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A range of evidence from animal, clinical and epidemiological studies indicates that highly polyunsaturated acyl chains play important roles in development, cognition, vision and other aspects of neurological function. In a number of these studies n3 polyunsaturated fatty acids (PUFAs) appear to be more efficacious than n6 PUFAs. In a previous study of retinal rod outer segments obtained from rats raised on either an n3 adequate or deficient diet, we demonstrated that the replacement of 22:6n3 by 22:5n6 in the n3 deficient rats led to functional deficits in each step in the visual signaling process (Niu et al., 2004). In this study, we examined rhodopsin and phosphodiesterase function and acyl chain packing properties in membranes consisting of phosphatidylcholines with sn-1=18:0, and sn-2=22:6n3, 22:5n6, or 22:5n3 in order to determine if differences in function are due to the loss of one double bond or due to differences in double bond location. At 37 °C the n6 lipid shifted the equilibrium between the active metarhodopsin II (MII) state and inactive metarhodopsin I (MI) state towards MI. In addition, 22:5n6 reduced the rates of MII formation and MII-transducin complex formation by 2- and 6-fold, respectively. At a physiologically relevant level of rhodopsin light stimulation, the activity of phosphodiesterase was reduced by 50% in the 22:5n6 membrane, relative to either of the n3 membranes. Activity levels in the two n3 membranes were essentially identical. Ensemble acyl chain order was assessed with time-resolved fluorescence measurements of the membrane probe diphenylhexatriene (DPH). Analysis in terms of the orientational distribution of DPH showed that acyl chain packing in the two n3 membranes is quite similar, while in the 22:5n6 membrane there was considerably less packing disorder in the bilayer midplane. These results demonstrate that the n3 bond configuration uniquely optimizes the early steps in signaling via a mechanism which may involve acyl chain packing deep in the bilayer., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.

The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had approximately 80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (G(t)) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency.

Objective: To assess the effects of n-3 polyunsaturated phospholipid acyl chains on the initial steps in G-protein-coupled signaling., Study Design: Isolated components of the visual signal transduction system, rhodopsin, G protein (G(t)), and phosphodiesterase (PDE), were reconstituted in membranes containing various levels of n-3 polyunsaturated phospholipid acyl chains. In addition, rod outer segment disk membranes containing these components were purified from rats raised on n-3-deficient and n-3-adequate diets. The conformation change of rhodopsin, coupling of rhodopsin to G(t), and PDE activity were each measured separately., Results: The ability of rhodopsin to form the active metarhodopsin II conformation and bind G(t) were both compromised in membranes with reduced levels of n-3 polyunsaturated acyl chains. The activity of PDE, directly related to the integrated cellular response, was reduced in all membranes lacking or deficient in n-3 polyunsaturated acyl chains. PDE activity in membranes containing 22:5n-6 PC was 50% lower than in membranes containing either 22:6n-3 PC or 22:5n-3 PC., Conclusions: The earliest events in G-protein-coupled signaling; receptor conformation change, receptor-G-protein binding, and PDE activity are reduced in membranes lacking n-3 polyunsaturated acyl chains. Efficient and rapid propagation of G-protein-coupled signaling requires polyunsaturated n-3 phospholipid acyl chains.

The effect of phospholipid acyl chain and cholesterol composition on G protein-coupled signaling was studied in native rod outer segment (ROS) disk and reconstituted membranes by measuring several steps in the visual transduction pathway. The cholesterol content of disk membranes was varied from 4 to 38 mol% cholesterol with methyl-beta-cyclodextrin. The visual signal transduction system [rhodopsin, G protein (G(t)), and phosphodiesterase (PDE)] was reconstituted with membranes containing various levels of phospholipid acyl chain unsaturation, with and without cholesterol. ROS membranes from rats raised on n-3 fatty acid-deficient and -adequate diets were also studied. The ability of rhodopsin to form the active metarhodopsin II conformation and bind G(t) was diminished by a reduction in the level of DHA (22:6n-3) acyl chains or an increase in membrane cholesterol. DHA acyl chain containing phospholipids minimized the inhibitory effects of cholesterol on the rate of rhodopsin-G(t) coupling. The activity of PDE, which is a measure of the integrated signal response, was reduced in membranes lacking or deficient in DHA acyl chains. PDE activity in membranes containing docosapentaenoic acid (DPA, 22:5n-6) acyl chains, which replace DHA in n-3 fatty acid deficiency, was 50% lower than in DHA-containing membranes. Our results indicate that efficient and rapid propagation of G protein-coupled signaling is optimized by DHA phospholipid acyl chains.

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.

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.

The visual transduction system was used as a model to investigate the effects of membrane lipid composition on receptor-G protein coupling. Rhodopsin was reconstituted into large, unilamellar phospholipid vesicles with varying acyl chain unsaturation, with and without cholesterol. The association constant (K(a)) for metarhodopsin II (MII) and transducin (G(t)) binding was determined by monitoring MII-G(t) complex formation spectrophotometrically. At 20 degrees C, in pH 7.5 isotonic buffer, the strongest MII-G(t) binding was observed in 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC), whereas the weakest binding was in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) with 30 mol% cholesterol. Increasing acyl chain unsaturation from 18:0,18:1PC to 18:0,22:6PC resulted in a 3-fold increase in K(a). The inclusion of 30 mol% cholesterol in the membrane reduced K(a) in both 18:0,22:6PC and 18:0,18:1PC. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. These findings, if extended to other members of the superfamily of G protein-coupled receptors, suggest that a loss in efficiency of receptor-G protein binding is a contributing factor to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency.

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.

In order to understand the role of the high levels of docosahexaenoic acid (DHA) in neuronal and retinal tissue, a study of the effect of membrane lipid composition on the visual pathway, a G protein-coupled system, was undertaken. The level of metarhodopsin II (MII) formation was determined to be a function of phospholipid acyl-chain unsaturation, with the highest levels seen in DHA-containing bilayers. Similarly, the rate of coupling of MII to the retinal G protein, Gt, to form a MII-Gt complex, was enhanced in DHA bilayers relative to less unsaturated phospholipids. Complex formation initiates the first stage of amplification in the visual pathway. The activation of the cGMP phosphodiesterase (PDE), the effector enzyme, represents the integrated pathway function. DHA-containing bilayers were found to support PDE levels comparable to those of the rod outer segment (ROS) disk membranes. Inclusion of 30 mol% cholesterol in the reconstituted bilayers had an inhibitory effect on each step in the visual pathway studied. Inclusion of cholesterol reduced MII formation and PDE activity and increased the lag time between the appearance of MII and the formation of the MII-Gt complex. However, signaling in DHA bilayers was far less affected by the addition of cholesterol than in bilayers containing less unsaturated phospholipids. These studies point up the importance of DHA acyl chains in promoting optimal function in G protein-coupled signaling pathways. The results reported here suggest that visual and cognitive deficits observed in n-3 deficiency may result from decreased efficiency in related neurotransmitter and visual signaling pathways in the absence of DHA.

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.

The combined effects of ethanol and osmolytes on both the extent of formation of metarhodopsin II (MII), which binds and activates transducin, and on acyl chain packing were examined in rod outer segment disc membranes. The ethanol-induced increase in MII formation was amplified by the addition of neutral osmolytes. This enhancement was linear with osmolality. At 360 milliosmolal, the osmolality of human plasma, 50 mM ethanol was 2.7 times more potent than at 0 osmolality, demonstrating the importance of water activity in in vitro experiments dealing with ethanol potency. Ethanol disordered acyl chain packing, and increasing osmolality enhanced this acyl chain disordering. Prior osmotic stress data showed a release of 35 +/- 2 water molecules upon MII formation. Ethanol increases this number to 49 water molecules, suggesting that ethanol replaces 15 additional water molecules upon MII formation. Amplification of ethanol effects on MII formation and acyl chain packing by osmolytes suggests that ethanol increases the equilibrium concentration of MII both by disordering acyl chain packing and by disrupting rhodopsin-water hydrogen bonds, demonstrating a direct effect of ethanol on rhodopsin. At physiologically relevant levels of osmolality and ethanol, about 90% of ethanol's effect is due to disordered acyl chain packing.

Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with 15N at Leu5 and Gly9, results in residual dipolar contributions to the 1J(NH) splittings for these two residues. Both residues show 1J(NH) splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu5 and Gly9 make angles of 48+/-4 degrees and 40+/-8 degrees, respectively, with the disk normal. These 'transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.

The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic bilayer structural properties of symmetrically substituted phosphatidylcholines (PCs) with acyl chains containing no, one, four, or six double bonds and mixed-chain phosphatidylcholines with a saturated sn-1 chain and one, four, or six double bonds in the sn-2 chain. Both the Brownian rotational diffusion (BRD) model and the wobble-in-cone model were fit to all differential polarization data, and the descriptions of the data provided by the BRD model were found to be statistically superior. Global analysis of differential polarization data revealed two statistically equivalent solutions. The solution corresponding to a bimodal orientational distribution function, f(theta), was selected based on the effects of temperature on f(theta) and previous measurements on fixed, oriented bilayers. The overall equilibrium acyl chain order in these bilayers was analyzed by comparing the orientational probability distribution for DPH, f(theta) sin theta, with a random orientational distribution. Orientational order decreased and probe dynamics increased in mixed-chain species as the unsaturation of the sn-2 chain was increased. The degree of orientational order dropped dramatically in the dipolyunsaturated species compared with the mixed-chain phosphatidylcholines, which contained a polyunsaturated sn-2 chain. In terms of both orientational order and probe dynamics, the differences between the highly polyunsaturated species and the monounsaturated species were much greater than the differences between the monounsaturated species and a disaturated PC.

The visual pigment rhodopsin is a prototypical member of the G protein-coupled receptor superfamily. In this study, we have investigated the effect of a series of n-alcohols on the formation of metarhodopsin II (MII), the photoactivated conformation of rhodopsin, which binds and activates transducin. When rhodopsin was photolyzed in the presence of several n-alcohols, increased MII formation was observed in the order ethanol > butanol > hexanol, whereas longer chain n-alcohols inhibited MII formation with decanol > octanol. The magnitude of the stimulatory effects was greater in a more highly unsaturated phospholipid. Alcohols, which enhanced MII formation also increased phospholipid acyl chain packing free volume, while those that decreased this bilayer property inhibited MII formation. An apparent discontinuity in the effect of these alcohols results when their potency is calculated in terms of the total aqueous alcohol concentration. In sharp contrast, a continuous variation in their behavior is observed, when their potency is calculated in terms of the amount of alcohol partitioned in the membrane. Our findings strongly support a lipid-mediated mechanism of action for alcohols on rhodopsin and, by analogy, for other G protein-coupled receptors.

Visual transduction is one of the best characterized G protein--coupled signalling systems. In addition, about 50% of the disk membrane phospholipid acyl chains are 22:6n-3, making this system ideal for determining the role of polyunsaturation in modulating membrane-signalling systems. The extent of formation of metarhodopsin II (MII), the G protein--activating photointermediate of rhodopsin, was studied in phospholipid vesicles composed of a variety of phosphatidylcholines, differing in their acyl chain composition at the sn-2 position. The amount of MII formed increased progressively with the level of acyl chain unsaturation at the sn-2 position. The effect of added cholesterol was to reduce the amount of MII formed. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by a fractional volume parameter fv derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship among sn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in fv with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.

The role of phospholipids in modulating the effect of ethanol on membrane receptor activation was investigated by studying the extent of metarhodopsin II (MII) formation in vesicles formed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine) and in native rod outer segment disk membranes as a function of ethanol concentration. The equilibrium concentration of MII, the G protein-activating form of photoactivated rhodopsin, was found to increase as a function of ethanol concentration in all three bilayers. Phospholipid composition had a marked effect on ethanol potency, with the presence of polyunsaturated phospholipid acyl chains increasing ethanol potency by 40%. The effects of ethanol on lipid acyl chain packing in POPC and PDPC were investigated using frequency domain anisotropy decay measurements of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene. Enhanced formation of MII due to the presence of ethanol was correlated with the effects of ethanol on acyl chain packing properties. These findings support a phospholipid-mediated mechanism for the action of ethanol in modulating integral membrane receptor conformation.

The superfamily of membrane-bound receptors, which function in signal transduction by activating a guanine nucleotide binding protein or G-protein in response to agonist binding, shares a number of structural and mechanistic properties. Among these similarities is downregulation of functional activity via receptor phosphorylation. In this study, the effects of intermediate levels of phosphorylation (greater than or equal to 4 added phosphates per receptor molecule) on receptor conformational equilibria are examined by comparing the photochemical properties of phosphorylated and unphosphorylated rhodopsins which were incorporated separately into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles. Postflash spectra reflecting the contributions of metarhodopsins I, II, and III (meta I, meta II, and meta III) were obtained from these samples. Deconvolution of appropriate difference spectra allowed a determination of the concentration of the photointermediates of interest. Meta II is the form of photolyzed rhodopsin which binds and activates the visual G-protein (Gt); thus, its relative abundance at equilibrium and temporal stability are important parameters in determining the efficiency of visual signal transduction. The effects of pH and temperature on the meta I in equilibrium with meta II equilibrium constant (Keq) and the rate of decay of meta II to meta III were examined for the reconstituted phosphorylated and unphosphorylated rhodopsin samples. Keq was essentially unaffected by phosphorylation when measured at pH 7.0 and 8.0 and 20 and 37 degrees C. The decay time (lifetime) of meta II----meta III had a value of approximately 4.7 min in both phosphorylated and unphosphorylated samples.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, Gt. The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta II and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20 degrees C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta II of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate Gt was measured by monitoring the level of catalyzed exchange of Gt-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta II and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)

The photochemical intermediate metarhodopsin II (meta II; lambda max = 380 nm) is generally identified with rho*, the conformation of photolyzed rhodopsin which binds and activates the visual G-protein, Gt [Emeis, D., & Hoffman, K.P. (1981) FEBS Lett. 136, 201-207]. Purified bovine rhodopsin was incorporated into vesicles consisting of dimyristoylphosphatidylcholine (DMPC), and the rapid formation of a photochemical intermediate absorbing maximally at 380 nm was quantified via both flash photolysis and equilibrium spectral measurements. Kinetic and equilibrium spectral measurements performed above the Tm of DMPC showed that Gt, in the absence of GTP, enhances the production of the 380-nm-absorbing species while reducing the concentration of the 478-nm-absorbing species, metarhodopsin I (meta I), in a manner similar to that observed in the native rod outer segment disk membrane. This Gt-induced shift in the equilibrium concentration of photointermediates indicated that the species with an absorbance maximum at 380 nm was meta II. The presence of rho* in the DMPC bilayer was established via measurements of photolysis-induced exchange of tritiated GMPPNP, a nonhydrolyzable analogue of GTP, on Gt. Above Tm, the metarhodopsin equilibrium is strongly shifted toward meta I relative to the native rod outer segment disk membrane; however, at 37 degrees C, 40% of the photointermediates are in the form of meta II. The formation of meta II above Tm is slowed by a factor of ca. 2 relative to the disk membrane. Below Tm, the equilibrium is shifted still further toward meta I, and meta II forms ca. 7 times slower than in the disk membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of lipid ordering on the kinetics and extent of metarhodopsin II (meta II) formation was evaluated in bovine rhodopsin which had been reconstituted into phosphatidylcholine vesicles containing 0, 15, and 30 mol% cholesterol. The rate of establishment of the dynamic equilibrium between metarhodopsin I (meta I) and the two kinetically distinguished forms of meta II in the branched meta II model [meta IIfast and meta IIslow; Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990) Biochemistry (preceding paper in this issue)] is derived from kinetic measurements of rhodopsin photolysis in these vesicle systems at several temperatures. Values of the meta I in equilibrium with meta IItotal equilibrium constant, Keq, are calculated from the derived model-dependent rate constants, and are shown to be equivalent to those derived from rapidly acquired absorbance spectra. The presence of 30 mol% cholesterol reduces Keq by approximately 50% between 10 and 37 degrees C. Analysis of the model-dependent parameters in terms of delta H and delta S reveals that cholesterol raises the free energy of meta IIslow, relative to meta I, by increasing delta H whereas it raises the relative free energy of meta IIfast by making delta S meta IIfast relative to meta I less positive. The reduction in Keq by both temperature and cholesterol is found to be directly correlated with a parameter that reflects the free volume available for molecular motion in the hydrophobic core of the bilayer [Straume, M., & Litman, B. J. (1988) Biochemistry 27, 7723-7733].(ABSTRACT TRUNCATED AT 250 WORDS)

Flash photolysis experiments designed to monitor the establishment of the metarhodopsin I to metarhodopsin II equilibrium are interpreted according to a branched model in which two spectrally indistinguishable but kinetically distinguishable forms of metarhodopsin II are postulated to exist in equilibrium with a common pool of metarhodopsin I. This interpretation arises from the consistent requirement for at least three exponentials for a valid description of the observed growth of absorbance at 380 nm following bleaching of bovine rhodopsin in rod outer segment disk membranes. Analysis of the 380-nm transient absorbance data permitted direct determination of the five physically interpretable individual rate constants of the model. This analysis represents a more explicit interpretation of kinetic data than that employed in earlier experiments of this kind, which involved estimating only apparent rates and apparent amplitudes of discrete multiexponential functions. The 380-nm absorbance contributions of all relevant species contributing to the observed dynamic absorbance change were accounted for simultaneously during nonlinear least-squares estimation of the model rate parameters. Analysis of deconvoluted equilibrium spectra acquired from samples identical with those used in the kinetics experiments confirmed the metarhodopsin I-metarhodopsin II equilibrium constants, Keq, derived from the dynamic analyses. It is shown that Keq varies from 1.28 at 10 degrees C to 7.3 at 37 degrees C and that approximately 90% of the metarhodopsin II present is in the form of metarhodopsin IIslow over the temperature range 10-37 degrees C. A physical interpretation of this decay model is discussed in the context of a distribution of metarhodopsin II structural and energetic states.

The result of mixing varying concentrations of the nonionic detergent octyl glucoside (OG) with small unilamellar vesicles (SUV) of egg phosphatidylcholine (PC) made by sonication depends on the ratio between OG and PC in the mixed aggregates. When this molar ratio (Re) is lower than 1.4, the detergent partitions between the PC vesicles and the aqueous medium with a partition coefficient of K = 0.033 mM-1. As a consequence of introduction of OG into the bilayers, the vesicles grow in size. The resultant vesicles have a mean diameter that is an increasing function of Re and is independent of the total PC concentration. Experiments in which the vesicles were loaded with high molecular weight dextran prior to being exposed to OG suggest that the mechanism responsible for the size growth involves lipid transfer rather than fusion. Mixtures with Re values within the range of 1.4-3.2 separate into two macroscopic phases: The lower phase is clear but very viscous. It contains constant OG and PC concentrations and is characterized by an Re value of 3.2, independent of the composition of the whole dispersion. The upper phase contains vesicles of varying concentrations of OG and PC, but a constant Re of 1.4. When the saturating level of 1.4 OG molecules per PC molecule is approached, the concentration of OG monomers in the aqueous medium reaches the value of 16.6 +/- 0.3 mM, which is the apparent cmc of OG in the lipid-containing medium. OG-PC mixed micelles contain at least 3.2 OG molecules per PC molecule. The mixed micelles present at Re = 3.2 apparently have the shape of oblate ellipsoids with a minor axis of about 2 nm and two major axes of about 25 nm. The surface area of the mixed micelles at this point is just sufficient for them to undergo conversion into the smallest possible spherical vesicles of a radius of 12 nm. At Re values above 3.2, the major axis of the mixed micelles becomes smaller as Re increases, while at values of Re below 3.2 the micelles would have been expected to grow very rapidly with decreasing Re. This may explain the partial vesicle closure occurring below Re = 3.2.

Recombinant membranes were prepared from phospholipid-free rhodopsin and egg phosphatidylcholine (PC) under a wide variety of conditions employing an octyl beta-D-glucoside (OG) dialysis procedure. Two bands were consistently observed after sucrose density centrifugation of these recombinants. The major band, which was protein rich, had a molar phospholipid:protein ratio that was in the range of 30:1 to 50:1, even when the molar phospholipid:protein ratio of the solubilized solution prior to OG removal was as high as 300:1. Similar results were obtained when dioleoyl-PC, 1-palmitoyl-2-oleoyl-PC, disk lipids, or diphytanoyl-PC was used instead of egg PC. These results can be explained in terms of a lower stability of the OG-phospholipid micelles relative to the OG-phospholipid-rhodopsin micelles. Of the phospholipids that were used in the OG dialysis procedure, only saturated dimyristoyl-PC produced a protein-rich recombinant band with a phospholipid:protein ratio close to that of the initial solubilized solution. In contrast to the results obtained by using OG, when solubilized disks supplemented with egg PC were reconstituted from sodium cholate or dodecyltrimethyl-ammonium bromide, the resulting recombinant membranes had initial and final phospholipid:protein ratios which were similar.

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.

Equilibrium and dynamic structural properties of minimally to highly unsaturated acyl chain, large, unilamellar phosphatidylcholine (PC) vesicles have been characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Fluorescence lifetimes and equilibrium and dynamic rotational properties of these probes were analyzed by limited-frequency phase-modulation fluorometry in egg PC, palmitoyloleoyl-PC (POPC), dioleoyl-PC (DOPC), palmitoylarachidonoyl-PC (PAPC), and palmitoyldocosahexaenoyl-PC (P-22:6-PC) vesicles over a temperature range from 5 to 37 degrees C. DPH equilibrium orientational distributions were derived according to a model permitting bimodal orientational distributions in which the parallel probability maximum was aligned parallel to the bilayer normal and the orthogonal probability maximum was oriented parallel to the plane of the bilayer. TMA-DPH orientational distributions were derived according to the same model except that all probability was constrained to the parallel orientation. TMA-DPH fluorescence lifetimes were much more sensitive than those of DPH to variations in acyl chain composition and temperature although the same qualitative behavior was generally observed with both probes. Greater acyl chain unsaturation and higher sample temperatures each gave rise to shorter lifetimes consistent with increased water penetrability into the bilayers. Equilibrium order of the hydrocarbon core (as probed by DPH) and of the interfacial and head group regions of the bilayers (as probed by TMA-DPH) was reduced by increasing levels of unsaturation and by higher sample temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

The microviscosity of the hydrocarbon region of the bovine retinal rod outer segment disk membrane was determined by measuring the anisotropy of fluorescence from the probe 1,6-diphenyl-1,3,5-hexatriene. The microviscosity ranged from 1.4 P at 40 degreeC to 15 P at O degree C, and no phase transition was observed in this temperature range. Bleaching of rhodopsin in the disk membrane produced no change in the microviscosity within the limits of error of our measurement (not equal to 5%). The presence of retinal in the disk membrane caused string quenching of the probe fluorescence. Removal of retinal from bleached disks produced a threefold increase in total fluorescence intensity and only a 10% drop in fluorescnece anisotropy, suggesting that large lateral gradients in microviscosity do not exist in the disk membrane. Bilayers prepared from extracted disk lipids had a microviscosity which was about one-fourth that of the intact disk membrane, demonstrating that rhodopsin hinders the mobility of the hydrocarbon chains of the disk phospholipids or egg phosphatidylcholine had identical microviscosities despite the much higher degree of unsaturation of the disk phospholipids.

The dependence of phospholipid vesicle size on lipid composition is investigated by photon correlation spectroscopy. For each lipid composition prolonged ultracentrifugation was used to isolate a nearly uniform population of minimum-sized vesicles. The residual size variations in the samples were sufficient to cause polydispersity that made comparisons between samples difficult. Analyses of the data by the method of cumulants and by a method for approximating the particle size distributions directly are presented. The latter method made possible unambiguous comparisons that revealed small but systematic dependences of vesicle size on composition in vesicles containing mixtures of egg phosphatidylcholine and phosphatidylethanolamine, egg phosphatidylcholine and beef brain sphingomyelin, and in single lipid vesicles of egg phosphatidylcholine, dioleylphosphatidylcholine, and beef brain sphingomyelin. These size dependences are quantified within the resolution limits of the technique and their implications are discussed.

The solubilization of large, unilamellar egg phosphatidylcholine vesicles by the nonionic detergent octyl glucoside (OG) was investigated by nuclear magnetic resonance (NMR), fluorescence anisotropy, turbidity, electron microscopy, and centrifugation followed by compositional analysis. The solubilization process is well described by the three-stage model previously proposed for other detergents. In stage I, the OG partitions between the bilayer and aqueous phases with a molar partition coefficient of 59 +/- 6. The presence of OG in the bilayers produces a small "fluidizing" effect, as indicated by changes in the NMR and fluorescence anisotropy parameters. A rearrangement that forms large mixed bilayers occurs in the latter part of stage I. Stage II, the conversion of detergent-saturated bilayers into mixed micelles, begins at a ratio of total OG concentration minus the critical micelle concentration to total phosphatidylcholine concentration of approximately 1.5 and continues until this ratio reaches about 3.0. The correction for the critical micelle concentration of the OG is necessary for comparison of experimental results obtained at different lipid concentrations. The mixed bilayer-mixed micelle interconversion is quantified by the centrifugation experiments and by 31P NMR. The agreement between the two methods is excellent. Advantages of the NMR method are discussed. In stage III, which was not studied in detail here, all of the phosphatidylcholine is present as mixed micelles. Evidence is presented that the various structures present in the dispersions are in equilibrium with one another during these experiments.

Solubilization of retinal rod outer segment disk membranes in octyl glucoside was employed to prepare rhodopsin samples with varying amounts of associated disk phospholipid. Flash photolysis studies were carried out on these samples to determine the dependence of the meta I to meta II transition kinetics on the level of associated phospholipid. The rate constant for the formation of meta II increased from 6.9 X 10(3) to 19.5 X 10(3) s-1 as the molar ratio of phospholipid per rhodopsin fell from 35 to 5. The activation free energy for this process had a linear dependence on the level of phospholipid, with a slope of 24 cal/mol of rhodopsin-associated phospholipid. A variety of evidence suggests that rhodopsin undergoes a reversible conformation change during the meta I to meta II transition. No evidence was found for an enhanced effect on the activation free energy for this conformation change at the level of associated phospholipid which corresponds to the formation of a phospholipid boundary layer around rhodopsin.

The suitability of octyl and decyl-beta-D-glucoside as solubilizing agents for the bovine retinal rod outer segment disc membrane was investigated and compared to that of hexadecyltrimethylammonium bromide, N,N-dimethyldodecylamine oxide, Emulphogene BC-720 and digitonin. The properties measured included the thermal stability of rhodopsin, regenerability of bleached rhodopsin by addition of 11-cis-retinal, and the rate of denaturation of bleached rhodopsin as measured by changes in the ultraviolet CD spectrum. Denaturing tendencies of the detergents were also evaluated by observing their effects on the absorption and CD spectra of sperm whale metmyoglobin. Our results demonstrate that octyl glucoside is superior to the other detergents, with the possible exception of digitonin, by the above criteria. Unlike digitonin, however, octyl glucoside affords rapid solubilization of the disc membrane and is itself highly soluble. Decyl glucoside has properties equivalent or superior to octyl glucoside, but salts and buffers interfere with its ability to solubilize the disc membrane. The well defined chemical composition, ease of removal by dialysis, and non-denaturing properties of the alkyl glucosides make them attractive detergents for membrane research.

Rhodopsin is phosphorylated in a light-dependent manner by a kinase intrinsic to the rod outer segment. We have used chromatofocusing to separate six phosphorylated species of rhodopsin and have recovered in the pH gradient fractions 60-80% of the initial phosphorylated sample loaded on the column. The isolated species of rhodopsin coincide with the species that are observed in isoelectric focusing gels in the pH range 6.1-4.7. Unphosphorylated rhodopsin focuses at a pI of 6.0. Two species having two phosphates per rhodopsin with isoelectric points of 5.45 and 5.40 have been isolated. The phosphate to rhodopsin ratios for the remaining species are 3.8, 5.0, 6.1, and 8.2 with isoelectric points of 5.16, 4.99, 4.85, and 4.73, respectively. The chromatofocusing profile suggests that there may be multiple forms of rhodopsin with the same number of phosphates among some of the other phosphorylated forms of rhodopsin.