Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges

The vacuolar pH and the trans-tonoplast ΔpH modifications induced by the activity of the two proton pumps H⁺-ATPase and H⁺-PPase and by the proton exchanges catalyzed by the Na⁺/H⁺ and Ca²⁺/H⁺ antiports at the tonoplast of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were measured using the ³¹P NMR technique. The H⁺-ATPase induced an intravacuolar acidification as large as 0.8 pH unit, building a trans-tonoplast ΔpH up to 2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar acidification were monitored simultaneously to estimate kinetically the apparent stoichiometry between the vectorial proton pumping and the hydrolytic activity of the H⁺-ATPase. A ratio of H⁺ translocated/ATP hydrolyzed of 1.97 ± 0.06 (mean ± standard error) was calculated. Pyrophosphate-treated vacuoles were also acidified to a significant extent. The H⁺-PPase at 2 millimolar PPi displayed hydrolytic and vectorial activities comparable to those of the H⁺-ATPase, building a steady state ΔpH of 2.1 pH units. Vacuoles incubated in the presence of 10 millimolar Na⁺ were alkalinized by 0.4 to 0.8 pH unit. It has been shown by using ²³Na NMR that sodium uptake was coupled to the H⁺ efflux and occurred against rather large concentration gradients. For the first time, the activity of the Ca²⁺/H⁺ antiport has been measured on isolated intact vacuoles. Ca²⁺ uptake was strongly inhibited by NH₄Cl or gramicidin. Vacuoles incubated with 1 millimolar Ca²⁺ were alkalinized by about 0.6 pH unit and this H⁺ efflux was associated to a Ca²⁺ uptake as demonstrated by measuring the external Ca²⁺ concentration with a calcium specific electrode. Steady state accumulation ratios of Ca²⁺ as high as 100 were reached for steady state external concentrations about 200 micromolar. The rate of Ca²⁺ uptake appeared markedly amplified in intact vacuoles when compared to tonoplast vesicles but the antiport displayed a much lower affinity for calcium. The different behavior of intact vacuoles compared to vesicles appears mainly to be due to differences in the surface to volume ratio and in the rates of dissipation of the pH gradient. Despite its low affinity, the Ca²⁺/H⁺ antiport has a high potential capacity to regulate cytoplasmic concentration of calcium.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

Effect of Ca and Calmodulin on DeltapH Formation in Tonoplast Vesicles from Corn Roots

The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (ΔpH) were studied in tonoplast membrane vesicles from corn (Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca²⁺ and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H⁺ efflux or due to inhibition of the H⁺-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed ΔpH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed ΔpH by 40% after the H⁺-ATPase was inhibited with NO₃⁻. This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H⁺-ATPase by direct inhibition.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.

     

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

The GTP-driven component of Ca²⁺ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca²⁺-translocating ATPase and assess its utility as a probe for this transport system. Uptake of ⁴⁵Ca²⁺ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca²⁺ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca²⁺-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of ⁴⁵Ca²⁺-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven ⁴⁵Ca²⁺ uptake represents the capacity of the plasma membrane Ca²⁺-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-³²P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca²⁺-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca²⁺-ATPases present in animal cells.     

Calcium and proton transport in membrane vesicles from barley roots

Ca²⁺ uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of ⁴⁵Ca²⁺ was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca²⁺ uptake coincided with the peak of proton transport by the tonoplast H⁺-ATPase. Depending on the concentration of Ca²⁺ in the assay, Ca²⁺ uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca²⁺ uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that ⁴⁵Ca uptake was driven by the low affinity, high capacity tonoplast Ca²⁺/nH⁺ antiporter and also by a high affinity, lower capacity Ca²⁺-ATPase. The Ca²⁺-ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca²⁺ transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-man-nosyl synthase activities. A small shoulder of Ca²⁺ uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca²⁺-ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca²⁺ concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca²⁺ created 800- to 2000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca²⁺ on ATP-generated pH gradients are discussed and the suggestion is made that Ca²⁺ dissipates pH gradients by a different mechanism than is responsible for Ca²⁺ uptake into tonoplast vesicles.     

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

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

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

ATP-dependent Ca2+ transport in wheat root plasma membrane vesicles

Plasma membrane preparations of high purity were obtained from roots of dark-grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. These preparations mainly contained sealed, right-side-out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside-out (cytoplasmic side out) vesicles increased to ca 30%. Inside-out and right-side-out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two-phase system. In this way, highly purified, sealed, inside-out (>60% inside-out) vesicles were isolated and subsequently used for characterization of the Ca²⁺ transport system in the wheat plasma membrane. The capacity for ⁴⁵Ca²⁺ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside-out plasma membrane vesicles) were all enriched in the inside-out vesicle fraction as compared to the right-side-out fraction. This confirms that the ATP-binding site of the ⁴⁵Ca²⁺ transport system, similar to the H⁺-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The ⁴⁵Ca²⁺ uptake was MgATP-dependent with an apparent K_m for ATP of 0.1 mM and a high affinity for Ca²⁺ [K_m(Ca²⁺/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the ⁴⁵Ca²⁺ uptake seen with ATP. The ⁴⁵Ca²⁺ uptake was stimulated by monovalent cations; K^? and Na⁺ being equally efficient. Vanadate inhibited the ⁴⁵Ca²⁺ accumulation with half-maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K⁺-stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half-maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca²⁺ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP-binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca²⁺ within the cell.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample

A continuous spectrophotometric assay of H⁺-ATPase activity was developed by combining two well-known methods for measuring proton pumping and ATPase activity. Proton uptake into plasma membrane vesicles from Avena sativa L. (cv Rhiannon) was monitored as the absorbance decrease at 495 nm of the ΔpH probe acridine orange. Simultaneously, ATPase activity was measured by following the absorbance decrease at 340 nanometers by coupling ATP hydrolysis enzymatically to the oxidation of NADH. This H⁺-ATPase assay is convenient for determining the relative relationship between ATP hydrolysis and proton pumping.     

Kinetics of Ca/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L

Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were used to study the kinetics of a Ca²⁺/H⁺ antiport across this membrane. Ca²⁺-dependent H⁺ fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ΔpH-dependent Ca²⁺ influx was measured radiometrically. Both H⁺ efflux and Ca²⁺ influx displayed saturation kinetics and an identical dependence on external calcium with apparent K_m values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg²⁺ but was inhibited by La³⁺ > Mn²⁺ > Cd²⁺. The apparent K_m for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested.