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Influence of Ca2+ on the surface behavior of phosphatidic acid and its mixture with diacylglycerol pyrophosphate at different pHs
Chemistry and Physics of Lipids 228 (2020) 104887
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Influence of Ca2+ on the surface behavior of phosphatidic acid and its mixture with diacylglycerol pyrophosphate at different pHs
T
Micaela Peppino Marguttia, Natalia Wilkeb,c, Ana Laura Villasusoa,d,* a
Universidad Nacional de Río Cuarto, FCEFQyN, Departamento de Biología Molecular, Río Cuarto, Argentina Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Química Biológica Ranwel Caputto, Córdoba, Argentina c CONICET, Universidad Nacional de Córdoba, Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Córdoba, Argentina d CONICET, Universidad Nacional de Río Cuarto, Instituto de Biotecnologia Ambiental y Salud, (INBIAS), Río Cuarto, Argentina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Diacylglycerol pyrophosphate Phosphatidic acid Membrane packing Glycerophospholipid monolayers Calcium
The signaling lipids phosphatidic acid (PA) and diacylglycerol pyrophosphate (DGPP) are involved in regulating the stress response in plants. PA and DGPP are anionic lipids consisting of a negatively charged phosphomonoester or pyrophosphate group attached to diacylglycerol, respectively. Changes in the pH modulate the protonation of their head groups modifying the interaction with other effectors. Here, we examine in a controlled system how the presence of Ca2+ modulates the surface organization of dioleyl diacylglycerol pyrophosphate (DGPP) and its interaction with dioleoyl phosphatidic acid (DOPA) at different pHs. Both lipids formed expanded monolayers at pH 5 and 8. At acid and basic pHs, monolayers formed by DOPA or DGPP became denser when Ca2+ was added to the subphase. At pH 5, Ca2+ also induced an increase of surface potential of both lipids. Conversely, at pH 8 the effects induced by the presence of Ca2+ on the surface potential were reversed. Mixed monolayers of DOPA and DGPP showed a non-ideal behavior. The addition of even tiny amounts of DGPP to DOPA films caused a reduction of the mean molecular area. This effect was more evident at pH 8 compared to pH 5. Our finding suggests that low amounts of DGPP in an film enriched in DOPA could lead to a local increase in film packing with a concomitant change in the local polarization, further regulated by local pH. This fact may have implications for the assigned role of PA as a pH-sensing phospholipid or during its interaction with proteins.
1. Introduction Phosphatidic acid (PA) is the glycerophospholipid with the simplest chemical structure in biological membranes. Phosphatidic acids (PAs) are minor components of biological membrane. However, PAs are key molecules in lipid metabolism, as they are used in the synthesis of the other phospholipids, galactolipids and triglycerides (Testerink and Munnik, 2011). In addition, PA functions as a signaling lipid involved in regulation of vesicular trafficking, secretion, pH sensing and stress response in eukaryotic cell (Pokotylo et al., 2018; Li et al., 2019). In plants, PA´s composition is also modulated in response to stress associated with water deficit, temperature, saline conditions, starvation, free radicals, and interactions with microbes (Liu et al., 2013). In quiescent cells, PA levels are very low, and the concentration increases mainly through the pathways driven by phospholipase D via hydrolysis of phosphatidylcholine (PC) or by phospholipase C which catalyzes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2) realizing inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG) followed by diacylglycerol kinase action (Hong et al., 2016). Despite PA behaves as a signal lipid, its local accumulation could disturb the homeostasis of cellular membrane (Kooijman and Burger, 2009). PA molecules have an inverted conical shape with a relatively small headgroup in comparison with the hydrophobic part of the molecule, which promotes negative spontaneous curvature (Kooijman et al., 2005). Thus, the tightly regulation of PA levels is an important event for maintaining the integrity of cellular membrane (Arisz et al., 2018). The dephosphorylation of PA to diacylglycerol (DAG) by phosphatidate phosphatase, or its phosphorylation to diacylglycerol pyrophosphate (DGPP) by phosphatidate kinase (PAK, see Fig. 1) are two important pathways involved in the attenuation of PA level (Racagni et al., 2008; Villasuso et al., 2013; Peppino Margutti et al., 2018). DGPP is also a minor phospholipid associated with stress response in plant.
⁎ Corresponding author at: Departamento de Biología Molecular-INBIAS-FCEFQN, Universidad Nacional de Río Cuarto, Ruta Nac. 36 - Km. 601, X5804BYA Río Cuarto, Córdoba, Argentina. E-mail address: [email protected] (A.L. Villasuso).
https://doi.org/10.1016/j.chemphyslip.2020.104887 Received 21 October 2019; Received in revised form 13 December 2019; Accepted 30 January 2020 Available online 03 February 2020 0009-3084/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structure of 1,2-dioleoyl-sn-glycerol 3 phosphate (DOPA) and dioleoylglycerol pyrophosphate (DGPP). Arrows indicate the dephosphorylation of DGPP to DOPA by lipid phosphatidate phosphatase (LPP), or DOPA phosphorylation to DGPP by phosphatidate kinase (PAK).
2003). This is because PA, which bears a single negative charge at neutral pH, is a doubly ionizable molecule. Calcium binding displaces the second proton, leaving a neutral PA:Ca(II) complex (Laroche et al., 1991). In addition, it is known that the interaction of calcium ions with others lipid membranes rigidifies and orders lipid bilayers and that the binding mode depends on calcium concentration (Binder and Zschornig, 2002; Garidel and Blume, 2005; Melcrova et al., 2016) Previously, we have demonstrated that the packing and electrostatic properties of pure lipids or mixed films composed of DGPP and POPA were affected by the subphase pH (Villasuso et al., 2010). DGPP in Langmuir monolayers interacts with cations such as zinc modulating the film packing properties (Villasuso et al., 2014). To gain insights into the dynamic properties of PA and DGPP, here we focus on the effect of calcium and pH on the interfacial behavior of pure and mixed lipids films. The main objective was to investigate the interaction of both DGPP and DOPA, the PA with the same hydrocarbon chain than DGPP, with calcium. In this regards, Langmuir technique offers the possibility of forming a well-defined monolayer of the amphiphilic molecules under study, with a unique control of the area per molecule. Although Langmuir monolayer is a highly simplify system, we circumscribe our conclusions to artificial membranes, that may give an insight of what happens in a very small region and at short times in cells. Our data show that DOPA is less sensitive to pH changes than DGPP. At acid and basic pHs, DOPA formed liquid-expanded monolayers with a detectable reorganization during the compression. Analyses of mixed films of DGPP and DOPA indicated that the interaction between lipids was stronger than those in the pure films, thus forming more compact monolayers. In contrast, effects promoted by Ca2+ were more evident at basic pHs, where the films were more charged and loosely packed than at acid pHs. This fact suggests that the biosynthesis of low amounts of DGPP in an environment enriched in DOPA could evoke a complex reorganization of film packing affecting to local electrostatics.
DGPP synthesis is temporary and it is always related with variations of the amount of PA (Paradis et al., 2011). DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. It was shown that, depending on pH, the pyrophosphate moiety of DGPP could display 2 or 3 negative charges making it a highly polar molecule (Villasuso et al., 2010; Strawn et al., 2012). The ionization of the pyrophosphate group is important in the electrostatic interactions between DGPP and proteins as well as with bivalent cations (Villasuso et al., 2014; Astorquiza et al., 2016). Although, several aspects of DGPP are still unknown, it has been shown that the ionization properties of the phosphomonoester of DGPP mimic those of PA following the electrostatic-hydrogen bond switch model (Strawn et al., 2012). However, DGPP is not a cone shaped lipid, i.e., it is not capable of imparting negative curvature stress to the membrane that could facilitate the insertion of hydrophobic protein domains in the membrane (Strawn et al., 2012). Thus, the charge and packing properties of DGPP might contribute with the turn-off of the PA-signal. Accordingly, it was hypothesized that the synthesis of DGPP would attenuate the disruptive effect of PA accumulation (Kooijman and Burger, 2009; Strawn et al., 2012). Consequently, it is necessary to understand the behavior of the species in controlled and simple systems, in order to go further to increasingly complex systems. Without this information, it will be more difficult to unravel the intricate behavior of lipids in living cells. On the other hand, dynamic changes in the proportion of these minor lipids occur together with alterations in calcium levels (Tang et al., 2006). The concentration of Ca2+ in the extracellular space is about 2 mM, while its intracellular levels are much lower and range amongst cell organelles from 100 nM in the cytosol to 600 μM in the endoplasmic reticulum (Xu et al., 2014). Besides, calcium signaling is connected with rapid spikes of Ca2+ concentration caused by an influx of the Ca2+ into the cytosol via calcium channels (Berridge et al., 2003). These spikes are spatially and temporally modulated by the calcium protein-based buffers. In addition, calcium ions strongly interact with the negatively charged component of plasma membrane as phospholipids (Melcrova et al., 2016). This aspect has been little explored for DGPP, while the interaction of calcium ions with PA has been proposed to occur with a 1:1 stoichiometry (Shoemaker and Vanderlick,
2. Material and methods Langmuir monolayers of the individual lipids and their binary mixtures were spread from premixed solutions in chloroform/methanol 2
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observed at about 35 Ų at both pHs. This non-monotonically behavior of the compressibility was not detected in our previous paper (Villasuso et al., 2010). In 2010 we used a different Langmuir balance, with another data acquisition system that averaged data in a longer time lapse as the compression isotherm is recorded. This subtle decrease in compressibility modulus was probably smoothed due to this averaging. We will discuss this phenomenon later. Fig. 2C shows the π-Am and SP-Am compression isotherms for DOPA on subphases at pH 5 (blue line) and at pH 8 (red line). Contrary to DGPP, no shift in the lift-off or collapse area with pH was observed for DOPA films. At both pHs, DOPA formed liquid-expanded monolayers with a detectable reorganization during the compression at 30 mN m−1, 55 Å2 and about 250 mV (see arrows). The compressibility moduli ranged from 20 mN/m to 100 mN/m (Fig. 2D), with a sharp decrease at 30 mN/m due to the film rearrangement, which was more marked at pH 8 than at pH 5. The monolayer electrostatics shows a subtle but reproducible change upon a pH decrease, SP goes from 340 mV (pH 8) to 300 mV (pH 5) at high compactation, which may result from molecular changes and from a decrease in the double layer potential upon lipid neutralization. We find very interesting the rearrangement observed in DOPA monolayers, and also detected in the compressibility plots of DGPP films, though very smooth. As far as we know, such a subtle rearrangement with a concomitant kink in the π-Am and SP-Am compression isotherms for films in a liquid-expanded state has not been reported before. Demel et al. (1992) and Kulig et al. (2019) reported the compression isotherm for DOPA on subphases of different composition and pH, and no kink in the isotherm were reported by them. This may be due to the differences in the subphase conditions or because they used a lower time resolution for data acquisition. These authors did not show curves of compressibility modulus or surface potential compression isotherms, where the rearrangement was more evident. Since the rearrangement is observed in both, DOPA and DGPP, we infer that it is related with a part of the molecule that they have in common. Furthermore, since the rearrangement was clearly detected in the SP profiles, electrostatics modifications were involved. We propose that the observed behavior is a consequence of a rearrangement of ions around the phosphate moiety attached to the glycerol back-bound, which is shared by both lipids. This charge movement would be less marked in DGPP, were the phosphate is less accessible to the subphase, and also at acid pHs, where the molecular charge is lower. Since the monolayer packing properties are affected by the interactions with neighboring lipids (Maggio, 2004), we have previously studied the interfacial behavior of mixtures of DGPP with POPA. We found that the lipids show a non-ideal behavior, with negative excess areas, indicating that the intermolecular interactions in the mixtures are more favorable than those in the films composed of the pure lipids (Villasuso et al., 2010). Since in this previous work we used POPA, the mixture was composed by lipids with different hydrocarbon chains. Here we did the study using DOPA, the PA with the same hydrocarbon chain than DGPP in order to understand what could happen locally, during the action of PAK or LPP (see Fig. 1). At these conditions, DGPP forms in a PA-enriched region and thus, our study may give an insight of what happens in a very small region and at short times in cells. π-Am and SP-Am compression isotherms of DOPA and DGPP films at different proportions were performed at pH 5 and 8, Fig. 3 shows representative experiments. Mixed films of DGPP with its precursor DOPA formed liquid-expanded films, with the rearrangement at 30 mN/m being less evident as the DGPP proportions increased. A shift towards lower Am values was detected when DGPP was added to the DOPA films at both pHs and at all π values. This deviation from the ideal behavior indicates that the interaction between DOPA and DGPP was stronger than those in the pure films, thus forming more compact monolayers. Fig. 3E shows the degree of condensation at 30 mN/m, a surface pressure at which molecular density of the films is similar to that of bilayers (Brockman et al., 2003; Brown and Brockman, 2007).
(2:1, v/v) onto the surface of different subphases at a mean molecular area larger than the lift off area. Before isometric compression of the film, the solvent was allowed to evaporate for 5 min. Surface pressure was measured with a platinized-Pt Wilhelmy plate, and surface potential with the Kelvin method using a commercial Langmuir balance (Minitrough from KSV Instruments). All experiments were performed at 24 ± 1 °C. Temperature was maintained within ± 1 °C with a refrigerated Haake F3C thermo circulator and air conditioning the room. Dioleoylglycerol pyrophosphate (DGPP) and 1,2-dioleoyl-sn-glycerol 3 phosphate (DOPA) were purchased from Avanti polar lipids, inc. (Alabaster, AL, USA). The subphase composition was 150 mM NaCl, 5 mM EDTA, pH 8 or 5, with or without CaCl2 at the indicated concentration. EDTA was added to the subphases in order to chelate trace metals, and also as buffer in the case of studies at pH 8, in order to avoid acidification by CO2 dissolution. The pH was stable during the time of the experiment. The subphase was prepared with ultra-pure water (18.2 MΩ) obtained from an Osmoion purifier (Apema, Argentina). Solvents were of the highest available purity from Merck (Darmstadt, Germany). Absence of surface-active impurities in the subphase and in the spreading solvents was routinely controlled. For this, the clean interface was compressed at the beginning of each working day whilst surface pressure and surface potential were determined, and both parameter remained constant during compression within tens of mV. The vibrating electrode was always placed very close to the interface (20–50 mm) and the whole system was located in a Faraday box connected to ground, thus minimizing spurious signals. At least triplicate monolayer isotherms were obtained and averaged at a compression rate of 0.45–0.60 nm2 molecule−1 min−1. In order to discard effects of lipid oxidation on the compression isotherms we performed compression isotherms after waiting different times (0, 10, 30 or 60 min). Besides, we checked that the isotherm for a recompressed monolayer after 5 min of equilibration of the expanded film was similar to the first compression isotherm. We did not find significant changes of the compression isotherms in either case. The percent of monolayer condensation was calculated as Aexc / Aid × 100 . Here Aexc = Areal − Aid , Areal is the experimental mean molecular area obtained from the compression isotherms of the mixtures and Aid is the ideal mean molecular area calculated with the mean molecular areas of the pure lipids as: Aid = ADOPA xDOPA + ADGPP xDGPP . ADOPA and ADGPP were obtained from the compression isotherm of the pure lipids and xDOPA and xDGPP are the molar fractions. Compressibility modulus, also known as in-plane elasticity, was calculated for the pure monolayers and for each mixture at the different conditions as, κ = −Am (∂π/∂Am)τ. The excess in surface potential (SP) was evaluated from the deviation of the SP values of the mixtures from the ideal value, calculated as the weighted sum of the values of the pure films: SPid = SPDOPA xDOPA + SPDGPP xDGPP (Brown and Brockman, 2007) 3. Results 3.1. Compression isotherms of DOPA and DGPP at acid and basic pHs Polar head group of DGPP may bear from 1 to 3 negative charges, depending on pH (Strawn et al., 2012). Taking the pyrophosphoric acid pKa’s in consideration, and the range of physiological pHs in plants, in a previous work we described the surface behavior of DGPP on subphases at pH 5 (pyrophosphate moiety with 1 or 2 negative charges) and at pH 8 (pyrophosphate moiety with 2 or 3 negative charges). We found that decreasing the pH promotes a shift of the compression isotherm to lower mean molecular areas (Am) and surface potentials (SP) at all surface pressures (π), with negligible changes in the compressibility modulus (Villasuso et al., 2010). Compression isotherms for DGPP at pH 5 and 8 are shown in Fig. 2A and B, being similar to those previously reported within experimental errors. A sharp but very subtle decrease in the compressibility modulus was 3
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Fig. 2. Compression isotherms for monolayers of DGPP and DOPA on subphases at pH 5 and 8. Lateral pressure (left scale) and SP (right scale) as a function of the mean molecular area for DGPP (A) or DOPA monolayers (C). Compressibility modulus for the isotherms shown in A and C. Subphase composition: 0.15 M NaCl +5 mM EDTA, pH 5 (blue line) and pH 8 (red line). All curves show a single representative experiment from a set of triplicates.
Am values were still lower than the ideal one. Regarding the film electrostatics, slight deviations from the weighted sum were observed at all surface pressures for DGPP percent higher than 2 %, with tendencies depending on pH, see Fig. 3C and D. Fig. 3F shows the excess SP (compared to the weighted sum) at 30 mN/ m and at pH 5 or 8. Interesting the deviations were positive at pH 5 and
Interesting, the shift to lower Am values was very high even at very low DGPP contents, with a minimum value of percent of condensation around 2 % DGPP. Furthermore, the degree of condensation markedly depended on pH, being more favorable the mixtures at slightly basic conditions. See that the condensation was doubly at pH 8 compared to pH 5. For higher amounts of DGPP the condensation decreased, but the
Fig. 3. Compression isotherms for mixed monolayers of DGPP and DOPA on subphases at acid and basic pHs. Lateral pressure (upper panels) and Surface potential (lower panels) as a function of the mean molecular area for mixed monolayers of DOPA-DGPP monolayers at acidic pH (A and C) and basic pH (B and C). All curves show a single representative experiment from a set of triplicates. Percent of condensation at 30 mN/m (E) and Surface Potential excess at 30 mN/m (F) as a function of DGPP mole fraction was calculated as it was indicated Material and Methods. The data in Fig. 3F correspond to the average ± SD (SD values from 2 to 10 mV). 4
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Fig. 4. Effect of calcium on compression isotherms for mixed monolayers of DGPP and DOPA on subphases at different pHs. Lateral pressure as a function of the mean molecular area for monolayers of DGPP and DOPA at acidic pH (A and C) and basic pH (B and C).
Fig. 5. Effect of calcium on surface potential density at acid and basic pH. Surface Potential as a function of the mean molecular area for monolayers of of DGPP and DOPA at acidic pH (A and C) and basic pH (B and C).
3.2. Ca(II)-regulation of the surface behavior of DOPA and DGPP at acid and basic pHs
negative at pH 8. The results shown up to now suggest that the biosynthesis of low amounts of DGPP in an environment enriched in DOPA could lead to a local increase in membrane compactation with a concomitant change in the local polarization, further regulated by local pH.
There is great interest in the study of the effect of calcium and their binding with lipid membranes. The effect of this divalent cation on several lipids has been well documented; however, no studies have shown the impact of calcium at different pHs on DGPP films. Fig. 4 5
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interactions, leading to loosely packed monolayers (Brown and Brockman, 2007). Here we analyzed the effect of neutralization by pH and by Ca2+ of monolayers composed of DGPP, it´s precursor DOPA, and mixtures of both lipids. Fig. 7 summarizes the effect of pH and of CaCl2 at 30 mN m−1, where the lipid´s density is comparable to bilayers (Brockman et al., 2003; Brown and Brockman, 2007). The results found at a lower surface pressure can be found in S1. We found, on one hand, that DOPA film density remain unchanged upon acidification, DGPP increased its density and the mixture decreased it (Fig. 7A). The observed compression isotherms of DGPP at pH 5 and 8 are in agreement with an increased electrostatic repulsion at pH 8 where a higher net charge (about −2.2 at pH 8 and about −1.8 at pH 5) is expected, according to the pKa’s values for the pyrophosphate acid (Villasuso et al., 2010). Contrary to DGPP, DOPA did not increase the molecular density upon a pH (and concomitantly a charge) decrease. In similar experiments, it was observed that DPPA monolayers are influenced by pH as a consequence of the change in the ionization of the lipid (Minones et al., 2002). At pH 3, DPPA molecules are uncharged showing a more rigid monolayer structure compared to monolayer spread on subphases at higher pHs. It was proposed that hydrogen bonding between protonated phosphatidic acid groups of adjacent molecules could lead to a more stable and coherent monolayer (Minones et al., 2002). This kind of bonding is only possible for close phosphatidic moieties, which may be present in DPPA, where the Van der Waals interaction between hydrocarbon chains is high, and not in DOPA, where the double bonds disrupts the hydrocabon chain order leading to a decreased interaction. By contrast to the behavior at pH 3, DPPA monolayer at pH 6 exhibits a larger molecular area due to the ionization of the first phosphate of phosphatidic acid, decreasing the possibility for intermolecular hydrogen bonding. The main species for DOPA at pH 5 is the -1charged, and at pH 8, the -2-charged species (Moncelli et al., 1994). However, it has been proposed that in 0.1 M NaCl solutions the phosphate moiety is chelated by Na+, forming neutral species at acid pHs and also at basic ones (Zhang et al., 2018). Related to this, Minones et al. (2002) reported changes in the film density caused by NaCl addition, further reinforcing the importance of Na+-lipid interactions in the film behavior. Also, a restructuration of this NA+-PA− complex may be involved in the rearrangement observed at 30 mN/m. This complex formation, together with the not so close distance between adjacent polar head-groups compared to saturated lipids, may give account of the unresponsively of the DOPA films upon pH, unlike DPPA. In this regards, it has recently been demonstrated that interactions between PA and PC molecules are not favorable compared to pure lipids for systems containing lipids with saturated and unsaturated acyl tails (strongly positive values of the excess free energy of mixing), and very favorable for systems in which all lipid tails are saturated (negative values of the excess free energy of mixing) (Kulig et al., 2019). This fact would indicate that the interaction between anionic and neutral lipids, the tails and also the head groups modulate the film packing. The molecular density in Langmuir films depends on the lateral interactions and on the possible orientation of molecules with its neighbors. All these depend on molecular shape, polar headgroup charge and hydration, hydrogen bonding, electrostatics and Van der Waals interactions, and hydrocarbon chain motion. Therefore, the film density in a mixture is an emergent property that does not necessarily correlate with the behavior of the pure films. This is the case of DGPP/ DOPA (5:95) films. While acid pHs neutralized and compacted DGPP films, and induced no change in DOPA films, the mixed films became more loosely packed upon acidification (see Fig. 7A). This may be due to a change in the coordination of the phosphate group, being less efficient the chelation by Na+ at acid pH than at pH 8. In this regard, as already mentioned the reorganization observed at pH 5 was less evident than at pH 8, probably due to a reduced affinity of the subphase ions with the lipid monolayer.
shows that the presence of CaCl2 in the subphase at pH 5 or 8 caused a shift of the whole isotherm of DGPP and DOPA to smaller Am values at concentrations and degrees that depend on the lipid, the pH and the surface pressure, similarly to the reported effects of Zn2+ on DGPP and POPA (Villasuso et al., 2014). This is expected considering that the binding of this ion neutralize, at least partially, the lipid monolayer. It is important to point out here that, despite the used CaCl2 concentration was mM, the concentration of free Ca2+ ions was much lower and depended on pH due to the chelation by EDTA. For 1 mM CaCl2 and 5 mM EDTA, free Ca2+ values of 20 μM and 1 nM are expected at pH 5 and 8, respectively. These values are within the physiological levels (Xu et al., 2014; Melcrova et al., 2016). We also performed experiments with CaCl2 concentrations higher than 1 mM (maximal 10 mM). However, we decided to show the result with CaCl2 concentration between 0.1–1 mM since these values are within the physiological levels. In this regards, it is important to mention that in cells, a great variety of species that are able to chelate calcium are present, thus decreasing the availability of the free-bivalent ion. At these conditions, competition for calcium with other species rather than a direct interaction with freecalcium is more likely to occur. In order to explore the effect of Ca2+ in the electrostatics of these monolayers, the surface potential was determined in the presence of increasing amounts of CaCl2. At pH 5, the presence of the bivalent ion induced an increase in SP for both, DOPA and DGPP monolayers (Fig. 5, left panels), while at pH 8, a decrease was observed (Fig. 5, right panels). Interesting, the rearrangement observed in DOPA compression isotherms (both, π-Am and SP-Am) persisted in the presence of CaCl2, suggesting that the bivalent ion bounded to the lipid in a different region than that affected by the rearrangement. In general, the effects promoted by Ca2+ were more evident at basic pHs, where the films were more charged and loosely packed (Villasuso et al., 2010) than at acid pHs. It was shown that the effective molecular shape of PA is highly influenced by pH, temperature, and the presence of divalent cations (Minones et al., 2002; Kooijman et al., 2005; Kooijman and Burger, 2009; Strawn et al., 2012; Villasuso et al., 2014). At pH 7 in the presence of magnesium ion, PA acquires a cylindrical shape and thus stabilizes the lamellar topography. However, once subjected to an acid environment, the head group of PA decreases its effective size, and the lipid undergoes a shape change to a cone-like shape, thus affecting the membrane by promoting an increase of negative membrane curvature (Kooijman and Burger, 2009). DGPP formation after stimulus takes place after a transient increase of the PA levels (Racagni et al., 2008). Therefore, as already mentioned, a temporary and local accumulation of DGPP and its precursor at the membrane interface is expected under stress. Since it has been shown that the effect of Zn2+ on POPA/DGPP mixtures depends on the mixture composition and subphase pH (Villasuso et al., 2010; 2014), we studied the packing properties of mixed films of DGPP and DOPA in the presence of Ca2+ on subphases at acid and basic pHs. Mixtures composed of DGPP/DOPA 5:95 were chosen for the analysis because, on one hand, it is a DGPP proportion of physiological relevance, since this lipid is minority in cells. On the other, the highest condensation was observed in this range of lipid proportions. Fig. 6 shows the compression isotherm for a DOPA/DGPP 95:5 mixture at pH 5 and 8 with increasing CaCl2 concentrations. Similar to the pure lipids, the cation decreased Am values at both pHs (Fig. 6A and B), whilst SP increased at acid pHs and decreased at basic ones (Fig. 6C and D). 4. Discussion The shape of the compression isotherms of monolayers for a particular lipid species depend on the length and unsaturation of the hydrocarbon chain and on the bulkiness and charge of the polar headgroup. Long and saturated hydrocarbon chains would interact through Van der Waals attractions, promoting more condensed monolayers. By contrast, ionization of the lipid head groups should result in repulsive 6
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Fig. 6. Effect of calcium on surface pressure and surface potential as a function of average molecular area for DOPA and DGPP mixture. Lateral pressure (upper panel) and surface potential (lower panel) as a function of the mean molecular area for mixed monolayers of 95 %DOPA-5 %DGPP at indicated pH and CaCl2 concentration.
Fig. 7. Effect of neutralization by pH and by Ca2+ of DGPP, DOPA, and mixtures of both lipids. Effect of Ca2+ on mean molecular area (A) and surface potential (C) for pure and mixed lipid. Percent of condensation (B) and surface potential change (D) as a function of CaCl2 concentration for pure and mixture of DOPA and DGPP. The lateral pressures are 30 mN m−1. The values are shown as average ± SD of three independent experiments for each condition at 30 mN/m. Asterisks (*) represent significant differences at pH 5 or pH 8 according to Student's test (p < 0.05). Scale starts at 225 mV.
7
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lipid has been evaluated with different model systems, and it was found that some divalent cations at high concentration induce the formation of clusters of phospholipids. In the case of the precursor of second messenger PIP2, only Ca2+, but not Mg2+, Zn2+ or polyamines induces a surface pressure drop that coincides with the formation of PIP2 clusters (Wang et al., 2012). Studies of the interaction between PA and Ba2+ suggested that PA is extremely effective in binding divalent ions through its oxygen atoms, with a broad distribution of binding constants and exhibiting the phenomenon of charge inversion (a total number of bound counterion charges that exceeds the negative PA charge) (Faraudo and Travesset, 2007). The authors predict that a PArich domains undergo a drastic reorganization when divalent cations as Ca2+ and Ba2+ reach micromolar concentrations (i.e., typical physiological conditions), as PA lipids become doubly charged by releasing their protons. Although restricted to PA, those results could be qualitatively similar for other phospholipids, as DGPP, which play somewhat similar roles as PA. Here, a marked increase on SP was observed at pH 5 upon an increase in CaCl2 concentration (Fig. 7C and D). On the contrary, at pH 8 Ca2+ ions induced a low depolarization of the interface with the pure lipids. Interestingly, the mixture showed an different behavior, an increase of SP was observed when CaCl2 was present in the subphase at both, acid and basic pHs (Fig. 7D).
Regarding the Ca(II)-mediated neutralization, the area reduction was maximal at basic pHs, where the lipid-ion interaction is expected to be favored, due to the negative charge on the polar headgroups (Fig. 7A and B). It has to be considered that at acid pHs, the free-Ca(II) concentration is higher than at basic pH due to the decrease in the concentration of EDTA4− (For 1 mM CaCl2, free-Ca2+ = 20 μM at pH 5 and 1 nM at pH 8), strengthening the observation that the interaction between the bivalent ion and the lipids was very favored at basic pHs compared to acid ones. Furthermore, the areas at pH 8 in the presence of CaCl2 showed the lowest values for each monolayer composition (Fig. 7A), indicating that neutralization by Ca2+ ions was more efficient for membrane compactation than protonation. It may be that a Ca2+ ion interacts with more than one lipid, thus promoting membrane condensation more effectively. Surprisingly, at basic pHs the mixture was less responsive to Ca2+ than the pure lipids (Fig. 7B, red circles compared with red triangles). This is probably a consequence that the mean molecular area for the mixtures was already very low in the absence of the bivalent ion, and thus with reduced possibilities of further compactation. Therefore, at pH 8 the behavior of the mixture upon CaCl2 addition was not intermediate between the pure components but different properties emerge regarding lipid compactation. The dielectric constant decreases abruptly from about 80 in the bulk aqueous medium to values in the range of 3 to 10 in the polar head group-hydrocarbon interface, depending on the characteristics of the polar head groups (Montich et al., 1985). Thus, the ionization degree of the lipid polar headgroup strongly impacts on membrane electrostatic potential with an effect on the distribution and ionization of nearby charged components such as lipid dipoles that can act as sensitive sensors of the surface electrostatics (Seelig et al., 1987). The surface potential is a resultant of a complex combination of the double layer potential, and contributions of hydrocarbon chain dipoles, of water dipoles from the polar head group hydration shell, and intrinsic polar headgroup dipoles, as well as from the electrostatic field brought about from the relative position of mobile ions interacting specifically with the polar head-group moieties. We observed, on one hand, that the surface potentials of DOPA and of DGPP in the absence of CaCl2 were lower at acid pHs than at basic ones, as a consequence of the lower surface charge of the films formed by both molecules at pH 5 compared to 8. Thus, despite the Na+ ions prevented density changes with pH in DOPA monolayers, depolarization of the interface occurred due to a pH decrease. Similar to what was described for the film density, the mixture showed a behavior that did not correlate with that of the pure lipids. In the mixed monolayers, no change in SP occurred when pH changed (Fig. 7C). Additionally, the film electrostatics of the mixture did not depict the values expected for an ideal mixture (SI 2). Compared to the pure lipids, we found that mixtures of DGPP with DOPA presented lower values of SP when mixed at pH 8, and higher values at pH 5 (SI 2, C and D), indicating a complex modulation of the membrane electrostatics. Since the mixtures were more condensed than ideal behavior (SI 2 A and B), the accessibility of the ions (Na+ and H+) of the subphase to the polar headgroups was reduced in the mixture, probably leading to the non-ideal behavior in the surface potential. Besides, the negative charge on the lipids signifies an anionic surface with a corresponding distribution of mobile ions in the immediate aqueous milieu (the double layer potential) which is also included in the surface potential measured. It was previously observed in stearic acid monolayers that an increase of the negative charge in the monolayer may decrease the double layer potential by values as high as 200 mV (Vega Mercado et al., 2011). Therefore, a decrease of the double layer potential when the lipid becomes ionized may result in a lower surface potential even if the overall lipid dipole is being increased in the negatively charged molecule (Vega Mercado et al., 2011), and thus the behavior of this parameter is not straightforward to predict nor to explain. The effect of divalent cations on the interfacial behavior of anionic
5. Conclusion Our results indicate that the film properties of DGPP and its precursor DOPA were sensitive to Ca2+ in a pH-dependent manner, and thus depended on the ionization state of the molecules forming the film. Low proportions of DGPP in the DOPA film further modulated this effect. The changes in film density promoted by Ca2+ were more marked on subphases at pH 8, while at pH 5 the influence of the cation in the film surface properties was attenuated. The non-ideal behavior of the mixed monolayer, which was sensitive to the ionic composition of the aqueous milieu, indicates a complex regulation of film properties with composition. It has been shown that changes in oscillations of Ca2+ usually occur with pH variations during stress response (Li et al., 2019). Thus, modifications in the PA and DGPP proportion, coupled with pH and Ca2+-level variations could be part of the response to stress in plant. Declaration of Competing Interest The authors declare no competing interests. Acknowledgments This work was supported by SECyT-UNRC, SECyT-UNC, FONCYT (PICT 2015-1108 and 2015-0662), and CONICET. MPM is a fellowship of CONICET. NW and ALV are Career Investigators of CONICET. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chemphyslip.2020. 104887. References Arisz, S.A., Heo, J.Y., Koevoets, I.T., Zhao, T., Van Egmond, P., Meyer, J., Zeng, W., Niu, X., Wang, B., Mitchell-Olds, T., Schranz, M.E., Testerink, C., 2018. Diacylglycerol acyltransferase 1 contributes to freezing tolerance. Plant Physiol. https://doi.org/10. 1104/pp.18.00503. Astorquiza, P.L., Usorach, J., Racagni, G., Villasuso, A.L., 2016. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone. Plant Physiol. Biochem. 101, 88–95. https://doi.org/10.1016/j. plaphy.2016.01.012.
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S0006-3495(94)80990-7. Montich, G.G., Bustos, M.M., Maggio, B., Cumar, F.A., 1985. Micropolarity of interfaces containing anionic and neutral glycosphingolipids as sensed by Merocyanine 540. Chem. Phys. Lipids 38, 319–326. https://doi.org/10.1016/0009-3084(85)90026-X. Paradis, S., Villasuso, A.L., Aguayo, S.S., Maldiney, R., Habricot, Y., Zalejski, C., Machado, E., Sotta, B., Miginiac, E., Jeannette, E., 2011. Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves. Plant Physiol. Biochem. 49, 357–362. https://doi.org/10.1016/j.plaphy.2011.01.010. Peppino Margutti, M., Gaveglio, V.L., Reyna, M., Pasquare, S.J., Racagni, G., Villasuso, A.L., 2018. Differential phosphatidic acid remodelling and metabolism in leaves and roots of barley induced by chilling temperature. Plant Physiol. Biochem en revisión. Pokotylo, I., Kravets, V., Martinec, J., Ruelland, E., 2018. The phosphatidic acid paradox: too many actions for one molecule class? Lessons from plants. Prog. Lipid Res. 71, 43–53. https://doi.org/10.1016/j.plipres.2018.05.003. Racagni, G., Villasuso, A.L., Pasquare, S.J., Giusto, N.M., Machado, E., 2008. Diacylglycerol pyrophosphate inhibits the alpha-amylase secretion stimulated by gibberellic acid in barley aleurone. Physiol. Plant. 134, 381–393. https://doi.org/10. 1111/j.1399-3054.2008.01148.x. Seelig, J., Macdonald, P.M., Scherer, P.G., 1987. Phospholipid head groups as sensors of electric charge in membranes. Biochemistry 26, 7535–7541. https://doi.org/10. 1021/bi00398a001. Shoemaker, S.D., Vanderlick, T.K., 2003. Calcium modulates the mechanical properties of anionic phospholipid membranes. J. Colloid Interface Sci. 266, 314–321. https://doi. org/10.1016/S0021-9797(03)00582-4. Strawn, L., Babb, A., Testerink, C., Kooijman, E.E., 2012. The physical chemistry of the enigmatic phospholipid diacylglycerol pyrophosphate. Front. Plant Sci. 3, 40. https://doi.org/10.3389/fpls.2012.00040. Tang, D., Dean, W.L., Borchman, D., Paterson, C.A., 2006. The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity. Cell Calcium 39, 209–216. https://doi.org/10.1016/j.ceca.2005.10.010. Testerink, C., Munnik, T., 2011. Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J. Exp. Bot. 62, 2349–2361. https://doi.org/ 10.1093/jxb/err079. Vega Mercado, F., Maggio, B., Wilke, N., 2011. Phase diagram of mixed monolayers of stearic acid and dimyristoylphosphatidylcholine. Effect of the acid ionization. Chem. Phys. Lipids 164, 386–392. https://doi.org/10.1016/j.chemphyslip.2011.05.004. Villasuso, A.L., Wilke, N., Maggio, B., Machado, E., 2010. The surface organization of diacylglycerol pyrophosphate and its interaction with phosphatidic acid at the airwater interface. Chem. Phys. Lipids 163, 771–777. https://doi.org/10.1016/j. chemphyslip.2010.09.002. Villasuso, A.L., Di Palma, M.A., Aveldano, M., Pasquare, S.J., Racagni, G., Giusto, N.M., Machado, E.E., 2013. Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells. Plant Physiol. Biochem. 65, 1–8. https://doi.org/10.1016/j.plaphy.2013.01.005. Villasuso, A.L., Wilke, N., Maggio, B., Machado, E., 2014. Zn(2+)-dependent surface behavior of diacylglycerol pyrophosphate and its mixtures with phosphatidic acid at different pHs. Front. Plant Sci. 5, 371. https://doi.org/10.3389/fpls.2014.00371. Wang, Y.-H., Collins, A., Guo, L., Smith-Dupont, K.B., Gai, F., Svitkina, T., Janmey, P.A., 2012. Divalent cation-induced cluster formation by polyphosphoinositides in model membranes. J. Am. Chem. Soc. 134, 3387–3395. https://doi.org/10.1021/ ja208640t. Xu, N., Francis, M., Cioffi, D.L., Stevens, T., 2014. Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on “detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells”. Am. J. Physiol., Cell Physiol. 306, C636–638. https://doi.org/10.1152/ajpcell.00046.2014. Zhang, T., Brantley, S.L., Verreault, D., Dhankani, R., Corcelli, S.A., Allen, H.C., 2018. Effect of pH and Salt on Surface pKa of Phosphatidic Acid Monolayers. Langmuir 34, 530–539. https://doi.org/10.1021/acs.langmuir.7b03579.
Berridge, M.J., Bootman, M.D., Roderick, H.L., 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529. https://doi.org/ 10.1038/nrm1155. Binder, H., Zschornig, O., 2002. The effect of metal cations on the phase behavior and hydration characteristics of phospholipid membranes. Chem. Phys. Lipids 115, 39–61. Brockman, H.L., Applegate, K.R., Momsen, M.M., King, W.C., Glomset, J.A., 2003. Packing and electrostatic behavior of sn-2-docosahexaenoyl and -arachidonoyl phosphoglycerides. Biophys. J. 85, 2384–2396. https://doi.org/10.1016/S00063495(03)74662-1. Brown, R.E., Brockman, H.L., 2007. Using monomolecular films to characterize lipid lateral interactions. Methods Mol. Biol. 398, 41–58. https://doi.org/10.1007/978-159745-513-8_5. Demel, R.A., Yin, C.C., Lin, B.Z., Hauser, H., 1992. Monolayer characterstics and thermal behaviour of phosphatidic acids. Chem. Phys. Lipids 60, 209–223. https://doi.org/ 10.1016/0009-3084(92)90073-X. Faraudo, J., Travesset, A., 2007. Phosphatidic acid domains in membranes: effect of divalent counterions. Biophys. J. 92, 2806–2818. https://doi.org/10.1529/biophysj. 106.092015. Garidel, P., Blume, A., 2005. 1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) monolayers: influence of temperature, pH, ionic strength and binding of alkaline earth cations. Chem. Phys. Lipids 138, 50–59. https://doi.org/10.1016/j. chemphyslip.2005.08.001. Hong, Y., Zhao, J., Guo, L., Kim, S.-C., Deng, X., Wang, G., Zhang, G., Li, M., Wang, X., 2016. Plant phospholipases D and C and their diverse functions in stress responses. Prog. Lipid Res. 62, 55–74. https://doi.org/10.1016/j.plipres.2016.01.002. Kooijman, E.E., Burger, K.N., 2009. Biophysics and function of phosphatidic acid: a molecular perspective. Biochim. Biophys. Acta 1791, 881–888. https://doi.org/10. 1016/j.bbalip.2009.04.001. Kooijman, E.E., Carter, K.M., Van Laar, E.G., Chupin, V., Burger, K.N., De Kruijff, B., 2005. What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special? Biochemistry 44, 17007–17015. https://doi.org/10.1021/bi0518794. Kulig, W., Korolainen, H., Zatorska, M., Kwolek, U., Wydro, P., Kepczynski, M., Rog, T., 2019. Complex behavior of phosphatidylcholine-phosphatidic acid bilayers and monolayers: effect of acyl chain unsaturation. Langmuir 35, 5944–5956. https://doi. org/10.1021/acs.langmuir.9b00381. Laroche, G., Dufourc, E.J., Dufourcq, J., Pezolet, M., 1991. Structure and dynamics of dimyristoylphosphatidic acid/calcium complexes by deuterium NMR, infrared, and Raman spectroscopies and small-angle x-ray diffraction. Biochemistry 30, 3105–3114. https://doi.org/10.1021/bi00226a018. Li, W., Song, T., Wallrad, L., Kudla, J., Wang, X., Zhang, W., 2019. Tissue-specific accumulation of pH-sensing phosphatidic acid determines plant stress tolerance. Nat. Plants 5, 1012–1021. https://doi.org/10.1038/s41477-019-0497-6. Liu, Y., Su, Y., Wang, X., 2013. Phosphatidic acid-mediated signaling. Adv. Exp. Med. Biol. 991, 159–176. https://doi.org/10.1007/978-94-007-6331-9_9. Maggio, B., 2004. Favorable and unfavorable lateral interactions of ceramide, neutral glycosphingolipids and gangliosides in mixed monolayers. Chem. Phys. Lipids 132, 209–224. https://doi.org/10.1016/j.chemphyslip.2004.07.002. Melcrova, A., Pokorna, S., Pullanchery, S., Kohagen, M., Jurkiewicz, P., Hof, M., Jungwirth, P., Cremer, P.S., Cwiklik, L., 2016. The complex nature of calcium cation interactions with phospholipid bilayers. Sci. Rep. 6, 38035. https://doi.org/10.1038/ srep38035. Minones Jr., J., Rodriguez Patino, J.M., Minones, J., Dynarowicz-Latka, P., Carrera, C., 2002. Structural and topographical characteristics of dipalmitoyl phosphatidic acid in Langmuir monolayers. J. Colloid Interface Sci. 249, 388–397. https://doi.org/10. 1006/jcis.2002.8285. Moncelli, M.R., Becucci, L., Guidelli, R., 1994. The intrinsic pKa values for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in monolayers deposited on mercury electrodes. Biophys. J. 66, 1969–1980. https://doi.org/10.1016/
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Influence of Ca2+ on the surface behavior of phosphatidic acid and its mixture with diacylglycerol pyrophosphate at different pHs
T
Micaela Peppino Marguttia, Natalia Wilkeb,c, Ana Laura Villasusoa,d,* a
Universidad Nacional de Río Cuarto, FCEFQyN, Departamento de Biología Molecular, Río Cuarto, Argentina Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Química Biológica Ranwel Caputto, Córdoba, Argentina c CONICET, Universidad Nacional de Córdoba, Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Córdoba, Argentina d CONICET, Universidad Nacional de Río Cuarto, Instituto de Biotecnologia Ambiental y Salud, (INBIAS), Río Cuarto, Argentina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Diacylglycerol pyrophosphate Phosphatidic acid Membrane packing Glycerophospholipid monolayers Calcium
The signaling lipids phosphatidic acid (PA) and diacylglycerol pyrophosphate (DGPP) are involved in regulating the stress response in plants. PA and DGPP are anionic lipids consisting of a negatively charged phosphomonoester or pyrophosphate group attached to diacylglycerol, respectively. Changes in the pH modulate the protonation of their head groups modifying the interaction with other effectors. Here, we examine in a controlled system how the presence of Ca2+ modulates the surface organization of dioleyl diacylglycerol pyrophosphate (DGPP) and its interaction with dioleoyl phosphatidic acid (DOPA) at different pHs. Both lipids formed expanded monolayers at pH 5 and 8. At acid and basic pHs, monolayers formed by DOPA or DGPP became denser when Ca2+ was added to the subphase. At pH 5, Ca2+ also induced an increase of surface potential of both lipids. Conversely, at pH 8 the effects induced by the presence of Ca2+ on the surface potential were reversed. Mixed monolayers of DOPA and DGPP showed a non-ideal behavior. The addition of even tiny amounts of DGPP to DOPA films caused a reduction of the mean molecular area. This effect was more evident at pH 8 compared to pH 5. Our finding suggests that low amounts of DGPP in an film enriched in DOPA could lead to a local increase in film packing with a concomitant change in the local polarization, further regulated by local pH. This fact may have implications for the assigned role of PA as a pH-sensing phospholipid or during its interaction with proteins.
1. Introduction Phosphatidic acid (PA) is the glycerophospholipid with the simplest chemical structure in biological membranes. Phosphatidic acids (PAs) are minor components of biological membrane. However, PAs are key molecules in lipid metabolism, as they are used in the synthesis of the other phospholipids, galactolipids and triglycerides (Testerink and Munnik, 2011). In addition, PA functions as a signaling lipid involved in regulation of vesicular trafficking, secretion, pH sensing and stress response in eukaryotic cell (Pokotylo et al., 2018; Li et al., 2019). In plants, PA´s composition is also modulated in response to stress associated with water deficit, temperature, saline conditions, starvation, free radicals, and interactions with microbes (Liu et al., 2013). In quiescent cells, PA levels are very low, and the concentration increases mainly through the pathways driven by phospholipase D via hydrolysis of phosphatidylcholine (PC) or by phospholipase C which catalyzes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2) realizing inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG) followed by diacylglycerol kinase action (Hong et al., 2016). Despite PA behaves as a signal lipid, its local accumulation could disturb the homeostasis of cellular membrane (Kooijman and Burger, 2009). PA molecules have an inverted conical shape with a relatively small headgroup in comparison with the hydrophobic part of the molecule, which promotes negative spontaneous curvature (Kooijman et al., 2005). Thus, the tightly regulation of PA levels is an important event for maintaining the integrity of cellular membrane (Arisz et al., 2018). The dephosphorylation of PA to diacylglycerol (DAG) by phosphatidate phosphatase, or its phosphorylation to diacylglycerol pyrophosphate (DGPP) by phosphatidate kinase (PAK, see Fig. 1) are two important pathways involved in the attenuation of PA level (Racagni et al., 2008; Villasuso et al., 2013; Peppino Margutti et al., 2018). DGPP is also a minor phospholipid associated with stress response in plant.
⁎ Corresponding author at: Departamento de Biología Molecular-INBIAS-FCEFQN, Universidad Nacional de Río Cuarto, Ruta Nac. 36 - Km. 601, X5804BYA Río Cuarto, Córdoba, Argentina. E-mail address: [email protected] (A.L. Villasuso).
https://doi.org/10.1016/j.chemphyslip.2020.104887 Received 21 October 2019; Received in revised form 13 December 2019; Accepted 30 January 2020 Available online 03 February 2020 0009-3084/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structure of 1,2-dioleoyl-sn-glycerol 3 phosphate (DOPA) and dioleoylglycerol pyrophosphate (DGPP). Arrows indicate the dephosphorylation of DGPP to DOPA by lipid phosphatidate phosphatase (LPP), or DOPA phosphorylation to DGPP by phosphatidate kinase (PAK).
2003). This is because PA, which bears a single negative charge at neutral pH, is a doubly ionizable molecule. Calcium binding displaces the second proton, leaving a neutral PA:Ca(II) complex (Laroche et al., 1991). In addition, it is known that the interaction of calcium ions with others lipid membranes rigidifies and orders lipid bilayers and that the binding mode depends on calcium concentration (Binder and Zschornig, 2002; Garidel and Blume, 2005; Melcrova et al., 2016) Previously, we have demonstrated that the packing and electrostatic properties of pure lipids or mixed films composed of DGPP and POPA were affected by the subphase pH (Villasuso et al., 2010). DGPP in Langmuir monolayers interacts with cations such as zinc modulating the film packing properties (Villasuso et al., 2014). To gain insights into the dynamic properties of PA and DGPP, here we focus on the effect of calcium and pH on the interfacial behavior of pure and mixed lipids films. The main objective was to investigate the interaction of both DGPP and DOPA, the PA with the same hydrocarbon chain than DGPP, with calcium. In this regards, Langmuir technique offers the possibility of forming a well-defined monolayer of the amphiphilic molecules under study, with a unique control of the area per molecule. Although Langmuir monolayer is a highly simplify system, we circumscribe our conclusions to artificial membranes, that may give an insight of what happens in a very small region and at short times in cells. Our data show that DOPA is less sensitive to pH changes than DGPP. At acid and basic pHs, DOPA formed liquid-expanded monolayers with a detectable reorganization during the compression. Analyses of mixed films of DGPP and DOPA indicated that the interaction between lipids was stronger than those in the pure films, thus forming more compact monolayers. In contrast, effects promoted by Ca2+ were more evident at basic pHs, where the films were more charged and loosely packed than at acid pHs. This fact suggests that the biosynthesis of low amounts of DGPP in an environment enriched in DOPA could evoke a complex reorganization of film packing affecting to local electrostatics.
DGPP synthesis is temporary and it is always related with variations of the amount of PA (Paradis et al., 2011). DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. It was shown that, depending on pH, the pyrophosphate moiety of DGPP could display 2 or 3 negative charges making it a highly polar molecule (Villasuso et al., 2010; Strawn et al., 2012). The ionization of the pyrophosphate group is important in the electrostatic interactions between DGPP and proteins as well as with bivalent cations (Villasuso et al., 2014; Astorquiza et al., 2016). Although, several aspects of DGPP are still unknown, it has been shown that the ionization properties of the phosphomonoester of DGPP mimic those of PA following the electrostatic-hydrogen bond switch model (Strawn et al., 2012). However, DGPP is not a cone shaped lipid, i.e., it is not capable of imparting negative curvature stress to the membrane that could facilitate the insertion of hydrophobic protein domains in the membrane (Strawn et al., 2012). Thus, the charge and packing properties of DGPP might contribute with the turn-off of the PA-signal. Accordingly, it was hypothesized that the synthesis of DGPP would attenuate the disruptive effect of PA accumulation (Kooijman and Burger, 2009; Strawn et al., 2012). Consequently, it is necessary to understand the behavior of the species in controlled and simple systems, in order to go further to increasingly complex systems. Without this information, it will be more difficult to unravel the intricate behavior of lipids in living cells. On the other hand, dynamic changes in the proportion of these minor lipids occur together with alterations in calcium levels (Tang et al., 2006). The concentration of Ca2+ in the extracellular space is about 2 mM, while its intracellular levels are much lower and range amongst cell organelles from 100 nM in the cytosol to 600 μM in the endoplasmic reticulum (Xu et al., 2014). Besides, calcium signaling is connected with rapid spikes of Ca2+ concentration caused by an influx of the Ca2+ into the cytosol via calcium channels (Berridge et al., 2003). These spikes are spatially and temporally modulated by the calcium protein-based buffers. In addition, calcium ions strongly interact with the negatively charged component of plasma membrane as phospholipids (Melcrova et al., 2016). This aspect has been little explored for DGPP, while the interaction of calcium ions with PA has been proposed to occur with a 1:1 stoichiometry (Shoemaker and Vanderlick,
2. Material and methods Langmuir monolayers of the individual lipids and their binary mixtures were spread from premixed solutions in chloroform/methanol 2
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observed at about 35 Ų at both pHs. This non-monotonically behavior of the compressibility was not detected in our previous paper (Villasuso et al., 2010). In 2010 we used a different Langmuir balance, with another data acquisition system that averaged data in a longer time lapse as the compression isotherm is recorded. This subtle decrease in compressibility modulus was probably smoothed due to this averaging. We will discuss this phenomenon later. Fig. 2C shows the π-Am and SP-Am compression isotherms for DOPA on subphases at pH 5 (blue line) and at pH 8 (red line). Contrary to DGPP, no shift in the lift-off or collapse area with pH was observed for DOPA films. At both pHs, DOPA formed liquid-expanded monolayers with a detectable reorganization during the compression at 30 mN m−1, 55 Å2 and about 250 mV (see arrows). The compressibility moduli ranged from 20 mN/m to 100 mN/m (Fig. 2D), with a sharp decrease at 30 mN/m due to the film rearrangement, which was more marked at pH 8 than at pH 5. The monolayer electrostatics shows a subtle but reproducible change upon a pH decrease, SP goes from 340 mV (pH 8) to 300 mV (pH 5) at high compactation, which may result from molecular changes and from a decrease in the double layer potential upon lipid neutralization. We find very interesting the rearrangement observed in DOPA monolayers, and also detected in the compressibility plots of DGPP films, though very smooth. As far as we know, such a subtle rearrangement with a concomitant kink in the π-Am and SP-Am compression isotherms for films in a liquid-expanded state has not been reported before. Demel et al. (1992) and Kulig et al. (2019) reported the compression isotherm for DOPA on subphases of different composition and pH, and no kink in the isotherm were reported by them. This may be due to the differences in the subphase conditions or because they used a lower time resolution for data acquisition. These authors did not show curves of compressibility modulus or surface potential compression isotherms, where the rearrangement was more evident. Since the rearrangement is observed in both, DOPA and DGPP, we infer that it is related with a part of the molecule that they have in common. Furthermore, since the rearrangement was clearly detected in the SP profiles, electrostatics modifications were involved. We propose that the observed behavior is a consequence of a rearrangement of ions around the phosphate moiety attached to the glycerol back-bound, which is shared by both lipids. This charge movement would be less marked in DGPP, were the phosphate is less accessible to the subphase, and also at acid pHs, where the molecular charge is lower. Since the monolayer packing properties are affected by the interactions with neighboring lipids (Maggio, 2004), we have previously studied the interfacial behavior of mixtures of DGPP with POPA. We found that the lipids show a non-ideal behavior, with negative excess areas, indicating that the intermolecular interactions in the mixtures are more favorable than those in the films composed of the pure lipids (Villasuso et al., 2010). Since in this previous work we used POPA, the mixture was composed by lipids with different hydrocarbon chains. Here we did the study using DOPA, the PA with the same hydrocarbon chain than DGPP in order to understand what could happen locally, during the action of PAK or LPP (see Fig. 1). At these conditions, DGPP forms in a PA-enriched region and thus, our study may give an insight of what happens in a very small region and at short times in cells. π-Am and SP-Am compression isotherms of DOPA and DGPP films at different proportions were performed at pH 5 and 8, Fig. 3 shows representative experiments. Mixed films of DGPP with its precursor DOPA formed liquid-expanded films, with the rearrangement at 30 mN/m being less evident as the DGPP proportions increased. A shift towards lower Am values was detected when DGPP was added to the DOPA films at both pHs and at all π values. This deviation from the ideal behavior indicates that the interaction between DOPA and DGPP was stronger than those in the pure films, thus forming more compact monolayers. Fig. 3E shows the degree of condensation at 30 mN/m, a surface pressure at which molecular density of the films is similar to that of bilayers (Brockman et al., 2003; Brown and Brockman, 2007).
(2:1, v/v) onto the surface of different subphases at a mean molecular area larger than the lift off area. Before isometric compression of the film, the solvent was allowed to evaporate for 5 min. Surface pressure was measured with a platinized-Pt Wilhelmy plate, and surface potential with the Kelvin method using a commercial Langmuir balance (Minitrough from KSV Instruments). All experiments were performed at 24 ± 1 °C. Temperature was maintained within ± 1 °C with a refrigerated Haake F3C thermo circulator and air conditioning the room. Dioleoylglycerol pyrophosphate (DGPP) and 1,2-dioleoyl-sn-glycerol 3 phosphate (DOPA) were purchased from Avanti polar lipids, inc. (Alabaster, AL, USA). The subphase composition was 150 mM NaCl, 5 mM EDTA, pH 8 or 5, with or without CaCl2 at the indicated concentration. EDTA was added to the subphases in order to chelate trace metals, and also as buffer in the case of studies at pH 8, in order to avoid acidification by CO2 dissolution. The pH was stable during the time of the experiment. The subphase was prepared with ultra-pure water (18.2 MΩ) obtained from an Osmoion purifier (Apema, Argentina). Solvents were of the highest available purity from Merck (Darmstadt, Germany). Absence of surface-active impurities in the subphase and in the spreading solvents was routinely controlled. For this, the clean interface was compressed at the beginning of each working day whilst surface pressure and surface potential were determined, and both parameter remained constant during compression within tens of mV. The vibrating electrode was always placed very close to the interface (20–50 mm) and the whole system was located in a Faraday box connected to ground, thus minimizing spurious signals. At least triplicate monolayer isotherms were obtained and averaged at a compression rate of 0.45–0.60 nm2 molecule−1 min−1. In order to discard effects of lipid oxidation on the compression isotherms we performed compression isotherms after waiting different times (0, 10, 30 or 60 min). Besides, we checked that the isotherm for a recompressed monolayer after 5 min of equilibration of the expanded film was similar to the first compression isotherm. We did not find significant changes of the compression isotherms in either case. The percent of monolayer condensation was calculated as Aexc / Aid × 100 . Here Aexc = Areal − Aid , Areal is the experimental mean molecular area obtained from the compression isotherms of the mixtures and Aid is the ideal mean molecular area calculated with the mean molecular areas of the pure lipids as: Aid = ADOPA xDOPA + ADGPP xDGPP . ADOPA and ADGPP were obtained from the compression isotherm of the pure lipids and xDOPA and xDGPP are the molar fractions. Compressibility modulus, also known as in-plane elasticity, was calculated for the pure monolayers and for each mixture at the different conditions as, κ = −Am (∂π/∂Am)τ. The excess in surface potential (SP) was evaluated from the deviation of the SP values of the mixtures from the ideal value, calculated as the weighted sum of the values of the pure films: SPid = SPDOPA xDOPA + SPDGPP xDGPP (Brown and Brockman, 2007) 3. Results 3.1. Compression isotherms of DOPA and DGPP at acid and basic pHs Polar head group of DGPP may bear from 1 to 3 negative charges, depending on pH (Strawn et al., 2012). Taking the pyrophosphoric acid pKa’s in consideration, and the range of physiological pHs in plants, in a previous work we described the surface behavior of DGPP on subphases at pH 5 (pyrophosphate moiety with 1 or 2 negative charges) and at pH 8 (pyrophosphate moiety with 2 or 3 negative charges). We found that decreasing the pH promotes a shift of the compression isotherm to lower mean molecular areas (Am) and surface potentials (SP) at all surface pressures (π), with negligible changes in the compressibility modulus (Villasuso et al., 2010). Compression isotherms for DGPP at pH 5 and 8 are shown in Fig. 2A and B, being similar to those previously reported within experimental errors. A sharp but very subtle decrease in the compressibility modulus was 3
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Fig. 2. Compression isotherms for monolayers of DGPP and DOPA on subphases at pH 5 and 8. Lateral pressure (left scale) and SP (right scale) as a function of the mean molecular area for DGPP (A) or DOPA monolayers (C). Compressibility modulus for the isotherms shown in A and C. Subphase composition: 0.15 M NaCl +5 mM EDTA, pH 5 (blue line) and pH 8 (red line). All curves show a single representative experiment from a set of triplicates.
Am values were still lower than the ideal one. Regarding the film electrostatics, slight deviations from the weighted sum were observed at all surface pressures for DGPP percent higher than 2 %, with tendencies depending on pH, see Fig. 3C and D. Fig. 3F shows the excess SP (compared to the weighted sum) at 30 mN/ m and at pH 5 or 8. Interesting the deviations were positive at pH 5 and
Interesting, the shift to lower Am values was very high even at very low DGPP contents, with a minimum value of percent of condensation around 2 % DGPP. Furthermore, the degree of condensation markedly depended on pH, being more favorable the mixtures at slightly basic conditions. See that the condensation was doubly at pH 8 compared to pH 5. For higher amounts of DGPP the condensation decreased, but the
Fig. 3. Compression isotherms for mixed monolayers of DGPP and DOPA on subphases at acid and basic pHs. Lateral pressure (upper panels) and Surface potential (lower panels) as a function of the mean molecular area for mixed monolayers of DOPA-DGPP monolayers at acidic pH (A and C) and basic pH (B and C). All curves show a single representative experiment from a set of triplicates. Percent of condensation at 30 mN/m (E) and Surface Potential excess at 30 mN/m (F) as a function of DGPP mole fraction was calculated as it was indicated Material and Methods. The data in Fig. 3F correspond to the average ± SD (SD values from 2 to 10 mV). 4
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Fig. 4. Effect of calcium on compression isotherms for mixed monolayers of DGPP and DOPA on subphases at different pHs. Lateral pressure as a function of the mean molecular area for monolayers of DGPP and DOPA at acidic pH (A and C) and basic pH (B and C).
Fig. 5. Effect of calcium on surface potential density at acid and basic pH. Surface Potential as a function of the mean molecular area for monolayers of of DGPP and DOPA at acidic pH (A and C) and basic pH (B and C).
3.2. Ca(II)-regulation of the surface behavior of DOPA and DGPP at acid and basic pHs
negative at pH 8. The results shown up to now suggest that the biosynthesis of low amounts of DGPP in an environment enriched in DOPA could lead to a local increase in membrane compactation with a concomitant change in the local polarization, further regulated by local pH.
There is great interest in the study of the effect of calcium and their binding with lipid membranes. The effect of this divalent cation on several lipids has been well documented; however, no studies have shown the impact of calcium at different pHs on DGPP films. Fig. 4 5
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interactions, leading to loosely packed monolayers (Brown and Brockman, 2007). Here we analyzed the effect of neutralization by pH and by Ca2+ of monolayers composed of DGPP, it´s precursor DOPA, and mixtures of both lipids. Fig. 7 summarizes the effect of pH and of CaCl2 at 30 mN m−1, where the lipid´s density is comparable to bilayers (Brockman et al., 2003; Brown and Brockman, 2007). The results found at a lower surface pressure can be found in S1. We found, on one hand, that DOPA film density remain unchanged upon acidification, DGPP increased its density and the mixture decreased it (Fig. 7A). The observed compression isotherms of DGPP at pH 5 and 8 are in agreement with an increased electrostatic repulsion at pH 8 where a higher net charge (about −2.2 at pH 8 and about −1.8 at pH 5) is expected, according to the pKa’s values for the pyrophosphate acid (Villasuso et al., 2010). Contrary to DGPP, DOPA did not increase the molecular density upon a pH (and concomitantly a charge) decrease. In similar experiments, it was observed that DPPA monolayers are influenced by pH as a consequence of the change in the ionization of the lipid (Minones et al., 2002). At pH 3, DPPA molecules are uncharged showing a more rigid monolayer structure compared to monolayer spread on subphases at higher pHs. It was proposed that hydrogen bonding between protonated phosphatidic acid groups of adjacent molecules could lead to a more stable and coherent monolayer (Minones et al., 2002). This kind of bonding is only possible for close phosphatidic moieties, which may be present in DPPA, where the Van der Waals interaction between hydrocarbon chains is high, and not in DOPA, where the double bonds disrupts the hydrocabon chain order leading to a decreased interaction. By contrast to the behavior at pH 3, DPPA monolayer at pH 6 exhibits a larger molecular area due to the ionization of the first phosphate of phosphatidic acid, decreasing the possibility for intermolecular hydrogen bonding. The main species for DOPA at pH 5 is the -1charged, and at pH 8, the -2-charged species (Moncelli et al., 1994). However, it has been proposed that in 0.1 M NaCl solutions the phosphate moiety is chelated by Na+, forming neutral species at acid pHs and also at basic ones (Zhang et al., 2018). Related to this, Minones et al. (2002) reported changes in the film density caused by NaCl addition, further reinforcing the importance of Na+-lipid interactions in the film behavior. Also, a restructuration of this NA+-PA− complex may be involved in the rearrangement observed at 30 mN/m. This complex formation, together with the not so close distance between adjacent polar head-groups compared to saturated lipids, may give account of the unresponsively of the DOPA films upon pH, unlike DPPA. In this regards, it has recently been demonstrated that interactions between PA and PC molecules are not favorable compared to pure lipids for systems containing lipids with saturated and unsaturated acyl tails (strongly positive values of the excess free energy of mixing), and very favorable for systems in which all lipid tails are saturated (negative values of the excess free energy of mixing) (Kulig et al., 2019). This fact would indicate that the interaction between anionic and neutral lipids, the tails and also the head groups modulate the film packing. The molecular density in Langmuir films depends on the lateral interactions and on the possible orientation of molecules with its neighbors. All these depend on molecular shape, polar headgroup charge and hydration, hydrogen bonding, electrostatics and Van der Waals interactions, and hydrocarbon chain motion. Therefore, the film density in a mixture is an emergent property that does not necessarily correlate with the behavior of the pure films. This is the case of DGPP/ DOPA (5:95) films. While acid pHs neutralized and compacted DGPP films, and induced no change in DOPA films, the mixed films became more loosely packed upon acidification (see Fig. 7A). This may be due to a change in the coordination of the phosphate group, being less efficient the chelation by Na+ at acid pH than at pH 8. In this regard, as already mentioned the reorganization observed at pH 5 was less evident than at pH 8, probably due to a reduced affinity of the subphase ions with the lipid monolayer.
shows that the presence of CaCl2 in the subphase at pH 5 or 8 caused a shift of the whole isotherm of DGPP and DOPA to smaller Am values at concentrations and degrees that depend on the lipid, the pH and the surface pressure, similarly to the reported effects of Zn2+ on DGPP and POPA (Villasuso et al., 2014). This is expected considering that the binding of this ion neutralize, at least partially, the lipid monolayer. It is important to point out here that, despite the used CaCl2 concentration was mM, the concentration of free Ca2+ ions was much lower and depended on pH due to the chelation by EDTA. For 1 mM CaCl2 and 5 mM EDTA, free Ca2+ values of 20 μM and 1 nM are expected at pH 5 and 8, respectively. These values are within the physiological levels (Xu et al., 2014; Melcrova et al., 2016). We also performed experiments with CaCl2 concentrations higher than 1 mM (maximal 10 mM). However, we decided to show the result with CaCl2 concentration between 0.1–1 mM since these values are within the physiological levels. In this regards, it is important to mention that in cells, a great variety of species that are able to chelate calcium are present, thus decreasing the availability of the free-bivalent ion. At these conditions, competition for calcium with other species rather than a direct interaction with freecalcium is more likely to occur. In order to explore the effect of Ca2+ in the electrostatics of these monolayers, the surface potential was determined in the presence of increasing amounts of CaCl2. At pH 5, the presence of the bivalent ion induced an increase in SP for both, DOPA and DGPP monolayers (Fig. 5, left panels), while at pH 8, a decrease was observed (Fig. 5, right panels). Interesting, the rearrangement observed in DOPA compression isotherms (both, π-Am and SP-Am) persisted in the presence of CaCl2, suggesting that the bivalent ion bounded to the lipid in a different region than that affected by the rearrangement. In general, the effects promoted by Ca2+ were more evident at basic pHs, where the films were more charged and loosely packed (Villasuso et al., 2010) than at acid pHs. It was shown that the effective molecular shape of PA is highly influenced by pH, temperature, and the presence of divalent cations (Minones et al., 2002; Kooijman et al., 2005; Kooijman and Burger, 2009; Strawn et al., 2012; Villasuso et al., 2014). At pH 7 in the presence of magnesium ion, PA acquires a cylindrical shape and thus stabilizes the lamellar topography. However, once subjected to an acid environment, the head group of PA decreases its effective size, and the lipid undergoes a shape change to a cone-like shape, thus affecting the membrane by promoting an increase of negative membrane curvature (Kooijman and Burger, 2009). DGPP formation after stimulus takes place after a transient increase of the PA levels (Racagni et al., 2008). Therefore, as already mentioned, a temporary and local accumulation of DGPP and its precursor at the membrane interface is expected under stress. Since it has been shown that the effect of Zn2+ on POPA/DGPP mixtures depends on the mixture composition and subphase pH (Villasuso et al., 2010; 2014), we studied the packing properties of mixed films of DGPP and DOPA in the presence of Ca2+ on subphases at acid and basic pHs. Mixtures composed of DGPP/DOPA 5:95 were chosen for the analysis because, on one hand, it is a DGPP proportion of physiological relevance, since this lipid is minority in cells. On the other, the highest condensation was observed in this range of lipid proportions. Fig. 6 shows the compression isotherm for a DOPA/DGPP 95:5 mixture at pH 5 and 8 with increasing CaCl2 concentrations. Similar to the pure lipids, the cation decreased Am values at both pHs (Fig. 6A and B), whilst SP increased at acid pHs and decreased at basic ones (Fig. 6C and D). 4. Discussion The shape of the compression isotherms of monolayers for a particular lipid species depend on the length and unsaturation of the hydrocarbon chain and on the bulkiness and charge of the polar headgroup. Long and saturated hydrocarbon chains would interact through Van der Waals attractions, promoting more condensed monolayers. By contrast, ionization of the lipid head groups should result in repulsive 6
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Fig. 6. Effect of calcium on surface pressure and surface potential as a function of average molecular area for DOPA and DGPP mixture. Lateral pressure (upper panel) and surface potential (lower panel) as a function of the mean molecular area for mixed monolayers of 95 %DOPA-5 %DGPP at indicated pH and CaCl2 concentration.
Fig. 7. Effect of neutralization by pH and by Ca2+ of DGPP, DOPA, and mixtures of both lipids. Effect of Ca2+ on mean molecular area (A) and surface potential (C) for pure and mixed lipid. Percent of condensation (B) and surface potential change (D) as a function of CaCl2 concentration for pure and mixture of DOPA and DGPP. The lateral pressures are 30 mN m−1. The values are shown as average ± SD of three independent experiments for each condition at 30 mN/m. Asterisks (*) represent significant differences at pH 5 or pH 8 according to Student's test (p < 0.05). Scale starts at 225 mV.
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lipid has been evaluated with different model systems, and it was found that some divalent cations at high concentration induce the formation of clusters of phospholipids. In the case of the precursor of second messenger PIP2, only Ca2+, but not Mg2+, Zn2+ or polyamines induces a surface pressure drop that coincides with the formation of PIP2 clusters (Wang et al., 2012). Studies of the interaction between PA and Ba2+ suggested that PA is extremely effective in binding divalent ions through its oxygen atoms, with a broad distribution of binding constants and exhibiting the phenomenon of charge inversion (a total number of bound counterion charges that exceeds the negative PA charge) (Faraudo and Travesset, 2007). The authors predict that a PArich domains undergo a drastic reorganization when divalent cations as Ca2+ and Ba2+ reach micromolar concentrations (i.e., typical physiological conditions), as PA lipids become doubly charged by releasing their protons. Although restricted to PA, those results could be qualitatively similar for other phospholipids, as DGPP, which play somewhat similar roles as PA. Here, a marked increase on SP was observed at pH 5 upon an increase in CaCl2 concentration (Fig. 7C and D). On the contrary, at pH 8 Ca2+ ions induced a low depolarization of the interface with the pure lipids. Interestingly, the mixture showed an different behavior, an increase of SP was observed when CaCl2 was present in the subphase at both, acid and basic pHs (Fig. 7D).
Regarding the Ca(II)-mediated neutralization, the area reduction was maximal at basic pHs, where the lipid-ion interaction is expected to be favored, due to the negative charge on the polar headgroups (Fig. 7A and B). It has to be considered that at acid pHs, the free-Ca(II) concentration is higher than at basic pH due to the decrease in the concentration of EDTA4− (For 1 mM CaCl2, free-Ca2+ = 20 μM at pH 5 and 1 nM at pH 8), strengthening the observation that the interaction between the bivalent ion and the lipids was very favored at basic pHs compared to acid ones. Furthermore, the areas at pH 8 in the presence of CaCl2 showed the lowest values for each monolayer composition (Fig. 7A), indicating that neutralization by Ca2+ ions was more efficient for membrane compactation than protonation. It may be that a Ca2+ ion interacts with more than one lipid, thus promoting membrane condensation more effectively. Surprisingly, at basic pHs the mixture was less responsive to Ca2+ than the pure lipids (Fig. 7B, red circles compared with red triangles). This is probably a consequence that the mean molecular area for the mixtures was already very low in the absence of the bivalent ion, and thus with reduced possibilities of further compactation. Therefore, at pH 8 the behavior of the mixture upon CaCl2 addition was not intermediate between the pure components but different properties emerge regarding lipid compactation. The dielectric constant decreases abruptly from about 80 in the bulk aqueous medium to values in the range of 3 to 10 in the polar head group-hydrocarbon interface, depending on the characteristics of the polar head groups (Montich et al., 1985). Thus, the ionization degree of the lipid polar headgroup strongly impacts on membrane electrostatic potential with an effect on the distribution and ionization of nearby charged components such as lipid dipoles that can act as sensitive sensors of the surface electrostatics (Seelig et al., 1987). The surface potential is a resultant of a complex combination of the double layer potential, and contributions of hydrocarbon chain dipoles, of water dipoles from the polar head group hydration shell, and intrinsic polar headgroup dipoles, as well as from the electrostatic field brought about from the relative position of mobile ions interacting specifically with the polar head-group moieties. We observed, on one hand, that the surface potentials of DOPA and of DGPP in the absence of CaCl2 were lower at acid pHs than at basic ones, as a consequence of the lower surface charge of the films formed by both molecules at pH 5 compared to 8. Thus, despite the Na+ ions prevented density changes with pH in DOPA monolayers, depolarization of the interface occurred due to a pH decrease. Similar to what was described for the film density, the mixture showed a behavior that did not correlate with that of the pure lipids. In the mixed monolayers, no change in SP occurred when pH changed (Fig. 7C). Additionally, the film electrostatics of the mixture did not depict the values expected for an ideal mixture (SI 2). Compared to the pure lipids, we found that mixtures of DGPP with DOPA presented lower values of SP when mixed at pH 8, and higher values at pH 5 (SI 2, C and D), indicating a complex modulation of the membrane electrostatics. Since the mixtures were more condensed than ideal behavior (SI 2 A and B), the accessibility of the ions (Na+ and H+) of the subphase to the polar headgroups was reduced in the mixture, probably leading to the non-ideal behavior in the surface potential. Besides, the negative charge on the lipids signifies an anionic surface with a corresponding distribution of mobile ions in the immediate aqueous milieu (the double layer potential) which is also included in the surface potential measured. It was previously observed in stearic acid monolayers that an increase of the negative charge in the monolayer may decrease the double layer potential by values as high as 200 mV (Vega Mercado et al., 2011). Therefore, a decrease of the double layer potential when the lipid becomes ionized may result in a lower surface potential even if the overall lipid dipole is being increased in the negatively charged molecule (Vega Mercado et al., 2011), and thus the behavior of this parameter is not straightforward to predict nor to explain. The effect of divalent cations on the interfacial behavior of anionic
5. Conclusion Our results indicate that the film properties of DGPP and its precursor DOPA were sensitive to Ca2+ in a pH-dependent manner, and thus depended on the ionization state of the molecules forming the film. Low proportions of DGPP in the DOPA film further modulated this effect. The changes in film density promoted by Ca2+ were more marked on subphases at pH 8, while at pH 5 the influence of the cation in the film surface properties was attenuated. The non-ideal behavior of the mixed monolayer, which was sensitive to the ionic composition of the aqueous milieu, indicates a complex regulation of film properties with composition. It has been shown that changes in oscillations of Ca2+ usually occur with pH variations during stress response (Li et al., 2019). Thus, modifications in the PA and DGPP proportion, coupled with pH and Ca2+-level variations could be part of the response to stress in plant. Declaration of Competing Interest The authors declare no competing interests. Acknowledgments This work was supported by SECyT-UNRC, SECyT-UNC, FONCYT (PICT 2015-1108 and 2015-0662), and CONICET. MPM is a fellowship of CONICET. NW and ALV are Career Investigators of CONICET. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chemphyslip.2020. 104887. References Arisz, S.A., Heo, J.Y., Koevoets, I.T., Zhao, T., Van Egmond, P., Meyer, J., Zeng, W., Niu, X., Wang, B., Mitchell-Olds, T., Schranz, M.E., Testerink, C., 2018. Diacylglycerol acyltransferase 1 contributes to freezing tolerance. Plant Physiol. https://doi.org/10. 1104/pp.18.00503. Astorquiza, P.L., Usorach, J., Racagni, G., Villasuso, A.L., 2016. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone. Plant Physiol. Biochem. 101, 88–95. https://doi.org/10.1016/j. plaphy.2016.01.012.
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