Study of visual pigment rhodopsin supramolecular organization in photoreceptor membrane by small-angle neutron scattering method with contrast variation

Supramolecular organization of rhodopsin in the photoreceptor membrane was investigated by small-angle neutron scattering method. The experiments, which were performed with mixtures of heavy/light water as solvent (contrast variation method), were aimed at obtaining information about the lipid and protein components of the photoreceptor disc membrane separately. It was shown that the packaging density of the rhodopsin molecules in the photoreceptor membrane was unusually high: the distance between the centers of the molecules was approximately 56 Å. The probability of the monomeric state of rhodopsin molecules in the photoreceptor membrane, according to the data obtained, is rather high.     

Rhodopsin photo-oxidation: oxygen consumption and spectrum of activity

The coincidence of action spectrum of SH-groups and lipid oxidation is shown in the photoreceptor photodamaging process. Dose dependence of the process is measured in the maximum of photooxidation (380 nm). Dose of 0.1 J/cm2 is the threshold of photodamaging of photoreceptor membrane. Oxygen consumption in photooxidation of rhodopsin SH-groups and lipids are determined.     

Lipid Metabolism in Photoreceptor Membranes: Regulation and Mechanisms

Lipid metabolism in photoreceptor rod outer segments has attracted considerable attention because of its importance in providing the appropriate environment for supporting an efficient phototransduction mechanism. Recent studies suggest that lipid metabolism in these membranes is involved in the generation of second messengers and in signal transduction mechanisms. Phospholipid turnover is tightly regulated by phosphorylation-dephosphorylation reactions and light, and provides, in turn, with molecules capable of activating protein kinases and cellular processes such as membrane fusion or light-adaptation. These findings suggest that photoreceptor membrane lipids are more than just important structural components of the visual cell rod outer segment.     

Transbilayer coupling mechanism for the formation of lipid asymmetry in biological membranes. Application to the photoreceptor disc membrane.

An equilibrium transmembrane asymmetry in charged lipids is shown to arise as a result of oriented, bipolar proteins in the membrane. The basic interaction giving rise to the asymmetry is between a lipid molecule and a transbilayer potential generated by the asymmetric charge distribution in the protein. Thus, a protein can generate a lipid asymmetry without a direct binding interaction between lipid and protein. The generation of an asymmetry in charged lipid by this mechanism can also lead to a concomitant asymmetry in neutral lipids if deviations from ideality in the lipid mixture are taken into account. It is shown that regular solution theory applied to the lipid phase predicts an asymmetry in all components of a ternary mixture as long as one component is electrostatically oriented according to the mechanism mentioned above. The resulting asymmetry is not strongly salt dependent. The mechanism quantitatively accounts for the experimentally determined phospholipid asymmetry in the rod outer segment disc membrane of the vertebrate photoreceptor.     

Inhibition of erythrocyte Ca2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca2+-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca2+-ATPase both free thiols and close proximity to unsaturated lipid.     

The cGMP-gated channel of bovine rod photoreceptors is localized exclusively in the plasma membrane

Although there have been several reports pertaining to the existence of the cGMP-gated channel in the disk membrane of rod photoreceptors, its density there relative to that of the photoreceptor plasma membrane is unknown. Using immunoblotting, immunohistochemical, and reconstitution techniques on purified disk and plasma membrane preparations, we found that the density of channels in the plasma membrane was at least 50-fold higher than that of the disk membrane. Purification of membrane fractions without prior digestion of cytoskeletal components by mild trypsinization was found to increase the amount of channel protein present in disk membrane preparations. We propose that the presence of the channel protein in rod disk membrane preparations is an artifact arising from fusion of plasma membrane components during permeabilization of the photoreceptor cell.     

Aromatic indolinic aminoxyls as antioxidants in cardiac sarcoplasmic reticulum lipid and protein oxidation

The results presented demonstrate the influence of aromatic indolinic aminoxyls: 1,2-dihydro-2-ethyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C2) and 1,2-dihydro-2-octadecyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C18) on oxidation of lipids and proteins of cardiac sarcoplasmic reticulum membranes. We have used doxorubicin and t-butyl hydroperoxide as agents inducing oxidative stress in isolated rat cardiac sarcoplasmic reticulum membrane system. Carbonyl groups were measured as the end product of membrane protein oxidation, and thiobarbituric acid reactive substances were assessed as a marker of lipid peroxidation. Inhibition of peroxidation of certain membrane components depends on the length of acyl chain. Aminoxyl IA-C2 inhibits the lipid peroxidation process while IA-C18 is an efficient protector against protein oxidation.     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.     

Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium

The ciliary base is marked by a transition zone in which Y-shaped cross- linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y- shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA- binding component seen in cytochemical experiments.     

Curvature and hydrophobic forces drive oligomerization and modulate activity of rhodopsin in membranes

G protein-coupled receptors (GPCRs) are essential components of cellular signaling pathways. They are the targets of many current pharmaceuticals and are postulated to dimerize or oligomerize in cellular membranes in conjunction with their functional mechanisms. We demonstrate using fluorescence resonance energy transfer how association of rhodopsin occurs by long-range lipid-protein interactions due to geometrical forces, yielding greater receptor crowding. Constitutive association of rhodopsin is promoted by a reduction in membrane thickness (hydrophobic mismatch), but also by an increase in protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of boundary lipids. The fluorescence data correlate with the pK(a) for the MI-to-MII transition of rhodopsin, where deprotonation of the retinylidene Schiff base occurs in conjunction with helical movements leading to activation of the photoreceptor. A more dispersed membrane environment optimizes formation of the MII conformation that results in visual function. A flexible surface model explains both the dispersal and activation of rhodopsin in terms of bilayer curvature deformation (strain) and hydrophobic solvation energy. The bilayer stress is related to the lateral pressure profile in terms of the spontaneous curvature and associated bending rigidity. Transduction of the strain energy (frustration) of the bilayer drives protein oligomerization and conformational changes in a coupled manner. Our findings illuminate the physical principles of membrane protein association due to chemically nonspecific interactions in fluid lipid bilayers. Moreover, they yield a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes influence their protein-mediated functions.     

Lateral heterogeneity of bacterial membranes]

Isolated membranes of M. lysodeikticus were rapidly frozen and disrupted in a Hughes press. After disruption the fragments were centrifuged at 144000 g for 1 hour and part of the supernatant just above the pellet was subjected to isopycnic centrifugation in a continuous sucrose density gradient. It was found that the material tested was a mixture of fragments differing in their buoyant densities. These fragments also differed in their protein/lipid ratios, cytochrome content and dehydrogenase activities calculated per protein and lipid as well as in proportion of the respiratory chain enzymes. The results obtained are indicative of lateral heterogeneity of the bacterial membrane and the existence of areas in the membrane having high concentration of the respiratory chain enzymes. The latter may suggest that the system of substrate oxidation is segregated in the membrane. It is assumed that there exists in the membrane an exchange of components between different electron-transporting chains operated due to their lateral diffusion.     

How a G protein binds a membrane

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.     

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.     

Rhodopsin Receptors of Phototaxis in Green Flagellate Algae

Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the signal transduction chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suitable approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique rhodopsin-type protein. It shares common chromophore properties with retinal proteins from archaea. However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual rhodopsins. The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinal-binding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential function of this protein as the phototaxis receptor has been questioned in recent time.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Formation of helical protein assemblies of IgG and transducin on varied lipid tubules

Helical protein arrays on lipid tubules are valuable assemblies for studying protein structure and protein-lipid interactions through electron microscopy and crystallography. We describe conditions for forming such arrays from two proteins, IgG and transducin, the photoreceptor G protein, using a variety of lipid surfaces. Anti-dinitrophenyl (DNP) IgG arrays formed on DNP-phosphatidylethanolamine (DNP-PE) mixed with either galactosyl-ceramide lipids or phosphatidylcholine (PC) display different pH sensitivities and dimensions, yet have similar helical symmetries. DNP-PE/PC mixtures formed small crystals and large well-ordered flattened tubules. The peripheral membrane protein transducin (G(t)) formed helical arrays either on a mixture of cationic and neutral lipids or on residual photoreceptor lipids. Despite differences in lipid composition, the G(t) arrays have similar structures and show similar sensitivity to activation and variations in ionic environment. G(t) activation causes the helical assemblies to collapse to small vesicles, a process resembling the vesiculation of activated dynamin-lipid tubules. In a preliminary three-dimensional reconstruction, the hapten-bound IgG appears to make two contacts to the central lipid tubule, presumably via the F(ab) domains. The ability to generate a three-dimensional reconstruction without tilts illustrates one advantage of helical structures for two-dimensional crystallography, especially for visualizing protein-lipid interactions.     

The presence of membranes or micelles induces structural changes of the myristoylated guanylate-cyclase activating protein-2

Guanylate cyclase-activating proteins (GCAPs) are neuronal Ca²⁺ sensors that play a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase. GCAPs are N-terminally myristoylated, and the role of the myristoyl moiety is not yet fully understood. While protein lipid chains typically represent membrane anchors, the crystal structure of GCAP-1 showed that the myristoyl chain of the protein is completely buried within a hydrophobic pocket of the protein, which stabilizes the protein structure. Therefore, we address the question of the localization of the myristoyl group of GCAP-2 in the absence and in the presence of lipid membranes as well as DPC detergents (as a membrane substitute amenable to solution state NMR). We investigate membrane binding of both myristoylated and nonmyristoylated GCAP-2 and study the structure and dynamics of the myristoyl moiety of GCAP-2 in the presence of POPC membranes. Further, we address structural alterations within the myristoylated N-terminus of GCAP-2 in the presence of membrane mimetics. Our results suggest that upon membrane binding the myristoyl group is released from the protein interior and inserts into the lipid bilayer.     

Alpha-synuclein can function as an antioxidant preventing oxidation of unsaturated lipid in vesicles

Alpha-synuclein, a presynaptic protein associated with Parkinson's disease, is found as both soluble cytosolic and membrane-bound forms. Although the function of alpha-synuclein is unknown, several observations suggest that its association with membranes is important. In the present study we investigated the effect of alpha-synuclein on lipid oxidation in membranes containing phospholipids with unsaturated fatty acids. The kinetics of lipid oxidation were monitored by the change in fluorescence intensity of the dye C11-BODIPY. We find that monomeric alpha-synuclein efficiently prevented lipid oxidation, whereas fibrillar alpha-synuclein had no such effect. Our data suggest that the prevention of unsaturated lipid oxidation by alpha-synuclein requires that it bind to the lipid membrane. The antioxidant function of alpha-synuclein is attributed to its facile oxidation via the formation of methionine sulfoxide, as shown by mass spectrometry. These findings suggest that the inhibition of lipid oxidation by alpha-synuclein may be a physiological function of the protein.     

Genistein abrogates pre-hemolytic and oxidative stress damage induced by 2,2'-Azobis (Amidinopropane)

The pre-hemolytic mechanism induced by free radicals initiated from water-soluble 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) and its reversal by genistein was investigated in human erythrocytes. The time course of K+ efflux compared to the occurrence of hemolysis suggests that AAPH-induced hemolysis occurs indirectly via pore formation and band 3 oxidation as expected. However, genistein inhibited hemolysis, LDH release and membrane protein oxidation but not K+ efflux. This indicated that erythrocyte protein oxidation possibly in the hydrophobic core plays a significant role in the membrane pre-hemolytic damage. Chemiluminescence (CL) analysis carried out in non-lysed erythrocytes treated with AAPH showed a dramatic increase in CL indicating both reduced levels of antioxidants and increased membrane lipid peroxide. The V0 value was also increased up to 6 times, denoting a high degree of membrane peroxidation very early in erythrocyte membrane damage. The whole process was inhibited by genistein in a dose-dependent manner. These results indicate that the genistein inhibited both hemolysis and pre-hemolytic damage and also hindered membrane lipid peroxide formation and protein oxidation. In addition, it is suggested that pre-hemolytic damage is mediated mainly by the oxidation of both phospholipid and protein located in the deeper hydrophobic region of the membrane.