An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Postprandial acid–base balance and ion regulation in freshwater and seawater-acclimated European flounder, <Emphasis Type="Italic">Platichthys flesus</Emphasis>

The effects of feeding on both acid–base and ion exchange with the environment, and internal acid–base and ion balance, in freshwater and seawater-acclimated flounder were investigated. Following voluntary feeding on a meal of 2.5–5% body mass and subsequent gastric acid secretion, no systemic alkaline tide or respiratory compensation was observed in either group. Ammonia efflux rates more than doubled from 489 ± 35 and 555 ± 64 μmol kg⁻¹ h⁻¹ under control conditions to 1,228 ± 127 and 1,300 ± 154 μmol kg⁻¹ h⁻¹ post-feeding in freshwater and seawater-acclimated fish, respectively. Based on predictions of gastric acid secreted during digestion, we calculated net postprandial internal base gains (i.e., HCO₃⁻ secreted from gastric parietal cells into the blood) of 3.4 mmol kg⁻¹ in seawater and 9.1 mmol kg⁻¹ in freshwater-acclimated flounder. However, net fluxes of ammonia, titratable alkalinity, Na⁺ and Cl⁻ indicated that branchial Cl⁻/HCO₃⁻ and Na⁺/H⁺ exchange played minimal roles in counteracting these predicted base gains and cannot explain the absence of alkaline tide. Instead, intestinal Cl⁻/HCO₃⁻ exchange appears to be enhanced after feeding in both freshwater and seawater flounder. This implicates the intestine rather than the gills as a potential route of postprandial base excretion in fish, to compensate for gastric acid secretion.     

Aqua bridge cleavage and metal ion extrusion by thiocyanate anions in a dicopper complex

Reaction of the imidazolidinyl phenolate-based ligand, H₃L [(2-(2′-hydroxyphenyl)-1,3-bis[4-(2-hydroxyphenyl)-3-azabut-3-enyl]-1,3-imidazolidine)] with Cu(ClO₄)₂·6H₂O produces an aqua-bridged cationic reactant complex [Cu₂(μ-H₂O)(μ-L)][ClO₄]·1.5H₂O (1·1.5H₂O). Solution phase interaction of 1·1.5H₂O with SCN⁻ anions in 1:1 molar ratio leads to [Cu₂(μ-L)(NCS)]·2H₂O (2·2H₂O) that does not possess anymore the reactive aqua bridge but instead a terminal SCN⁻ anion coordinated only to one Cu^II ion. Whereas in 1:2 molar ratio, partial extrusion of the Cu^II ions takes place to generate in situ [Cu(NCS)₃(OH₂)]⁻ anions. These complex anions then quantitatively replace anions in 1·1.5H₂O via ‘anion metathesis’ and concurrently remove the aqua bridge by coordination of linear MeCN to one of the Cu^II ions to give [Cu₂(μ-L)(CH₃CN)][Cu(NCS)₃(OH₂)] (3). The literature unknown [Cu(NCS)₃(OH₂)]⁻ anion forms an intimate H-bonded assembly with the cationic part of 3 to yield a novel [Cu₃] isosceles triangle. The precursor complex is known as antiferromagnetic whereas in 2·2H₂O, the Cu^II (S = 1/2) ions in a dinuclear entity exhibit ferromagnetic interactions (J/k_B = +15.0 K and g = 2.22) to yield an S_T = 1 spin ground state in good agreement with the M versus H data below 8 K.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes

Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (σ_max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol >ClO₄⁻ >I⁻ >SCN⁻ >Br⁻ >NO₃⁻ >Cl⁻ SO₄²⁻. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of σ_max. The binding sites were considered as defects in the package of lipid molecules in membranes.     

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

The mitochondrial inner membrane anion channel is inhibited by DIDS

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg²⁺ and H⁺. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC₅₀ values of 26, 44, and 88 M for DIDS, H₂-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl^– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC₅₀ which is severalfold higher than that for malonate. The IC₅₀ for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC₅₀ by about 50%, but under conditions where only the matrix pH is made alkaline the IC₅₀ is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl^– channels than previously thought, but also suggest that other Cl^– channels may be sensitive to some of the many regulators of IMAC which have been identified.     

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO -) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 μM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 μM, while inhibiting it at 1000 μM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro . Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 μM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro , infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.     

Superoxide Anion Production and Expression of gp91phox and p47phox Are Increased in Glomeruli and Proximal Tubules of Cisplatin‐Treated Rats

The chemotherapeutic drug cisplatin has some side effects including nephrotoxicity that has been associated with reactive oxygen species production, particularly superoxide anion. The major source of superoxide anion is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. However, the specific segment of the nephron in which superoxide anion is produced has not been identified. Rats were sacrificed 72 h after cisplatin injection (7.5 mg/kg), and kidneys were obtained to isolate glomeruli and proximal and distal tubules. Cisplatin induced superoxide anion production in glomeruli and proximal tubules but not in distal tubules. This enhanced superoxide anion production was prevented by diphenylene iodonium, an inhibitor of NADPH oxidase. Consistently, this effect was associated with the increased expression of gp91^phox and p47^phox, subunits of NADPH oxidase. The enhanced superoxide anion production in glomeruli and proximal tubules, associated with the increased expression of gp91^phox and p47^phox, is involved in the oxidative stress in cisplatin‐induced nephrotoxicity.