Optimization of receptor-G protein coupling by bilayer lipid composition I: kinetics of rhodopsin-transducin binding.

The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.     

Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains

The effects of ceramides with varying saturated N-linked acyl chains (C2-C14) on cholesterol displacement from sphingomyelin-rich domains and on the stability of ordered domains were studied. The bilayers examined were made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), D-erythro-N-palmitoyl-sphingomyelin (PSM), D-erythro-N-acyl-sphingosine, and cholesterol (60:15:15:10 mol%, respectively). Cholestatrienol (CTL) or D-erythro-N-trans-parinoyl-sphingomyelin (tParSM) were used as reporter molecules (at 1 mol%) for the ordered domains, and 1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phosphocholine (7SLPC) as a fluorescence quencher (30 mol%, replacing POPC) in the liquid-disordered phase. The results indicate that the ceramide had to have an N-linked acyl chain with at least 8 methylene units in order for it to displace cholesterol from the sphingomyelin-rich domains at the concentration used. The melting of the sphingomyelin-rich domain shifted to higher temperatures (compared to the ceramide-free control) with C2, C12 and longer chain ceramides, whereas C4-C10 ceramides led to domain melting at lower temperatures than control. This study shows that short-chain ceramides do not have the same effects on sterol- and sphingomyelin-rich domains as naturally occurring longer-chain ceramides have.     

Molecular organization of cholesterol in polyunsaturated phospholipid membranes: a solid state 2H NMR investigation.

We compared the molecular organization of equimolar [3alpha-2H1]cholesterol in 18:0-18:1PC (1-stearoyl-2-oleoylphosphatidylcholine), 18:0-22:6PC (1-stearoyl-2-docosahexaenoylphosphatidylcholine), 18:0-20:4PC (1-stearoyl-2-arachidonylphosphatidylcholine) and 20:4-20:4PC (1,2-diarachidonylphosphatidylcholine) bilayers by solid state 2H NMR. Essentially identical quadrupolar splittings (delta v(r) = 45 +/- 1 kHz) corresponding to the same molecular orientation characterized by tilt angle alpha0 = 16 +/- 1 degrees were measured in 18:0-18:1PC, 18:0-22:6PC and 18:0-20:4PC. A profound difference in molecular interaction with dipolyunsaturated 20:4-20:4PC, in contrast, is indicated for the sterol. Specifically, the tilt angle alpha0 = 22 +/- 1 degrees (derived from delta v(r) = 37 +/- 1 kHz) is greater and its membrane intercalation is only 15 mol%.     

Cholesterol hydroxyl group is found to reside in the center of a polyunsaturated lipid membrane

Cholesterol and saturated lipid species preferentially partition into liquid ordered microdomains, such as lipid rafts, away from unsaturated lipid species for which the sterol has less affinity in the surrounding liquid-disordered membrane. To observe how cholesterol interacts with unsaturated phospholipids, we have determined, from one-dimensional neutron scattering length density profiles, the depth of cholesterol in phosphatidylcholine (PC) bilayers with varying amounts of acyl chain unsaturation. Through the use of [2,2,3,4,4,6-(2)H(6)]-labeled cholesterol, we show that in 1-palmitoyl-2-oleoylphosphatidylcholine (16:0-18:1 PC), 1,2-dioleoylphosphatidylcholine (18:1-18:1 PC), and 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4 PC) bilayers the center of mass of the deuterated sites is approximately 16 A from the bilayer center. This location places the hydroxyl group of the sterol moiety at the hydrophobic/hydrophilic bilayer interface, which is the generally accepted position. In dramatic contrast, for 20:4-20:4 PC membranes the hydroxyl group is found, unequivocally, sequestered in the bilayer center. We attribute the change in location to the high disorder of polyunsaturated fatty acids (PUFA) that is incompatible with close proximity to the steroid moiety in its usual "upright" orientation.     

Cholesterol is found to reside in the center of a polyunsaturated lipid membrane

Previously, we reported neutron diffraction studies on the depth of cholesterol in phosphatidylcholine (PC) bilayers with varying amounts of acyl chain unsaturation [Harroun, T. A., et al. (2006) Biochemistry 45, 1227-1233]. The center of mass of the 2,2,3,4,4,6-D 6 deuterated sites on the sterol label was found to reside 16 A from the middle of the bilayer in 1-palmitoyl-2-oleoylphosphatidylcholine (16:0-18:1PC), 1,2-dioleoylphosphatidylcholine (18:1-18:1PC), and 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4PC). This location places cholesterol's hydroxyl group close to the membrane surface, indicative of the molecule in its commonly understood "upright" orientation. However, for dipolyunsaturated 20:4-20:4PC membranes the label, thus the hydroxyl group, was found sequestered in the center of the bilayer. We attributed the change in location to the high level of disorder of polyunsaturated fatty acids (PUFA) that is incompatible with proximity to the rigid steroid moiety in its usual upright orientation. From that study, the unresolved question was whether the molecule was inverted or lying flat with respect to the membrane plane, in the middle of the bilayer. We have followed up those results with additional neutron experiments employing [25,26,26,26,27,27-D 7]cholesterol, a deuterated analogue labeled in the tail. These diffraction measurements unequivocally show cholesterol lies flat in the middle of 20:4-20:4PC bilayers.     

Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species

Neuronal membranes contain high levels of phosphatidylserine (PS) and docosahexaenoic acid (22:6n-3, DHA). In this study, substrate preference in PS synthesis was determined to gain insight on the biochemical basis for concentrating PS in neuronal membranes where 22:6n-3 is highly enriched. We first established an in vitro assay method using unilamellar vesicles (LUV) of deuterium-labeled substrates and reversed-phase HPLC/electrospray ionization (ESI) mass spectrometry. The PS production by the incubation of deuterium-labeled substrate and microsomal fractions was monitored. We found that tissue-specific substrate preference exists in PS synthesis. Microsomes from the cerebral cortex synthesized PS from 18:0,22:6-PC most favorably among the PC substrates tested, followed by 18:0,22:5-PC, resulting in the PC substrate preference in the order of 18:0,22:6 > 18:0,22:5 > 18:0,20:4 = 18:0,18:1. Liver microsomes also preferred 18:0,22:6-PC as the substrate in PS synthesis but did not use 18:0,22:5-PC favorably. The 18:0,22:5-PC species was converted to PS at the similar extent as 18:0,20:4- or 18:0,18:1-PC species in the liver. Both brain and liver microsomes showed a preference for 18:0 over 16:0 as the sn-1 fatty acid. From these data it was deduced that preferential conversion of 18:0,22:6-PC to the corresponding PS species is at least partly responsible for concentrating PS in neuronal tissues where 22:6n-3 is particularly abundant. The distinctive preference for 18:0,22:5-PS observed with brain microsomes may help to maintain PS at a high level in the brain when 22:6n-3 is replaced by 22:5n-3 as in the case of n-3 fatty acid deficiency.     

Rapid flip-flop in polyunsaturated (docosahexaenoate) phospholipid membranes

The transbilayer movement (flip-flop) of 7-nitrobenz-2-oxa-1,3-diazol-4-yl phosphatidylethanolamine (NBD-PE) in phosphatidylcholine (PC) membranes containing various acyl chains was measured by dithionite quenching of NBD fluorescence. Of specific interest was docosahexaenoic acid (DHA), the longest and most unsaturated acyl chain commonly found in membranes. This molecule represents the extreme example of a family of important fatty acids known as omega-3s and has been clearly demonstrated to alter membrane structure and function. One important property that has yet to be reported is the effect of DHA on membrane phospholipid flip-flop. This study demonstrates that as the number of double bonds in the fatty acyl chains comprising the membrane increases, so does the rate of flip-flop of the NBD-PE probe. The increase is particularly marked in the presence of DHA. Half-lives t(1/2) of 0.29 and 0.086 h describe the process in 1-stearoyl-2-docosahexaenoylphosphatidylcholine and 1,2-didocosahexaenoylphosphatidylcholine, respectively, whereas in 1-stearoyl-2-oleoylphosphatidylcholine t(1/2)=11.5h. Enhanced permeability to dithionite with increasing unsaturation was also indicated by our results. We conclude that PC membranes containing DHA support faster flip-flop and permeability rates than those measured for other less-unsaturated PCs.     

Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration

Model membranes composed of cholesterol plus one of two phosphatidylcholines (PC), each containing a saturated and a dienoic acyl chain, have been studied by differential scanning calorimetry. The gel to liquid-crystalline phase transition temperature of 1-palmitoyl-2-linoleoyl PC was -19.5 degrees C and that of 1-stearoyl-2-linoleoyl PC was -13.7 degrees C. The addition of cholesterol to the phosphatidylcholines in aqueous dispersion resulted in the progressive removal of the phase transition as observed by differential scanning calorimetry. Per mole of sterol in the membrane, cholesterol was more effective at reducing the enthalpy change of the phase transitions of these bilayers containing dienoic phosphatidylcholines than it is in eliminating the transition of membranes made with other phospholipids that contain more saturated chains. No transitions in membranes made with palmitoyl-linoleoyl PC or stearoyl-linoleoyl PC could be detected calorimetrically when 17 mol% cholesterol was present.     

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

Membrane-rigidifying effects of anti-cancer dietary factors

Since several anti-cancer drugs interact with cell membrane lipids, the effects of anti-cancer dietary factors on liposomal membranes with different lipid composition were comparatively studied by measuring fluorescence polarization. Fluidity was imparted on both hydrophobic and hydrophilic regions of lipid bilayers by decreasing cholesterol and increasing unsaturated phosphatidylcholine in membranes. At 0.625-10 microM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-cancer drug, doxorubicin, rigidified the tumor cell model membranes consisting of 20 mol% cholesterol and 80 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 1.0, but not daidzein. They were more effective on the membrane core than the membrane surface. Quercetin showed a biphasic effect on the hydrophobic regions of membrane lipid bilayers to rigidify above 5 microM and fluidize below 2.5 microM. In contrast, anti-cancer dietary factors and doxorubicin were not or much less effective in rigidifying the normal cell model membranes consisting of 40 mol% cholesterol and 60 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 0.5. The membrane-rigidifying effects were greater depending on a decrease of the cholesterol/phosphatidylcholine ratio and an increase of the phosphatidylcholine unsaturation degree. Membrane-active dietary factors and doxorubicin inhibited the growth of mouse myeloma cells at 10-100 microM, while the growth inhibition by membrane-inactive daidzein was relatively weak. Anti-cancer dietary factors appear to act on more fluid membranes like tumor cells as well as doxorubicin to induce rigidification, especially in the hydrocarbon core of membrane lipids, which is determined by the composition of cholesterol and unsaturated phospholipids.     

Structural aspects of pressure effects on infrared spectra of mixed-chain phosphatidylcholine assemblies in D2O

The barotropic behavior of D2O dispersions of 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine, C(18):C(10)PC, a highly asymmetric phospholipid in which the length of the fully extended acyl chain at the sn-1 position of the glycerol backbone is twice as long as that at the sn-2 position, has been investigated by high-pressure Fourier transform infrared spectroscopy. This asymmetric phosphatidylcholine bilayer at room temperature displays a pressure-induced phase transition corresponding to the liquid-crystalline----gel phase transition at 1.4 kbar. A conformational ordering of the lipid acyl chains is observed to take place abruptly at the transition pressure of 1.4 kbar. However, the lamellar lipid molecules and their acyl chains remain to be orientationally disordered in the gel phase until the applied pressure reaches 5.5 kbar. In the gel phase of fully hydrated C(18):C(10)PC, the asymmetric lipid molecules assemble into mixed interdigitated bilayers with perpendicular orientation of the zigzag planes among neighboring acyl chains. The role of excess water played in the interchain structure and the behavior of excess water and bound water under high pressure are also discussed.     

Effect of cholesterol on the bond ordering in hydrated unsaturated lipid bilayers

Molecular dynamics computer simulations of hydrated bilayers of unsaturated phosphatidylcholines in which double bonds are in the states: 18:0/18:1(n-9)cis (PC), 18:0/18:2(n-6)cis (PC), 18:0/18:3(n-3)cis (PC), 18:0/20:4(n-6)cis (PC), and 18:0/22:6(n-3)cis in the presence of cholesterol (40 mol%) and its absence have been performed. The simulation have been performed at 303 K and 1 atm, under the conditions corresponding to the experimentally observed liquid-crystalline state of the bilayer from phosphatidylcholine. The C-C and C-H bond order parameter profiles with respect to the bilayer normal and the C-C bond orientation distribution functions have been calculated. The widths of the functions and positions of their maxima have been determined. The dependence of these characteristics on the type of the bond, the degree of unsaturation of the chain, the presence of cholesterol in the bilayer, and the bond order parameters have been analyzed.     

Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition

Lateral domain or raft formation in biological membranes is often discussed in terms of cholesterol-lipid interactions. Preferential interactions of cholesterol with lipids, varying in headgroup and acyl chain unsaturation, were studied by measuring the partition coefficient for cholesterol in unilamellar vesicles. A novel vesicle-cyclodextrin system was used, which precludes the possibility of cross-contamination between donor-acceptor vesicles or the need to modify one of the vesicle populations. Variation in phospholipid headgroup resulted in cholesterol partitioning in the order of sphingomyelin (SM) > phosphatidylserine > phosphatidylcholine (PC) > phosphatidylenthanolamine (PE), spanning a range of partition DeltaG of -1181 cal/mol to +683 cal/mol for SM and PE, respectively. Among the acyl chains examined, the order of cholesterol partitioning was 18:0(stearic acid),18:1n-9(oleic acid) PC > di18:1n-9PC > di18:1n-12(petroselenic acid) PC > di18:2n-6(linoleic acid) PC > 16:0(palmitic acid),22:6n-3(DHA) PC > di18:3n-3(alpha-linolenic acid) PC > di22:6n-3PC with a range in partition DeltaG of 913 cal/mol. Our results suggest that the large differences observed in cholesterol-lipid interactions contribute to the forces responsible for lateral domain formation in plasma membranes. These differences may also be responsible for the heterogeneous cholesterol distribution in cellular membranes, where cholesterol is highly enriched in plasma membranes and relatively depleted in intracellular membranes.     

Thermal history alters cholesterol effect on transition of 1-palmitoyl-2-linoleoyl phosphatidylcholine.

The effect of cholesterol on the bilayer phase behavior of heteroacid phosphatidylcholines with one unsaturated fatty acid depends on the nature of the unsaturated chain. Previous differential scanning calorimetry (DSC) studies showed that 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC) had a broad, weak transition at about -18 degrees C, which was effectively eliminated by less than 15 mol% cholesterol. Phospholipids with greater and lesser degrees of unsaturation displayed stronger phase transitions and less sensitivity to cholesterol. In this work, deuterium nuclear magnetic resonance has been used to examine the phase behavior of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC-d31) alone, and with 15 mol % cholesterol. The behavior is found to be sensitive to sample thermal history. Moderately fast cooling (1 degree/h) results in a continuous phase change from a fluid to an ordered phase in the pure lipid. Under similar cooling conditions, the sample containing cholesterol displays increased chain order and a continuous phase change with no apparent isothermal transition. However, when these systems are cooled at a reduced rate (0.3 degree/h), the continuous phase change is pre-empted by a sharp transition into a more ordered phase that gives a deuterium spectrum having intensity at a value of the quadrupole-splitting characteristic of a rigid lattice system. In the pure lipid, this transition effectively coincides with the center of the continuous phase change. Addition of 15 mol % cholesterol lowers the temperature of this sharp transition by about 3 degrees C. These observations provide some insights into the behavior of this system seen using differential scanning calorimetry. Results of deuteron transverse relaxation measurements under these conditions are also reported.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Structure and Dynamics of Cholesterol-Containing Polyunsaturated Lipid Membranes Studied by Neutron Diffraction and NMR

A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, (2)H-NMR and (13)C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 ? away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid-water interface. Addition of cholesterol partially redistributes DHA density from near the lipid-water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C(8)-C(18) is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin.     

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T(m)) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T(m) to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T(m) value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.     

Cholesterol dependent recruitment of di22:6-PC by a G protein-coupled receptor into lateral domains

Bovine rhodopsin was reconstituted into mixtures of didocosahexaenoylphosphatidylcholine (di22:6-PC), dipalmitoylphosphatidylcholine (di16:0-PC), sn-1-palmitoyl-sn-2-docosahexaenoylphosphatidylcholine (16:0, 22:6-PC) and cholesterol. Rhodopsin denaturation was examined by using high-sensitivity differential scanning calorimetry. The unfolding temperature was increased at lower levels of lipid unsaturation, but the highest temperature was detected for native disk membranes: di22:6-PC < 16:0,22:6-PC < di16:0,18:1-PC < native disks. The incorporation of 30 mol% of cholesterol resulted in 2-4 degrees C increase of denaturation temperature in all reconstituted systems examined. From the analysis of van't Hoff's and calorimetric enthalpies, it was concluded that the presence of cholesterol in di22:6-PC-containing bilayers induces a level of cooperativity in rhodopsin unfolding. Fluorescence resonance energy transfer (FRET), using lipids labeled at the headgroup with pyrene (Py) as donors and rhodopsin retinal group as acceptor of fluorescence, was used to study rhodopsin association with lipids. Higher FRET efficiencies detected for di22:6-PE-Py, compared to di16:0-PE-Py, in mixed di22:6-PC-di16:0-PC-cholesterol bilayers, indicate preferential segregation of rhodopsin with polyunsaturated lipids. The effective range of the rhodopsin-lipid interactions facilitating cluster formation exceeds two adjacent lipid layers. In similar mixed bilayers containing no cholesterol, cluster formation was absent at temperatures above lipid phase transition, indicating a crucial role of cholesterol in microdomain formation.