25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Control of membrane fusion by phospholipid head groups II. The role of phosphatidylethanolamine in mixtures with phosphatidate and phosphatidylinositol

Membrane fusion induced by Ca²⁺ and Mg²⁺ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca²⁺ or Mg²⁺ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca²⁺ or Mg²⁺. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca²⁺ in the presence of a Mg²⁺ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca²⁺, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (H_II) transition in membrane fusion, was examined by the temperature dependence of Ca²⁺-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes.     

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.     

Effect of calmodulin,Ca2+ and Mg2+ on the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes

Calmodulin-depleted isotonic erythrocyte ghosts contain 200 ng residual calmodulin/mg protein which is not removed by extensive washings at pCa²⁺ > 7. Specific activity and Ca²⁺-affinity of the (Ca²⁺ + Mg²⁺)ATPase increase at increasing calmodulin, with K_{0.5 Ca} of 0.38 μM at calmodulin concentrations corresponding to that in erythrocytes. High Ca²⁺ concentrations inhibit the enzyme. Specific activity and Ca²⁺-affinity of the enzyme decrease at increasing Mg²⁺ concentrations. The Ca²⁺ ? Mg²⁺ antagonism is likewise observed at inhibitory Ca²⁺ concentrations.     

A Novel Phosphorylated Lipid Counteracts Activation of the Renal Plasma Membrane (Ca2+ + Mg2+)ATPase by Endogenous Phosphatidylinositol-4-Phosphate

The plasma membrane (Ca²⁺ + Mg²⁺)ATPase is activated by acidic phospholipids in reconstituted systems. In this report it is shown that reversible phosphorylation of endogenous phosphatidylinositol regulates the renal plasma membrane (Ca²⁺ + Mg²⁺)ATPase, and that a novel phosphorylated lipid that can be isolated from the same membrane strongly counteracts the stimulatory effect of phosphatidylinositol-4-phosphate.     

Synergetic effects of Ca2+ and Cu2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant (K_H2O) of the coordinated H₂O, such as Ca²⁺ and Cu²⁺, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by ¹H-NMR and ESR using Ca²⁺ and / or Cu²⁺, which has a similar K_H2O to that of Ca²⁺. We found that Ca²⁺ did not hinder the molecular movements of PS. However, Cu²⁺ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca²⁺ and Cu²⁺, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.     

The phospholipid requirement of the (Ca + Mg)-ATPase from human platelets

The phospholipid requirement of the (Ca²⁺ + Mg²⁺)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Laser Raman study of internally perfused muscle fibers effect of Mg2+, ATP and Ca2+

Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca²⁺, Mg²⁺ and ATP. The comparaison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg²⁺ and Ca²⁺ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg²⁺ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca²⁺ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the α-helical content, or of the orientation, of some of the contractile proteins.     

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine?phosphatidylethanolamine?0. Acidic phospholipids increased V_max-Ca²⁺ and lowered the value of K_0.5-Ca²⁺ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K_0.5-Ca²⁺ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1–116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Δ74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca²⁺-ATPase provides new evidence that type 2B Ca²⁺-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.     

Ca2+-induced isotropic motion and phosphatidylcholine flip-flop in phosphatidylcholine-cardiolipin bilayers

Ca²⁺ induces a structural change in phosphatidylcholine-cardiolipin bilayers, which is visualised by freeze-fracturing as lipidic particles associated with the bilayer and is detected by ³¹P-NMR as isotropic motion of the phospholipids. In this structure a rapid transbilayer movement of phosphatidylcholine and a highly increased permeability towards Mn²⁺ are observed.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.