate and hydroxyethanesulfonate are slow processes (k = 0.005-0.008 h-1, 37 degrees C) at pH 7.4. A decrease of pH highly stimulates the flip. A major role of the anion exchanger (AE1), band 3, in this flip is indicated by (a) the strong inhibition of the flip (55-85%) by stilbene disulfonates and other inhibitors of anion transport, (b) the stimulation and loss of pH dependence of the flip after modification of band 3 by Woodward's reagent K and NaBH4, and (c) the stimulation of the flip after proteolytic cleavage of band 3 by papain. The flip of mono-anionic NBD-phospholipids with phosphate esters of glycerol, glycol, methanol, butanol and benzyl alcohol is much faster than that of their dianionic analogs (k = 0.04 to > 3.0 h-1, 37 degrees C). It is inhibited by stilbene disulfonates to a decreasing extent (35 to 0%) and is not affected by several reversible inhibitors of anion exchange. This indicates a minor component of band-3-mediated flip and a major component of nonmediated flip. The outward translocations (flop), from the inner to outer membrane leaflet, of both mono- and di-anionic phospholipids are very fast (1.0-5.9 h-1), ATP-dependent and inhibitable by vanadate, fluoride, SH-reagents or Mg(2+)-depletion of cells and thereby likely to be largely mediated by a 'floppase'. The stationary distributions of the NBD-labeled anionic phospholipids are asymmetric to an extent (outer to inner leaflet ratio 2-9) correlating with the ratio of the rates of the outward and the inward translocation. Thus, asymmetry is largely abolished by blockage of the floppase-mediated translocation.

Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes. A comparison with the binding to leukemia cells.

In the presence of albumin Merocyanine 540 (MC540) exhibits a very limited binding to the outer surface of the membrane of normal erythrocytes, whereas pronounced binding is observed to leukemia cells. To find out whether this difference is due to differences in the composition or structural organization of the cell membrane we analyzed effects of a number of covalent and non-covalent perturbations of the red cell membrane on the binding and fluorescence characteristics of membrane-bound MC540. It is shown that exposure of the cells to cationic chlorpromazine, neuraminidase or photodynamic treatment with AlPcS4 as sensitizer caused a limited increase (30-50%) of MC540 binding, together with a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield of membrane-bound MC540. Other forms of perturbation of the membrane structure, like hyperthermia (48 degrees C) and treatments that produce a decrease of phospholipid asymmetry in addition to accelerated flip-flop, did not result in increased MC540 binding, but did cause a red shift of the fluorescence emission maximum and an increase of the relative fluorescence quantum yield. These changes in fluorescence properties indicate a penetration of the dye into more hydrophobic regions in the membrane. MC540, bound to Brown Norway myelocytic leukemia cells, exhibited a red shift of the fluorescence emission maximum and an increased relative fluorescence quantum yield as compared to MC540 bound to untreated erythrocytes. These changes were of the same order of magnitude as in photodynamically treated red blood cells. Dye binding per surface area, however, was about 3-times higher with these leukemia cells than with photodynamically treated red blood cells. This demonstrates that certain perturbations of the erythrocyte membrane evoked a MC540 binding that became qualitatively comparable to the dye binding to leukemia cells, although dye binding per surface area was still significantly lower.

Band 3, an accidental flippase for anionic phospholipids?

The inward translocation of the monovalent anionic phospholipid 1-palmitoyl-sn-glycero-3-phosphomethanol in the membrane of human erythrocytes is a fast process (t/2 = 11 min, 37 degrees C). Translocation of the protonated uncharged phospholipid is not responsible for the fast flip rate, and mediation of translocation by the aminophospholipid flippase could be excluded. Involvement of the anion exchanger band 3 in this process was derived from its inhibition (40-70%) by several established inhibitors of band 3-mediated anion exchange and its acceleration after proteolysis of band 3 by external papain. The translocation of the dianionic NBD-labeled phosphatidic acid is 5-fold slower, but also affected by the inhibitors. Thus, the anion exchanger can act as a flippase, defined as a transporter accepting substrates from the lipid bilayer.

Alcohols produce reversible and irreversible acceleration of phospholipid flip-flop in the human erythrocyte membrane.

The slow, non-mediated transmembrane movement of the lipid probes lysophosphatidylcholine, NBD-phosphatidylcholine and NBD-phosphatidylserine in human erythrocytes becomes highly enhanced in the presence of 1-alkanols (C2-C8) and 1,2-alkane diols (C4-C8). Above a threshold concentration characteristic for each alcohol, flip rates increase exponentially with the alcohol concentration. The equieffective concentrations of the alcohols decrease about 3-fold per methylene added. All 1-alkanols studied are equieffective at comparable calculated membrane concentrations. This is also observed or the 1,2-alkane diols, albeit at a 5-fold lower membrane concentration. At low alcohol concentrations, flip enhancement is reversible to a major extent upon removal of the alcohol. In contrast, a residual irreversible flip acceleration is observed following removal of the alcohol after a treatment at higher concentrations. The threshold concentrations to produce irreversible flip acceleration by 1-alkanols and 1,2-alkane diols are 1.5- and 3-fold higher than those for flip acceleration in the presence of the corresponding alcohols. A causal role in reversible flip-acceleration of a global increase of membrane fluidity or membrane polarity seems to be unlikely. Alcohols may act by increasing the probability of formation of transient structural defects in the hydrophobic barrier that already occur in the native membrane. Membrane defects responsible for irreversible flip-acceleration may result from alterations of membrane skeletal proteins by alcohols.

Fast translocation of phosphatidylcholine to the outer membrane leaflet after its synthesis at the inner membrane surface in human erythrocytes.

The translocation rate of [14C]phosphatidylcholine to the outer membrane leaflet of human erythrocytes after its primary synthesis from lysophosphatidylcholine by acylation with 14C-labeled oleic or palmitic acid in the inner leaflet has been measured by following the time-dependent increase of cleavability of 14C-labeled phospholipids by external phospholipase A2 (5 min, 37 degrees C). Immediately after a short acylation time period of 10 min about 20% of the newly synthesized [14C]phosphatidylcholine are already detectable in the outer leaflet. After an incubation of 1 h at 37 degrees C following 10 min of acylation the fractions of labeled and native phosphatidylcholine accessible to the lipase are identical, which demonstrates that [14C]phosphatidylcholine has attained the same asymmetric distribution as its endogenous analogue. The calculated halftime of the outward translocation is about 20 min and its activation energy is low, 30 kJ/mol. Translocation is inhibited by a 5 min treatment with phenylglyoxal following acylation. A fast translocation is not observed for newly synthesized phosphatidylethanolamine. Results suggest a selective, protein-mediated outward translocation of newly synthesized phosphatidylcholine.

Echinocytosis and microvesiculation of human erythrocytes induced by insertion of merocyanine 540 into the outer membrane leaflet.

Echinocytosis and release of microvesicles from human erythrocytes treated with the impermeant fluorescent dye merocyanine 540 (MC540) has been correlated with the extent of dye binding to intact cells and ghosts. At 20 degrees C binding appeared to saturate at about 9.3.10(6) molecules per cell (3.6 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 2.7%. Stage 3 echinocytes were formed upon binding of (3-4).10(6) molecules of MC540/cell (about 1.3 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 1.0%. Negligible release of microvesicles was observed with MC540 at 20 degrees C. Binding of MC540 to permeable ghosts was approximately twice that to cells suggesting that there was no selective binding to the unsaturated (more fluid) phospholipids which are concentrated in the inner lipid leaflet of the membrane. At 37 degrees C apparent maximal binding of MC540 was about 3.2 mol/100 mol phospholipid and correlated with the maximal release of microvesicles from the cells as measured by release of phospholipid and acetylcholinesterase. These results are discussed in relation to the bilayer couple hypothesis of Sheetz and Singer (Proc. Natl. Acad. Sci. USA 71 (1974) 4457-4461).

Translocation of oleic acid across the erythrocyte membrane. Evidence for a fast process.

To clarify divergent views concerning the mechanism of fatty acid translocation across biomembranes this issue was now investigated in human erythrocytes. Translocation rates of exogenously inserted radioactive oleic acid across the membrane of native cells were derived from the time-dependent increase of the fraction of radioactivity becoming non-extractable by albumin. No accumulation of non-extractable unesterified oleic acid occurred. The rate of transfer was markedly suppressed by SH-reagents and by ATP-depletion. The suppression, however, resulted from a mere decrease of incorporation of oleic acid into phospholipids and was not accompanied by an increase of non-extractable unesterified oleic acid. These findings were reconcilable with the concept of a slow, possibly carrier-mediated fatty acid transfer as well as a very fast presumably, diffusional process not resolvable by the albumin extraction procedure. This ambiguity was resolved by using resealed ghosts, which are unable to incorporate oleic acid into phospholipids. In such ghosts all of the oleic acid inserted into the membrane remains extractable by albumin even after prolonged incubation. On the other hand, ghosts containing albumin accumulated non-extractable oleic acid. The rate of accumulation was beyond the time resolution of the albumin extraction procedure at 4 degrees C. Oleic acid uptake into albumin-containing ghosts became kinetically resolvable when the fatty acid was added as a complex with albumin. Correspondingly, time-resolvable release of oleic acid, originally complexed to internal albumin, into an albumin-containing medium was demonstrated at 4 degrees C. Rate and extent of these redistributions of oleic acid were dependent on the concentrations of internal and external albumin. This indicates limitation by the dissociation of oleic acid from albumin and not its translocation across the membrane. Translocation of oleic acid, which is probably a simple diffusive flip-flop process, must therefore occur with a half-time of less than 15 s. These findings raise doubts on the physiological role of presently discussed concepts of a carrier-mediated translocation of fatty acids across plasma membranes.

Nonmediated flip-flop of phospholipid analogues in the erythrocyte membrane as probed by palmitoylcarnitine: basic properties and influence of membrane modification.

The rules governing the transbilayer reorientation (flip-flop) of long-chain amphiphilic components in biological membranes were further elucidated by studying the flip-flop of palmitoylcarnitine in human erythrocytes. Flip rates were derived from the time-dependent decrease of extractability of palmitoylcarnitine by albumin after primary insertion of trace amounts of the labeled probe into the outer membrane layer. The flip rate (half time 2.6 hr at 37 degrees C in human erythrocytes) is fast enough to be measurable also in membranes exhibiting low flip rates such as that of ox erythrocytes. Flip rate constants for the inward and outward reorientation are similar and the probe equilibrates at a 1:1 ratio between the two layers. The flip is a simple, diffusion-like process. It is not inhibited but even enhanced by chemical modification of membrane proteins. It is also enhanced by insertion of channel-forming antibiotics into the membrane and by pre-exposure of the cells to temperatures exceeding 42 degrees C. The extent of this enhancement increases with the duration and the temperature of the pre-exposure. Since spectrin is denatured in this range of temperatures, the finding constitutes a new piece of evidence that the membrane skeleton is involved in the maintenance of bilayer stability and that a decrease of bilayer stability goes along with the formation of local defects acting as flip sites for phospholipids and related compounds. As a particularity, the flip is enhanced by lowering the pH and exhibits interindividual variability, phenomena not observed for the flip-flop of lysophosphatidylcholine. This suggests that generalizations on the kinetics of nonmediated flip-flop of membrane-intercalated amphiphiles may not be justified.