Cytofacial Leaflet Research Articles

Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a β-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a β-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.

Substrate Selectivity of a P4‐ATPase Flippase

Phospholipids, the primary structural components of cell membranes, are distributed unequally across the membrane. The sphingolipids, including sphingomyelin (SM) and glycosphingolipids, are enriched on the exofacial surface of the plasma membrane or the luminal surfaces of internal organelles, while the cytofacial leaflet is enriched in the aminophospholipids, phophatidylserine (PS) and phosphatidylethanolamine (PE). Contributing to maintenance of this asymmetry is a combination of slow passive transbilayer phospholipid movement, trapping by cytofacial proteins and the activity of a number of energy‐dependent transporters. The substrate specificity and directionality of these pumps defines the transbilayer asymmetry of phospholipids. Two transporter superfamilies, ABC transporters and P‐type ATPases, account for these activities and move lipids in either the exofacial (floppase) or cytofacial (flippase) directions, respectively. In previous studies we have characterized the biochemical requirements and substrate specificity for a PS specific flippase activity in human red blood cells. Transport is ATP‐dependent and sensitive to vanadate and sulfhydryl‐modification. This flippase is selective for the sn‐1,2‐isomer of PS. Transport is diminished by modification of the PS headgroup amine (with the exception of mono‐methylation), carboxyl and phosphate groups, while a variety of fatty acyl groups and the D‐serine analog are accommodated. Red blood cells express two members of the P4‐type ATPase subfamily, ATP8A1 and ATP11C, that are likely responsible for this activity. Here, we report on the heterologous expression, purification, and functional assaying of hexahistidine‐tagged constructs of Atp8a1 with and without its cognate accessory subunit, Cdc50a. Purified Atp8a1 is vanadate‐sensitive and dependent upon Cdc50a co‐expression and PS for robust ATPase activity, although lipid specificity is not dependent on the presence of Cdc50a. In addition to reconstituting the flippase activity of Atp8a1 and ATP11c in vitro, our efforts are focused on rational mutagenesis of Atp8a1 to identify the lipid transport path and the determinants of substrate specificity, directed by homology modeling and the lipid‐binding characteristics of the closest members of the P‐type superfamily. X‐ray crystal structures of the SERCA pump in conformation E2 (PDB 2AGV) display a PE glycerol backbone and head group tightly pinched by helices M2 and M4 with PE's acyl chains extending between helices M2 and M6. This PE‐binding site implies a mechanism for how the P4lipid‐binding site and transport pathway evolved from ancestor P‐types designed to move simple cations. With these clues, we aim to identify the PS binding site and deduce the origin of the sn‐1,2‐stereospecificity of Atp8a1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Does Membrane Asymmetry Affect Nanoparticle-Membrane Interactions

Plasma membrane damage is one of the primary mechanisms through which engineered nanomaterials cause cytotoxicity. Despite a number of studies, the role of the structural features of the plasma membrane, in particular membrane phospholipid asymmetry in regulating nanoparticle-membrane interactions has remained obscure. In the current study, the role of membrane phospholipid asymmetry, differences in the lipid composition of exofacial and cytofacial leaflet of the plasma membrane in regulating nanoparticle-membrane interactions was investigated. Vesicles mimicking the exofacial (Vexo) and cytofacial leaflets (Vcyto) of the plasma membrane of erythrocytes were exposed to silica nanoparticles (50 nm), with different surface functional groups, plain, or carboxyl-, amine-, and polyethylene glycol (PEG)-modified and the effect of particles on vesicle integrity was examined. Symmetric and asymmetric vesicles with similar outer leaflet, but different inner leaflet lipid compositions, as well as live erythrocytes, were also used to elucidate the role of membrane asymmetry and individual leaflets in regulating nanoparticle-membrane interaction. Vexo and Vcyto vesicles showed significant difference in their interactions with nanoparticles. Vexo vesicles showed drastic leakage of intravesicular content after exposure to plain and amine-modified particles while none of the particles caused significant leakage in Vcyto vesicles. FRET experiments revealed that nanoparticle-induced disruption by plain and amine-modified particles occurs via pore formation. In agreement, FRET assay and GUV images revealed significant localization of plain and amine-modified particles at the surface of the Vexo vesicles. However, no localization was observed with the Vcyto vesicles. Interestingly, symmetric and asymmetric vesicles were disrupted similarly by plain and amine-modified particles. Importantly, hemolysis was observed after the incubation of erythrocytes with plain and amine-modified particles, consistent with leakage assays using Vexo vesicles. In conclusion, these results suggest that the exofacial leaflet of the plasma membrane is the primary regulator of nanoparticle-induced membrane damage in erythrocytes.

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.

Cholesterol asymmetry in synaptic plasma membranes

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function.

Cholesterol Distribution, Not Total Levels, Correlate With Altered Amyloid Precursor Protein Processing in Statin-Treated Mice

There are now a number of studies that suggest that cholesterol might regulate the processing of the amyloid precursor protein to form the neurotoxic peptide Abeta. This research has opened the possibility that cholesterol-lowering drugs might be efficacious as anti-Abeta drugs for use in Alzheimer's disease. The use of HMG-CoA reductase inhibitors (commonly called statins) in vitro and in vivo has proven them to be Abeta-lowering agents, however, the mechanism of action of these drugs is not yet known. One possible mechanism is that they reduce Abeta levels indirectly by reducing cholesterol in the central nervous system (CNS). In this study, we administered three different statins (simvastatin, lovastatin, and atorvastatin) to nontransgenic mice. We found that all three compounds had similar effects on Abeta, reducing both Abeta40 and Abeta42. The statins decreased beta-cleaved C-terminal fragment (CTF) although having no effect on alpha-CTF levels. However, the drugs did not have a similar effect on cholesterol in the CNS. Only lovastatin significantly reduced total cholesterol in isolated plasma membranes. As cholesterol is not distributed evenly in the plasma membrane, we examined bilayer distribution of cholesterol and found that all three statins caused CNS cholesterol to translocate from the cytofacial leaflet to the exofacial leaflet. This data suggests that cholesterol distribution and not total cholesterol levels may be important to Abeta production in the CNS.

Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking.

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).

Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice

Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3-4-, 14-15-, and 24-25-month old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.

Hemileaflet susceptibility to oxidative damage in the intestinal brush-border membrane.

Oxidation of biological membranes is characteristic of many types of tissue injury, including those observed with inflammatory bowel disease. The lipid compositions of the inner and outer leaflets of biological membranes differ significantly, making one leaflet theoretically more susceptible to oxidative stress than the other. In this study, we evaluated the susceptibility of each membrane hemileaflet for peroxyl radical-mediated oxidation. In vitro peroxidation of intestinal brush-border membrane was initiated with the peroxyl radical-generator 2,2'-azobis-(2-amidinopropane)hydrochloric acid (AAPH). Oxidation events were monitored by following the oxidation-sensitive degradation of the lipid-soluble fluorescent probe cis-parinaric acid (PnA). The degradation patterns were clearly distinct in the inner and outer hemileaflet. PnA degradation in the inner hemileaflet was consistent with a slow first-order reaction, whereas degradation in the outer leaflet appeared as two first-order processes delayed in time. The results suggest that the sum of available antioxidants and endogenous substrates for oxidation are consumed more rapidly in the outer membrane hemileaflet, making this leaflet more susceptible to peroxidation compared with the cytofacial leaflet.

Expression of Liver Fatty Acid Binding Protein in L-Cells: Plasma Membrane Response to Ethanol

Expression of liver fatty acid binding protein (L-FABP) in transfected L-cell fibroblasts modifies plasma membrane structure and function [Incerpi et al., 1992, Arch. Biochem. Biophys. 298, 35-42], The effect of L-FABP expression on ethanol induced fluidization of plasma membranes was examined. Ethanol in vitro selectively fluidized the exofacial leaflet of the plasma membranes from L-cells expressing low amounts of L-FABP. In contrast, the plasma membranes from L-cells expressing high amounts of L-FABP were resistant to the actions of ethanol. Furthermore, diphenylhexatriene lifetime distributional analysis demonstrated that the plasma membrane exofacial leaflet had a lower range of apparent dielectric constants than the cytofacial leaflet for both low- and high-expression cells. Both the center of lifetime and the lifetime distributional width of diphenylhexatriene in the bulk plasma membrane versus the cytofacial leaflet were consistent with significantly lower apparent dielectric constant in the exofacial leaflet of high-expression versus low-expression cells. Ethanol in vitro preferentially increased the exofacial leaflet apparent dielectric properties of the plasma membranes from low-expression but not high-expression cells. In conclusion, ethanol appears to dehydrate the lipid headgroups of plasma membranes from high-expression cells, thereby conferring resistance to ethanol fluidization. In contrast, ethanol may not dehydrate the plasma membrane lipid head groups of low-expression cells, resulting in ethanol fluidizing the outer leaflet of the plasma membrane.

Cholesterol domains in biological membranes.

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intraplasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50-60% of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains alter transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca(2+)-ATPase and Na+, K(+)-ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.

Effects of exogenous phospholipids on platelet activation

Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and spiculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457–4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406–5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.

Chronic ethanol consumption alters transbilayer distribution of phosphatidylcholine in erythrocytes of Sinclair (S-1) miniature swine

Effects of chronic ethanol consumption on transbilayer distribution of phospholipids in the exofacial and cytofacial leaflets of erythrocytes from chronic ethanol-consuming Sinclair (S-1) miniature swine were examined. Phosphatidylcholine (PC) was predominantly located in the exofacial leaflet and phosphatidylethanolamine (PE) and phosphatidylserine (PS) located primarily in the cytofacial leaflet. Chronic ethanol consumption significantly increased PC content in the exofacial leaflet without changing bulk membrane PC composition. Ethanol-induced changes in PC distribution were specific for PC and not detected in PE or PS. There was also a significant decrease in sphingomyelin in the ethanol group. Sphingomyelin is primarily an exofacial phospholipid. The specific ethanol-induced changes in the exofacial leaflet are consistent with recent studies showing that the exofacial membrane leaflet is more susceptible to effects of ethanol as compared to the cytofacial leaflet. Such specificity of action provides a new way of viewing how ethanol alters membrane structure and function.

Characterization and modulation of rat small intestinal brush-border membrane transbilayer fluidity

In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets. Moreover, in vitro addition of benzyl alcohol (final concn 25 mM) preferentially fluidized the exofacial leaflet and concomitantly decreased leucine aminopeptidase activity but did not affect the activities of maltase, sucrase, alkaline phosphatase, or gamma-glutamyltranspeptidase. In vivo addition of the membrane-mobility agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanate] (A2C) (final concn 7.5 microM) preferentially fluidized the cytofacial leaflet and increased Na(+)-gradient-dependent D-glucose transport but not Na(+)-gradient-dependent L-leucine transport.

Differential modulation of human small intestinal brush-border membrane hemileaflet fluidity affects leucine aminopeptidase activity and transport of d-glucose and l-glutamate

The fluidity of the exofacial (outer) and cytofacial (inner) leaflets of human proximal small intestinal brush-border membrane vesicles was studied by selective quenching by trinitrophenyl groups, steady-state fluorescence polarization, and differential polarized phase fluorometry techniques, utilizing the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Differences in the hemileaflet's phospholipid composition were also analyzed by trinitrophenylation of aminophospholipids and phospholipase A 2 treatment of these preparations. The results of these studies demonstrated that the inner leaflet of these membranes was less fluid than its outer counterpart. Phosphatidylserine was located mainly in the inner hemileaflet, whereas phosphatidylethanolamine and phosphatidylcholine were more symmetrically distributed between the hemileaflets of this membrane. Moreover, in vitro addition of 2-[(2-methoxyethoxy)ethyl]- cis-8-(2-octylcyclopropyl)octanoate (final concentration, 7.5 μ m) preferentially fluidized the cytofacial leaflet and concomitantly increased Na +-gradient-dependent d-glucose uptake, but decreased Na +, K +-dependent l-glutamic acid uptake in these membrane vesicles. In vitro addition of benzyl alcohol (final concentration, 25 m m) preferentially fluidized the exofacial leaflet and decreased leucine aminopeptidase activity in these preparations. These results, therefore, demonstrate that the hemileaflets of human small intestinal brush-border membranes have different phospholipid compositions and fluidities. Alterations of either the exofacial or cytofacial leaflet fluidity, moreover, modulate protein-mediated activities in a distinct manner.

Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: effects of chronic ethanol consumption

Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85–94) and in vivo ((1989) J. Neurochem. 52, 1925–1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids.

Role of polyunsaturated fatty acids and lipid peroxidation in LM fibroblast plasma membrane transbilayer structure

The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospho-lipids by culture medium supplementation, decreased the limiting anisotropy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H 2O 2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbi-layer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20–28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72–76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.

Acute and chronic effects of ethanol on transbilayer membrane domains.

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.

Charged anesthetics selectively alter plasma membrane order.

Although indirect evidence supporting differential lipid fluidity in the two monolayers of plasma membranes has accumulated, unambiguous demonstration of this difference has been difficult to obtain. In the present study, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), selective quenching of fluorescence by trinitrophenyl groups, and differential polarized phase fluorescence techniques were used to directly examine the static (order) and dynamic (rotational rate) components of lipid motion in the exofacial and cytofacial leaflets of LM fibroblast plasma membranes. The limiting anisotropy (0.137), the order parameter (0.590), and the rotational relaxation time (1.20 ns) of DPH in the plasma membranes (inner plus outer leaflet) indicated rapid but restricted probe motion in the lipid environment. However, the statics and dynamics of DPH motion in the individual monolayers were significantly (p less than 0.025) different. The limiting anisotropy, order parameter, and rotational relaxation time of DPH in the cytofacial monolayer were 0.036, 0.08, and 0.16 ns, respectively, greater than calculated for the exofacial monolayer of the LM plasma membrane. At appropriate concentrations, phenobarbital and, to a lesser degree, pentobarbital preferentially reduced the limiting anisotropy of DPH calculated for the exofacial leaflet while prilocaine reduced the limiting anisotropy of DPH in the cytofacial leaflet of LM fibroblast plasma membranes. In contrast, the putative cytofacial anesthetic procaine failed to show any preference for either leaflet. Arrhenius plots of DPH fluorescence in LM plasma membranes showed a prominent characteristic break point near 30-32 degrees C. Phenobarbital, pentobarbital, and procaine did not affect this break point while prilocaine selectively abolished it. The break point was therefore assigned to the inner monolayer of the LM plasma membrane.

Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.