Molecular organization of the non-bilayer phospholipid arrangements that induce an autoimmune disease resembling human lupus in mice

Abstract

Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H_II), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn²⁺ cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H_II-preferring lipid, in the absence or presence of Mn²⁺, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H_II-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn²⁺, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.     

Ca2+-cardiolipin interaction in a model system Selectivity and apparent high affinity

The interaction of cardiolipin with Ca²⁺ was assessed by measuring the cardiolipin-mediated extraction of ⁴⁵Ca²⁺ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca²⁺ with high affinity [$\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(}\text{apparent}\text{)=0.70±0.17 μ}\text{M (S.D.)}$]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca²⁺ is insensitive to Na⁺, but is inhibited by divalent cations with $\text{Mn}{}^{\mathrm{2+}}\text{>}\text{Zn}{}^{\mathrm{2+}}\text{>}\text{Mg}{}^{\mathrm{2+}}$. In addition La³⁺ and Ruthenium red are particularly potent inhibitors of Ca²⁺ binding by cardiolipin. Cardiolipin-mediated extraction of Ca²⁺ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca²⁺-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.     

In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium

We report that a localized intracellular and extracellular Ca²⁺ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca²⁺-sensitive protein (yellow cameleon 3.0) report that intracellular Ca²⁺ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca²⁺ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca²⁺ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca²⁺ mobilization. Indomethacin and verapamil also inhibit the luminal Ca²⁺ increase. Intracellular Ca²⁺ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca²⁺ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca²⁺ and unevenly inhibits late-phase intracellular Ca²⁺ mobilization. Both modes of Ca²⁺ chelation slow gastric repair. In plasma membrane Ca-ATPase 1^+/− mice, but not plasma membrane Ca-ATPase 4^−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca²⁺ increase. We conclude that endogenous Ca²⁺, mobilized by signaling pathways and transmembrane Ca²⁺ transport, causes increased Ca²⁺ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.     

Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria

Phosphoenolpyruvate partially inhibits the accumulation of Ca²⁺ in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca²⁺ uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca²⁺ uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca²⁺ uptake completely while partially inhibiting NADH-supported Ca²⁺ uptake. The oxaloacetate inhibition of NADH-supported Ca²⁺ uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca²⁺ uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca²⁺ uptake.     

Ca-induced stimulation of the membrane binding of Escherichia coli SecA and its association with signal peptides of secretory proteins

Previously, it was found that Ca²⁺ stimulates the intrinsic Escherichia coli SecA ATPase activity [Kim et al., FEBS Lett. 493 (2001) 12-16]. Now, we suggest that Ca²⁺ is required for efficient interaction of SecA with membranes and the signal peptide of ribose-binding protein. When the amount of external Ca²⁺ was enhanced, the amounts of membrane-bound SecA and its lipid/ATPase activity increased. In the presence of entrapped Ca²⁺ in liposomes, the binding was also stimulated in a Ca²⁺ concentration-dependent manner. The effect of Ca²⁺ on the functional regulation of SecA was also evident in the presence of the signal peptides of secretory proteins, which the interaction of SecA with the signal peptide increased with increasing Ca²⁺ concentration in the presence of membranes. However, other divalent cations including Mg²⁺, Mn²⁺, and Zn²⁺ had inhibitory or no effect, suggesting a specific role of Ca²⁺ in SecA interaction with lipid bilayers and signal peptides.     

3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca-ATPase

3-O-Methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca²⁺-ATPase in the absence of Ca²⁺, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca²⁺, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca²⁺ concentration produces a bell-shaped curve with a maximum at 50 μM Ca²⁺. At optimal Ca²⁺ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca²⁺, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca²⁺ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca²⁺-ATPases.     

High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes

We studied the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_e) on adipocyte differentiation. Preadipocytes exposed to continuous [Ca²⁺]_e higher than 2.5 mmol/l accumulated little or no cytoplasmic lipid compared to controls in 1.8 mmol/l [Ca²⁺]_e. Differentiation was monitored by Oil Red O staining of cytoplasmic lipid and triglyceride assay of accumulated lipid, by RT-PCR analysis of adipogenic markers, and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). Elevated [Ca²⁺]_e inhibited expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and steroid regulatory binding element protein. High [Ca²⁺]_e significantly inhibited differentiation marker expression including adipocyte fatty acid binding protein, and GPDH. The decrease in Pref-1 expression that accompanied differentiation also was prevented by high [Ca²⁺]_e. Treatment of 3T3-L1 cells with high [Ca²⁺]_e did not significantly affect cell number or viability and did not trigger apoptosis. Levels of intracellular Ca⁺² remained unchanged in various [Ca²⁺]_e. Treatment of 3T3-L1 with pertussis toxin (PTX) partially restored lipid accumulation and increased differentiation markers in cells treated with 5 mmol/l [Ca²⁺]_e. ‘Classical’ parathyroid cell Ca²⁺ sensing receptors (CaSR) were not detected either by RT-PCR or by Western blotting. These results suggest that continuos exposure to high [Ca²⁺]_e inhibits preadipocyte differentiation and that this may involve a G-protein-coupled mechanism mediated by a novel Ca²⁺ sensor or receptor.     

Synthetic peptide K+ carrier with Ca2+ inhibition

Based on a proposed solution conformation of the Ca²⁺ ion complex of the repeat hexapeptide of elastin, l-Val-l-Ala-l-Pro-Gly-l-Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N-formyl; Me, N-methyl; Sar, N-methyl glycine). Both peptides bound Ca²⁺ preferentially but did not exhibit the properties of a Ca²⁺ carrier. They were however active as K⁺ carriers although K⁺ ion titration curves showed a much lower affinity for K⁺ than for Ca²⁺. The addition of Ca²⁺ or Mg²⁺ to the bilayer system inhibited the peptide K⁺ carrier activity. Three possible explanations of this interesting Ca²⁺ inhibition of carrier activity are irreversible complexation of Ca²⁺, mixed ligand complex formation involving Ca²⁺, lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.     

Effects of ruthenium red on Ca2+ uptake and ATPase of sarcoplasmic reticulum of rabbit skeletal muscle

Ruthenium red, a powerful inhibitor of Ca²⁺ transport by mitochondria, does not inhibit the active Ca²⁺ uptake by sarcoplasmic reticulum isolated from rabbit skeletal muscle promoted by 5 mM ATP-Mg in the presence or absence of potassium oxalate. Although concentrations of ruthenium red up to 100 μM do not affect the active uptake of Ca²⁺, 25 μM of the inorganic dye inhibit the passive binding of Ca²⁺ by about 50%. This inhibitory effect is observed in sarcoplasmic reticulum even after its lipid fraction is extracted with acetone.Although active Ca²⁺ uptake by sarcoplasmic reticulum is not inhibited by ruthenium red, in the absence of oxalate it inhibits significantly the Ca²⁺-dependent ATPase activity but not the Mg²⁺-ATPase. However, if potassium oxalate is present, the Ca²⁺-stimulated ATPase is not sensitive to the dye. It is not clear how oxalate functions to protect the Ca²⁺-ATPase against the inhibitor effect of ruthenium red.The high sensitivity to ruthenium red of the Ca²⁺ transport mechanism in mitochondria as compared to the Ca²⁺ transport in sarcoplasmic reticulum may be useful in determining the extent to which each organelle functions in the cell to regulate intracellular free Ca²⁺.     

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca²⁺ regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca²⁺ oscillation without changing the basal intracellular Ca²⁺ concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca²⁺ concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca²⁺ regulation may be a common feature among animal sperm.     

Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca²⁺ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, W_ex. By measuring W_ex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers.In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8·10^?8 cm²/s at 59°C.Addition of Ca²⁺ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca²⁺-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of W_ex on the Ca²⁺ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca²⁺ concentration clusters containing about 30 mol% lecithin are formed. At high lecithin or Ca²⁺ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn²⁺ using ESR spectroscopy.Polylysine leads to the same strong increase in the lecithin segregation as Ca²⁺. The transition of the phosphatidic acid bound by the polypeptide is shifted from T_t = 47.5° to T_t = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport

Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca²⁺ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca²⁺. A low affinity Ca²⁺ uptake system (K_m > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H⁺/Ca²⁺ antiport. A high affinity Ca²⁺ uptake system (K_m = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca²⁺ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca²⁺/H⁺ antiport activity could only by partially ascribed to an effect of low temperature on H⁺-ATPase activity, ATP-dependent H⁺ transport, passive H⁺ fluxes, or passive Ca²⁺ fluxes. These results suggest that low temperature directly affects Ca²⁺/H⁺ exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca²⁺/H⁺ exchange protein or by an indirect effect of temperature on lipid interactions with the Ca²⁺/H⁺ exchange protein.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Light-affected ca fluxes in protoplasts from vallisneria mesophyll cells

In Vallisneria gigantea Graebner mesophyll cells, red light irradiation induces cytoplasmic streaming by decreasing the Ca²⁺ concentration in the cytoplasm, while far-red light irradiation inhibits it by increasing the concentration (S Takagi, R Nagai 1985 Plant Cell Physiol 26: 941-951). To examine the effects of light irradiation on Ca²⁺ fluxes across the cell membrane, protoplasts are isolated from the mesophyll cells. Changes in Ca²⁺ concentration in a solution bathing the protoplasts are monitored by spectrophotometry, using the Ca²⁺ -sensitive dye murexide. Red light irradiation induces an increase in Ca²⁺ concentration, which means an efflux of Ca²⁺ from the protoplasts. Subsequent far-red light irradiation produces a rapid decrease in Ca²⁺ concentration down to the dark control level; however, this is not observed in the presence of the Ca²⁺ -channel blocker nifedipine. Vanadate inhibits both the streaming and the Ca²⁺ efflux induced by red light irradiation. The results suggest that red light and far-red light control Ca²⁺ movements across the cell membrane, which in turn regulate the streaming.     

Inhibition Mechanism of the Intracellular Transporter Ca2+-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca²⁺-ATPase activity and ATP-dependent Ca²⁺ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca²⁺-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca²⁺ transport was evaluated. Since Ca²⁺ transport was more sensitive to inhibition than Ca²⁺-ATPase activity the drugs under study caused Ca²⁺/P_i uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca²⁺-pump functioning. Dimethyl-celecoxib prevented Ca²⁺ binding by stabilizing the inactive Ca²⁺-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca²⁺–pump was exposed to the drug in the presence of Ca²⁺ before phosphorylation. This provided evidence of non-preferential interaction with the Ca²⁺-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca²⁺ was consistent with the mentioned effect on Ca²⁺ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca²⁺-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and dimethyl-celecoxib with the intracellular Ca²⁺ transporter at the inhibition site of hydroquinones.     

Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

Calcium regulation of Ca²⁺-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca²⁺ permeability. The molecular mechanisms associated with PC2 regulation by Ca²⁺ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2_hst) but not the in vitro translated protein (PC2_iv), functionally responds to changes in intracellular (cis) Ca²⁺. In this study we determined the regulatory effect(s) of Ca²⁺-sensitive and -insensitive actin-binding proteins (ABPs) on PC2_iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2_iv channel function in the presence of cis Ca²⁺, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2_iv channel function in the presence, but no effect in the absence of cis Ca²⁺. Gelsolin stimulated PC2_iv channel function in the presence, but not the absence of cis Ca²⁺. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2_iv channel function both in the presence and absence of Ca²⁺. The distinct effect(s) of the ABPs on PC2_iv channel function demonstrate that Ca²⁺ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca²⁺-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation.     

Influence of vanadate on calcium fluxes and net movement of calcium in intact squid axons

Intracellular vanadate at a concentration of 100 μM inhibits the uncoupled efflux of Ca²⁺ from intact axons but has little effect on the exchange fluxes and on the Ca²⁺-dependent Na⁺ efflux. External vanadate has no effect on the Ca²⁺ efflux. In addition and most importantly intracellular vanadate inhibits the Ca²⁺ efflux in the presence of external Na⁺ and Ca²⁺ suggesting that the uncoupled efflux is operative under physiological conditions. Measurements of the net movements of Ca²⁺ under near physiological conditions have confirmed this conclusion.     

Calcium inhibits diacylglycerol uptake by serum albumin

Serum albumin is an abundant protein in blood plasma, that is well-known for its ability to transport hydrophobic biomolecules and drugs. Recent hypotheses propose that serum albumin plays a role in the regulation of lipid metabolism in addition to its lipid transport properties. The present work explores the capacity of bovine serum albumin (BSA) to extract diacylglycerols (DAG) from phospholipid bilayers, and the inhibition of such interaction by divalent cations. Quantitative measurements using radioactive DAG and morphological evidence derived from giant unilamellar vesicles examined by confocal microscopy provide concurrent results. BSA extracts DAG from vesicles consisting of phosphatidylinositol/DAG. Long, saturated DAG species are incorporated more readily than the shorter-chain or unsaturated ones. Divalent cations hinder DAG uptake by BSA. For Ca²⁺, the concentration causing half-maximal inhibition is ≈ 10 μM; 90% inhibition is caused by 100 μM Ca²⁺. Sr²⁺ requires concentrations one order of magnitude higher, while Mg²⁺ has virtually no effect. As an example on how DAG uptake by BSA, and its inhibition by Ca²⁺, could play a regulating role in lipid metabolism, a PI-specific phospholipase C has been assayed in the presence of BSA and/or Ca²⁺. BSA activates the enzyme by removing the end-product DAG, but the activation is reverted by Ca²⁺ that inhibits DAG uptake.     

NMR analysis of free and lipid nanodisc anchored CEACAM1 membrane proximal peptides with Ca2+/CaM

CEACAM1, a homotypic transmembrane receptor with 12 or 72 amino acid cytosolic domain isoforms, is converted from inactive cis-dimers to active trans-dimers by calcium-calmodulin (Ca²⁺/CaM). Previously, the weak binding of Ca²⁺/CaM to the human 12 AA cytosolic domain was studied using C-terminal anchored peptides. We now show the binding of ¹⁵N labeled Phe-454 cytosolic domain peptides in solution or membrane anchored using NMR demonstrates a significant role for the lipid bilayer. Although binding is increased by the mutation Phe454Ala, this mutation was previously shown to abrogate actin binding. On the other hand, Ca²⁺/CaM binding is abrogated by phosphorylation of nearby Thr-457, a post-translation modification required for actin binding and subsequent in vitro lumen formation. Binding of Ca²⁺/CaM to a membrane proximal peptide from the long 72 AA cytosolic domain anchored to lipid nanodiscs was very weak compared to lipid free conditions, suggesting membrane specific effects between the two isoforms. NMR analysis of ¹⁵N labeled Ca²⁺/CaM with unlabeled peptides showed the C-lobe of Ca²⁺/CaM is involved in peptide interactions, and hydrophobic residues such as Met-109, Val-142 and Met-144 play important roles in binding peptide. This information was incorporated into transmembrane models of CEACAM1 binding to Ca²⁺/CaM. The lack of Ca²⁺/CaM binding to phosphorylated Thr-457, a residue we have previously shown to be phosphorylated by CaMK2D, also dependent on Ca²⁺/CaM, suggests stepwise binding of the cytosolic domain first to Ca²⁺/CaM and then to actin.