Ca2+-induced lateral phase separation in ternary mixtures of phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine inferred by calorimetry

Phase transition characteristics of ternary mixtures of dipalmitoylphosphatidic acid, dipalmitoylphosphatidylcholine, and phosphatidylethanolamine (dilauroyl-, dimyristoyl-, or dipalmitoyl-phosphatidylethanolamine) were examined by differential scanning calorimetry at various concentrations of calcium ions. In the absence of calcium ion, these ternary mixtures showed a broad phase transition, which suggested a high miscibility of these components. Addition of a low concentration of calcium ions showed a tendency to induce separation of the transition into a major one and a small one. As the concentration of calcium ions increased, the separation became more distinct and the transition enthalpy of the major transition decreased. At a Ca2+/dipalmitoylphosphatidic acid ratio (mol/mol) of 1.5, the major transition became similar to the transition of dipalmitoylphosphatidylcholine and the phosphatidylethanolamine binary mixture. On the other hand, in a binary mixture dipalmitoylphosphatidic acid and dipalmitoylphosphatidylcholine, the Ca2+-induced phase separation was distinct even at the lowest concentration of calcium ions used in the present experiment. The results indicate that a high concentration of calcium ion is required for inducing complete phase separation of the transition event in the ternary mixture because of its high miscibility. It is suggested that the phase separation revealed by spin-labeled phospholipid in ternary mixtures at a low Ca2+ concentration might be a phase separation in a local domain.     

Molecular species of phosphatidylinositol, phosphatidic acid and diacylglycerol in a phytohemagglutinin-stimulated T-cell leukemia line

Addition of phytohemagglutinin to JURKAT cells, a human T-cell leukemia line, induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (and may also be phosphatidylinositol 4-phosphate) and an accumulation of phosphatidic acid. The accumulation and disappearance of the various molecular species of phosphatidic acid, diacylglycerol and phosphatidylinositol (PtdIns) in response to phytohemagglutinin was studied in JURKAT cells. The cells were prelabeled with [2-3H]glycerol for 2 days and 3H-labeled lipids were isolated from the cells after incubation for 2 min at 37 degrees C in the absence or in the presence of phytohemagglutinin. The isolated 3H-labeled lipids were separated into individual molecular species by reverse-phase HPLC after conversion to their 1,2-[3H]diacylglycerol acetate derivatives either by acetolysis or by acetylation. Stimulation with phytohemagglutinin induced a 2-fold increase in [3H]phosphatidic acid. The molecular species of the accumulated [3H]phosphatidic acid consisted of polyenoic species, which were almost absent in the [3H]phosphatidic acid of the unstimulated cells. Stearoylarachidonoyl species of [3H]phosphatidic acid accumulated most prominently. Although an accumulation of [3H]diacylglycerol was hardly measurable in the phytohemagglutinin-stimulated cells, the HPLC analysis of the molecular species of [3H]diacylglycerol showed a 2-fold increase in the stearoylarachidonoyl species in the stimulated cells. Stimulation with phytohemagglutinin had almost no effect on the composition of molecular species of [3H]PtdIns. The stearoylarachidonyl species is the most abundant molecular species of PtdIns in JURKAT cells. These results suggest that the [3H]diacylglycerol moiety of [3']phosphatidic acid originates from inositol lipid(s). The results also suggest a rapid and preferential phosphorylation of the diacylglycerol formed by receptor-stimulated hydrolysis of inositol lipid(s).     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Increase in free Ca2+ level determined by quin-2 in spleen lymphocytes from irradiated rats

The output of lymphocytes, obtained on a ficoll-verografin gradient and intracellular free Ca2+ concentration were studied in the rat spleen 1, 3 and 6 days after X-ray irradiation in a dose of 0.5 Gy. The amount of lymphocytes decreased by 30% a day after irradiation and turned to the control level 3 days after exposure of animals. Free Ca2+ concentration remains abnormally high during all the examined period, increasing to 411 nM a day after irradiation and approaching to the control value 6 days after irradiation.     

Characterization of Ca2+ transport in rat renal brush-border membranes and its modulation by phosphatidic acid.

The Ca2+ transport process by isolated renal brush-border membranes was characterized and the influence of the acidic phospholipid phosphatidic acid (PtdA) on this transport process was assessed. Ca2+ uptake by brush-border membranes exhibited saturation kinetics. It was inhibitable by a variety of multivalent cations, as well as by Ca2+-entry inhibitors, including verapamil, Ruthenium Red and gentamicin. It was selective for Ca2+ compared with Mg2+. This process was also electrophoretic since generation of K+ and anion-diffusion potentials, negative inside the vesicle, increased Ca2+ uptake. Elevations in PtdA content of brush-border membranes by either exogenous addition or endogenous generation of PtdA by incubating brush-border membranes with MgATP2- elevated the rate of Ca2+ uptake. This ATP effect could not be attributed to (Ca2+ + Mg2+)-dependent ATPase or contaminating membrane fragments. PtdA also increased the magnitude and rate of Ca2+ efflux from brush-border membranes preloaded with Ca2+. These modulations in uptake and efflux were not observed with phosphatidylcholine or phosphatidylinositol. In summary, these results are consistent with the presence of an electrophoretic uniport system for Ca2+ in renal brush-border membranes, and demonstrate that PtdA uniquely among phospholipids tested appears to facilitate transmembrane flux of Ca2+ across this membrane preparation.     

Capacitive and ionic currents in BLM from phosphatidic acid in Ca2+-induced phase transition

The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm.     

Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37 °C, pH 7.2, 0.5 m m free Mg²⁺), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca²⁺ (pH 6.6–5.9, 0.3 m m Ca²⁺). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cation-lysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.     

The biosynthesis of phosphatidic acid and phosphatidylinositol in mammalian pancreas

1. The labelling of guinea-pig pancreas phospholipids in vivo after intraperitoneal injection of [(32)P]orthophosphate is described. 2. Acyl-CoA synthetase activity in pancreas homogenates has been studied. There is no absolute requirement for added fatty acids, indicating an adequate supply of endogenous fatty acids in these preparations. 3. Phosphatidic acid is formed in guinea-pig pancreas preparations by two distinct routes, namely the acylation of l-3-glycerophosphate and the phosphorylation of 1,2-diglyceride. Phosphatidic acid formed by either mechanism is converted into phosphatidylinositol by guinea-pig pancreas in vitro. 4. The enzymes of pancreas that convert phosphatidic acid into phosphatidylinositol via CDP-diglyceride have been characterized. 5. Addition of bovine serum albumin is necessary in assaying certain of these enzymes.     

Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid

Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced PtdIns-P2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by alpha-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.     

Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.     

Ca2+-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.     

Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities

Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhibition, similar to that observed in C6-2B cells, which predominantly express AC(6). Ca(2+) inhibition of AC(6) in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC(6) to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.     

Diacylglycerol, phosphatidic acid, and the converting enzyme, diacylglycerol kinase, in the nucleus

There exists phosphoinositide (PI) cycle in the nucleus, which is operated differentially from the classical PI cycle at the plasma membrane. Evidence has been accumulated that nuclear PIs and the related enzymes are closely involved in a variety of nuclear processes, although the details remain to be elucidated. In this mini review, some components of PI cycle, i.e., diacylglycerol, phosphatidic acid, and the converting enzyme, diacylglycerol kinase, in the nucleus are discussed with focusing on the lipid metabolism, cell cycle regulation, and animal models.     

Phytohemagglutinin induces rapid degradation of phosphatidylinositol 4,5-bisphosphate and transient accumulation of phosphatidic acid and diacylglycerol in a human T lymphoblastoid cell line, CCRF-CEM

The human T lymphoblastoid cell line designated CCRF-CEM responds to phytohemagglutinin with a 3.7-fold enhancement of the 32PO4 incorporation into phosphatidylinositol. In myo-[2-3H]inositol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a 3.3-fold accumulation of myo-[2-3H]inositol phosphate during 15 min incubation at 37 degrees C in the presence of 5 mM LiCl. Since Li+ is a potent inhibitor of myo-inositol-1-phosphatase, the results indicate that phytohemagglutinin induces the hydrolysis of inositol lipids in CCRF-CEM cells. In 32PO4-prelabeled CCRF-CEM cells, phytohemagglutinin induced a breakdown of 28% of [32P]phosphatidylinositol 4,5-bisphosphate 40-60 s after the stimulation. The decrease of [32P]phosphatidylinositol 4,5-bisphosphate was found as early as 10 s after the stimulation. This decrease was followed by an increased 32P-labeling of phosphatidic acid. In [2-3H]glycerol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid and [3H]diacylglycerol. The amount of [3H]phosphatidic acid in the stimulated cells was 3.7-times the control value at 2 min after the stimulation, whereas the amount of [3H]diacylglycerol in the stimulated cells was 1.5-times the control value at 5 min after the stimulation. In [3H8]arachidonate-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid; the amount was 2.5-times the control value at 2 min after the stimulation. Quinacrine (1 mM) caused 41% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation in [2-3H]glycerol-prelabeled cells. Stimulation in a Ca2+-free saline containing 1 mM EGTA caused 53% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation. The results presented in this paper indicate that a human T lymphoblastoid cell line, CCRF-CEM, responds to phytohemagglutinin with a rapid turnover of inositol lipids.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

Modulation of the mammalian target of rapamycin pathway by diacylglycerol kinase-produced phosphatidic acid

The protein known as mammalian target of rapamycin (mTOR) regulates cell growth by integrating different stimuli, such as available nutrients and mitogenic factors. The lipid messenger phosphatidic acid (PA) binds and positively regulates the mitogenic response of mTOR. PA generator enzymes are consequently potential regulators of mTOR. Here we explored the contribution to this pathway of the enzyme diacylglycerol kinase (DGK), which produces PA through phosphorylation of diacylglycerol. We found that overexpression of the DGKzeta, but not of the alpha isoform, in serum-deprived HEK293 cells induced mTOR-dependent phosphorylation of p70S6 kinase (p70S6K). After serum addition, p70S6K phosphorylation was higher and more resistant to rapamycin treatment in cells overexpressing DGKzeta. The effect of this DGK isoform on p70S6K hyperphosphorylation required the mTOR PA binding region. Down-regulation of endogenous DGKzeta by small interfering RNA in HEK293 cells diminished serum-induced p70S6K phosphorylation, highlighting the role of this isoform in the mTOR pathway. Our results confirm a role for PA in mTOR regulation and describe a novel pathway in which DGKzeta-derived PA acts as a mediator of mTOR signaling.     

Ca2+ and phosphatidylinositol 4,5-bisphosphate stabilize a Gbeta gamma-sensitive state of Ca V2 Ca 2+ channels

Direct interactions between G-protein betagamma subunits and N- or P/Q-type Ca(2+) channels mediate the inhibitory action of several neurotransmitters in the brain. Membrane potential, channel phosphorylation, or auxiliary subunit association tightly regulate these interactions and the consequent inhibition of Ca(2+) current. We now provide evidence that intracellular Ca(2+) concentration and phosphoinositides play a stabilizing role in this direct voltage-dependent inhibition. Lowering resting cytosolic Ca(2+) concentration in Xenopus oocytes expressing Ca(V)2Ca(2+) channels strongly decreased basal as well as phasic, agonist-dependent inhibition of Ca(2+) channels by G-proteins. Decreasing phosphoinositide levels also suppressed G-protein inhibition and completely occluded the effects of a subsequent injection of Ca(2+) chelator. Similar regulations are observed in mouse dorsal root ganglia neurons. Alteration of G-protein block by these agents is independent of protein phosphorylation, cytoskeleton dynamics, and GTPase or GDP/GTP exchange activity, suggesting a direct action at the level of the Ca(2+) channel/Gbetagamma-protein interaction. Moreover, affinity binding experiments of intracellular loops of the Ca(V)2.1 Ca(2+) channels to different phospholipids revealed specific interactions between the C-terminal tail of the channel and phosphoinositides. Taken together these data indicate that a Ca(2+)-sensitive interaction of the C-terminal tail of P/Q channels with the plasma membrane is important for G-protein regulation.     

Polymyxin B and Ca(2+) induce conductance fluctuations in the dipalmitoyl phosphatidic acid bilayer lipid membrane

Polymyxin B in micromolar concentrations induces current fluctuations in liquid crystalline bilayer lipid membranes from dipalmitoylphosphatidic acid identified as ion channels. The appearance of ion channels correlates with phase separation of the lipid in the presence of peptide polycations detected by differential scanning calorimetry. Ca2+ also induces the formation of ion channels in liquid crystalline bilayer lipid membranes from dipalmitoylphosphatidic acid followed by the phase transition of the phospholipid. The capacitive current, which indicates the possibility of structural transformations of bilayer-non-bilayer type (hexagonal phase II), precedes the formation of Ca(2+)-induced channels in bilayer lipid membranes from dipalmitoylphosphatidic acid.