<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

Differential expression of thermophilic phosphatases in the wild type and auxotrophic mutant strains of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

In the wild type strain (stock no. 1227) of Thermoactinomyces vulgaris, as reported earlier [Sinha and Singh (1980) Biochem. J. 190, 457–460], all phosphatase isoenzymes (three alkaline — AlpI, AlpII and AlpIII, and one acidic — Acp) are present. However, the auxotrophic mutants, the strains 1286 (thi ⁻), 1279 (nic ⁻, ura ⁻) and 1278 (thi ⁻, ura ⁻) exhibited two alkaline phosphatase isoenzymes (AlpII and AlpIII), but AlpI was lacking. In the strain 1261 (nic ⁻, thi ⁻), only AlpIII was expressed, and AlpI and AlpII isoenzymes were missing. The results suggest that the strains, which require either thiamine (1286 and 1278) or nicotinamide (1279) for their growth, were AlpI ⁻ mutants; and the strain (1261), which requires both thiamine and nicotinamide for its growth, was AlpI ⁻/AlpII ⁻ double mutant. There was no direct correlation between uracil auxotrophy and the expression of phosphatases. The uniform expression of AlpIII and Acp in all the strains, irrespective of their nutrient requirements, suggest that these constitutive phosphatases are species-specific. The specific activities of the thermophilic acid and alkaline phosphatases were maximum in the wild type strain (1227) of T. vulgaris. The next in phosphatase activity was the strain 1279 (an AlpI ⁻ mutant), followed by their decrease, in order, in the strains 1286 and 1278 (which were also AlpI ⁻ mutants); while least activity of these enzymes was observed in the obligate thermophile strain 1261 (AlpI ⁻/AlpII ⁻ double mutant).     

Ca<Superscript>2+</Superscript>-Dependence and Inhibition of Transformation by Trifluoperazine and Chlorpromazine in <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

Ca²⁺ enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl₂ upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01–0.04 mM along with optimal CaCl₂ concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca²⁺-stimulated transformation. The results of present investigation suggest the involvement of a Ca²⁺-dependent protein activator in the development of Ca²⁺-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete. Received: 21 May 2002 / Accepted: 21 June 2002     

Plant Ca<Superscript>2+</Superscript>-ATPases

Plant calcium pumps, similarly to animal Ca²⁺ pumps, belong to the superfamily of P-type ATPase comprising also the plasma membrane H⁺-ATPase of fungi and plants, Na⁺/K⁺ ATPase of animals and H⁺/K⁺ ATPase of mammalian gastric mucosa. According to their sensitivity to calmodulin the plant Ca²⁺-ATPases have been divided into two subgroups: type IIA (homologues of animal SERCA) and type IIB (homologues of animal PMCA). Regardless of the similarities in a protein sequence, the plant Ca²⁺ pumps differ from those in animals in their cellular localization, structure and sensitivity to inhibitors. Genomic investigations revealed multiplicity of plant Ca²⁺-ATPases; they are present not only in the plasma membranes and ER but also in membranes of most of the cell compartments, such as vacuole, plastids, nucleus or Golgi apparatus. Studies using yeast mutants made possible the functional and biochemical characterization of individual plant Ca²⁺-ATMPases. Plant calcium pumps play an essential role in signal transduction pathways, they are responsible for the regulation of [Ca²⁺] in both cytoplasm and endomembrane compartments. These Ca²⁺-ATPases appear to be involved in plant adaptation to stress conditions, like salinity, chilling or anoxia.     

Chlorpromazine and dimethyl sulfoxide modulate the catalytic activity of the plasma membrane Ca<Superscript>2+</Superscript>-ATPase from human erythrocyte

The plasma membrane Ca²⁺-ATPase (PMCA) removes Ca²⁺ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca²⁺ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca^{2 +}.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Role of SH Groups in the Functioning of Ca2+-Transporting ATPases Regulating Ca2+ Homeostasis and Exocytosis

Using a ^Ca2+-sensitive fluorescent indicator, Fura-2/AM, and a metallochromic dye, arsenazo, we measured the intracellular concentration of Ca²⁺ ([Ca²⁺] _i ) and the content of total calcium in isolated acinar cells of the rat submandibular salivary gland. It was shown that the influence of a mercaptide-forming compound, sodium p-chloromercuribenzoate (pChMB), increased both the [Ca²⁺] _i and content of total calcium but did not change the intensity of exocytosis. Such a situation is probably related to the fact that pChMB inhibits plasmalemmal Ca²⁺-ATPase (PMCA). The absence of changes in the exocytotic activity can be explained as follows: the influence of a pChMB-induced significant increase in the [Ca²⁺] _i is neutralized due to the functioning of Ca²⁺-ATPases of the endoplasmic reticulum (SERCA), which pump Ca²⁺ into the store. Incubation of a microsomal fraction with pChMB resulted in suppression of the specific PMCA and SERCA activities with apparent constants of inhibition (I ₅₀) 245 and 52 M, respectively. Dithiothreitol (DTT, 0.1 mM) increased the PMCA and SERCA activities (probably facilitating the access of substrate to the active centers of ATPases at the expense of a decrease in the number of disulfide bonds, which is followed by changes in the conformation of intracellular hydrophilic loops of their molecules). Dithiothreitol also recovered the suppression of PMCA and SERCA activities induced by pre-incubation with pChMB (by 45 and 32%, respectively); these activities did not, however, reach the initial levels. A probable interpretation of this fact is that DTT shields from the action of pChMB only superficial but not sterically less accessible SH groups. Limited proteolysis of the microsomes by -chymotrypsin decreased the specific PMCA and SERCA activities by 16 and 60%, respectively. Incubation of the microsomes in an -chymotrypsin-containing medium (15 sec) with subsequent addition of 150 M pChMB exerted almost no influence on the PMCA activity, whereas the SERCA activity dramatically increased (by 146%). This fact allows us to suggest that -chymotrypsin is capable of eliminating the inhibitory effect of pChMB on the SERCA activity; the mechanism of this effect remains unknown. Therefore, functionally important SH groups are present in the catalytic and active centers of both PMCA and SERCA; superficial SH groups dominate in the PMCA molecules, whereas SERCA is controlled by more deeply localized SH groups.     

Ca<Superscript>2+</Superscript>-ATPase in the symbiosome membrane from broad bean root nodules: further evidence for its functioning as ATP-driven Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchanger

To date, it has been established that the symbiosome membrane (SM), i.e., plant-derived membrane of symbiosomes, nitrogen-fixing compartments of legume root nodules, is equipped with Ca²⁺-ATPase transporting Ca²⁺ ions through the SM from the cytosol of infected cells into the symbiosome space (SS). Earlier in the experiments on the SM vesicles isolated from broad bean root nodules some data indicating the action of the Ca²⁺-ATPase as ATP-driven Ca²⁺/H⁺ antiporter were obtained. In the present work performed on isolated symbiosomes from the same plant object, further evidence in favor of calcium-proton countertransport mechanism of the pump operation was obtained. These were expressed in vanadate-sensitive alkalinization of the SS coupled with Ca²⁺ uptake by symbiosomes catalyzed by the SM Ca²⁺-ATPase, stimulation of the kinetics of the latter process in the response to artificial acidification of the SS and expectable modulation of ITP-hydrolyzing activity of this enzyme caused by the variation of pH within this compartment. The above findings are discussed in the framework of the model describing the mechanism of Ca²⁺-ATPase operation as an ATP-driven Ca²⁺/H⁺ exchanger and on this base allow us to put forward the hypothesis about the involvement of this enzyme in symbiosome signaling in a Ca²⁺- and pH-dependent manner.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

N-terminus of PutCAX2 from <Emphasis Type="Italic">Puccinellia tenuiflora</Emphasis> affects Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript> tolerance in yeast

Cation/H⁺ exchangers (CAXs) are membrane proteins that transport Ca²⁺ and other cations using the H⁺ gradient generated by H⁺-ATPase or H⁺-pyrophosphatase. This study reports the characterization of CAX2 from Puccinellia tenuiflora with respect to molecular and functional properties. PutCAX2 was cloned from a cDNA library of P. tenuiflora seedlings. The expression of PutCAX2 in shoots and roots was induced by Ca²⁺ and Ba²⁺ treatments. A green fluorescent protein (GFP) marker revealed that PutCAX2 was located on the endoplasmic reticulum (ER) membrane. Four yeast transformants were created using GFP fusion PutCAX2 and truncated PutCAX2s, and their growth in the presence of various cations (Fe³⁺, Al³⁺, Mn²⁺, Cu²⁺, Co²⁺, Ni²⁺, Mg²⁺, Zn²⁺, Na⁺, Li⁺, Ca²⁺, and Ba²⁺) was analyzed. The N-terminally truncated PutCAX2 (GFP-ΔNPutCAX2) and the N and C-terminally truncated PutCAX2 (GFP-ΔNCPutCAX2) transformants grew well in the presence of 100 and 150 mM Ca²⁺ or 8 and 20 mM Ba²⁺, whereas the GFP-PutCAX2 and C-terminally truncated PutCAX2 (GFP-ΔCPutCAX2) transformants did not show any tolerance to Ca²⁺ or Ba²⁺. The Ba²⁺ content in whole yeast cells expressing GFP-ΔNPutCAX2 or GFP-ΔNCPutCAX2 was lower than that in other yeast transformants. Moreover, the efflux experiment showed that the Ba²⁺ efflux rate of yeast cells expressing GFP-ΔNPutCAX2 and GFP-ΔNCPutCAX2 was higher than that of other yeast cells. To our knowledge, this is the first report on the molecular and functional characterization of a novel ER-localized CAX protein from a wild halophyte plant; the results suggest that the N-terminus of PutCAX2 acts as an auto-inhibitory domain, which affects the Ca²⁺ and Ba²⁺ tolerance of yeast.     

Mechanism of luminal Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca²⁺-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca²⁺ and Mg²⁺ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca²⁺—with a much higher affinity than Mg²⁺—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca²⁺-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca²⁺ at the vacuolar side. The specificity for Ca²⁺ compared to Mg²⁺ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca²⁺ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar     

Cloning and Characterization of a Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> Antiporter from Halophyte <Emphasis Type="Italic">Suaeda salsa</Emphasis> L.

We have isolated the cDNA of Ca²⁺/H⁺ antiporter which was designated as Suaeda salsa cation exchanger 1 (SsCAX1) from the C₃ halophyte S. salsa L. To ascertain the location of SsCAX1 in the cell, a peptide-specific antibody to SsCAX1 was prepared, and Western blotting analysis showed that it reacted only with a 48.8-kDa protein from S. salsa vacuolar membrane. Furthermore, SsCAX1 could resume yeast vacuolar Ca²⁺ transport mutants growth in high Ca²⁺ concentration (200 mM) culture medium. Northern blotting analysis showed that SsCAX1 expression was mainly found in the leaves and stems and slightly in the roots of S. salsa seedlings. Moreover, SsCAX1 expression levels and the protein amounts were significantly upregulated by CaCl₂ and NaCl treatment, respectively. In addition, the upregulation of the expression levels of V-H⁺-ATPase subunit c coordinated with the expression levels of the Ca²⁺/H⁺ antiporter under salinity. These results suggested that SsCAX1 from halophyte S. salsa might be a Ca²⁺ transporter at tonoplast and plays a key role in maintaining cytosolic Ca²⁺ homeostasis under saline condition.     

Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca<Superscript>2+</Superscript> sensitivity but not on Ca<Superscript>2+</Superscript> sparks or BK channels

Background

In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK) channels leading to lowered SMC [Ca²⁺]_i and vasodilation. Here we investigated whether Ca²⁺ sparks regulate SMC global [Ca²⁺]_i and diameter in the spiral modiolar artery (SMA) by activating BK channels.

Methods

SMAs were isolated from adult female gerbils, loaded with the Ca²⁺-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca²⁺ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca²⁺ signals and vascular diameter were analyzed.

Results

Ca²⁺ sparks and waves were observed in pressurized SMAs. Inhibition of Ca²⁺ sparks with ryanodine increased global Ca²⁺ and constricted SMA at 40 cmH₂O but inhibition of Ca²⁺ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH₂O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH₂O was abolished at 60 cmH₂O, consistent with a greater Ca²⁺-sensitivity of constriction at 40 cmH₂O than at 60 cmH₂O. When the Ca²⁺-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH₂O.

Conclusions

The results suggest that Ca²⁺ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca²⁺-sensitivity of constriction.

     

Ca<Superscript>2+</Superscript> signalling in cardiovascular disease: the role of the plasma membrane calcium pumps

The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca²⁺ from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca²⁺ signalling, depending on the cell type. In the cardiovascular system, Ca²⁺ is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca²⁺ homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.     

Effect of jasmonic acid on Ca<Superscript>+2</Superscript> transport through the plasmalemma of potato tuber cells

The rate of Ca²⁺ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10^?5–10^?10 M jasmonic acid on the accumulation of Ca⁺² in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca⁺²,Mg⁺²-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca⁺²,Mg⁺²-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.     

Dependence of the CO<Subscript>2</Subscript> Uptake in a Plant Cell on the Plasma Membrane H<Superscript>+</Superscript>-ATPase Activity: Theoretical Analysis

A decrease of the plasma membrane H⁺-ATPase activity in plant cells is associated with the formation of response to adverse factors and reception of signals. A theoretical analysis of the influence of the H⁺-ATPase activity on the flow of CO₂ into a plant cell has been conducted. With this purpose the model of transport processes and electrogenesis in the plant cell developed previously was used, which takes into account transport systems, including H⁺-ATPase, in the plasma membrane and tonoplast. The CO₂ fraction (\({P_{c{o_2}}}\)) in the total amount of inorganic carbon (C_i) in the external medium was used as an indicator of the CO₂ amount entering the plant cell; this parameter depends on the extracellular pH, which, in particular, is influenced by the H⁺-ATPase activity. Excitable and non-excitable cells were simulated. It was shown that a decrease of the H+-ATPase activity causes a \({P_{c{o_2}}}\)reduction in both variants of the model, and this reduction has an extremum: after passing through minimal values, \({P_{c{o_2}}}\)reaches a stationary level. The dynamics of \({P_{c{o_2}}}\)decrease may be related to the Ca²⁺ influx into the cytoplasm of the plant cell. The reduction of \({P_{c{o_2}}}\)depended on the extent of the H⁺-ATPase inactivation and on its initial activity. As a whole, it was shown that the inactivation of the H⁺-ATPase can affect the CO₂ uptake in a plant cell and thereby regulate photosynthetic processes.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional expression in yeast of an N-deleted form of<Emphasis Type="Italic"> At</Emphasis>-ACA8, a plasma membrane Ca<Superscript>2+</Superscript>-ATPase of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>, and characterization of a hyperactive mutant

A constitutively active form of At-ACA8, a plasma membrane Ca²⁺-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (74-At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca²⁺ transport systems. 74-At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. 74-At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca²⁺ uptake and Ca²⁺-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca²⁺-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the ⁷⁹⁴HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of 74-At-ACA8. The ⁷⁹⁴HEKK mutant was also about 6-fold more sensitive than 74-At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E₂ state during the catalytic cycle.Abbreviations aa Amino acids - CaM Calmodulin - EB Erythrosin B - ER Endoplasmic reticulum - EY Eosin Y - FITC Fluorescein isothiocyanate - PM Plasma membrane     

Carbonyl stress: malondialdehyde induces damage on rat hippocampal neurons by disturbance of Ca<Superscript>2+</Superscript> homeostasis

The objective of this study was to investigate the influences of carbonyl stress induced by malondialdehyde (MDA), a typical intermediate of lipid peroxidation, on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) alterations in cultured hippocampal neurons of rat. The microphotographic study clearly demonstrated that the hippocampal neurons became gradually damaged following exposure to different concentrations of MDA. Further study indicated that the plasma membrane Ca²⁺-ATPase (PMCA) activity was inhibited by MDA in a concentration- and time-dependent manner. The supplementation of 100 μM MDA was found to cause a notable early phase increase of [Ca²⁺]_i in hippocampal neuron cultures followed by a more pronounced late-phase elevation of [Ca²⁺]_i. Such effect of MDA was prevented by the addition of nimodipine, an inhibitor of L-type calcium channel or by an extracellular Ca²⁺ chelator EGTA. The identification of the calcium signalling pathways were studied by applying U73122, an inhibitor of PL-C, and H-89, an inhibitor of protein kinase A (PKA), showing the involvement of PL-C/IP3 pathway but not the PKA/cAMP pathway. These results suggested that MDA-related carbonyl stress caused damages of rat hippocampal neurons by triggering Ca²⁺ influx and influencing Ca²⁺ homeostasis in cultured neurons, and also MDA may act as a signalling molecule regulating Ca²⁺ release from intracellular stores.