Anion-cation interactions in the pore of neuronal background chloride channels

Background Cl channels in neurons and skeletal muscle are significantly permeable for alkali cations when tested with asymmetrical concentrations of the same salt. Both anion and cation permeation were proposed to require binding of an alkali cation with the pore (Franciolini, F., and W. Nonner. 1987. Journal of General Physiology. 90:453-478). We tested this hypothesis by bilaterally substituting large alkali cations for Na and found no significant changes of unitary conductance at 300 mM symmetrical concentrations. In addition, all organic cations examined were permeant in a salt gradient test (1,000 mM internal@300 mM external), including triethanolamine, benzyltrimethylamine, and bis-tris-propane (BTP, which is divalent at the tested pH of 6.2). Inward currents were detected following substitution of internal NaCl by the Na salts of the divalent anions of phosphoric, fumaric, and malic acid. Zero-current potentials in gradients of the Na and BTP salts of varied anions (propionate, F, Br, nitrate) that have different permeabilities under bi-ionic conditions, were approximately constant, as if the permeation of either cation were coupled to the permeation of the anion. These results rule out our earlier hypothesis of anion permeation dependent on a bound alkali cation, but they are consistent with the idea that the tested anions and cations form mixed complexes while traversing the Cl channel.     

The chemical composition of Ricinus phloem exudate

Summary The chemical composition of exudate obtained from incisions made in the bark of the stem of actively growing Ricinus plants has been determined. The exudate had a high dry matter content (100–125 mg/ml), a high sugar content (80–106 mg/ml) which was solely sucrose, reducing sugars being absent. The amino acid composition was mainly glutamic and aspartic acids and threonine with a total amino acid concentration of 35.2 mM. The exudate had a pH of 8.0–8.2. Potassium was the major cation (60–112 mM) with sodium present at a lower concentration (2–12 mM). Of the divalent cations, calcium was at a low concentration (0.5–2.3 mM) and magnesium relatively higher (4.5–5.4 mM). Chloride was the major inorganic anion (10–19 mM). Phosphate concentration was relatively high (3.7–5.7 mM) and low concentrations of sulphate (0.3–0.5 mM) and bicarbonate (1.7 mM) were also present. Nitrate was absent. The ionic balance was maintained by the presence of relatively large quantities (30–47 meq/l) of organic anions, mainly malate. Bioassays revealed auxin, gibberellin and cytokinin activities in chromatographed exudate. Adenosine triphosphate was found in the exudate (0.40–0.60 mM). The analysis is dicussed with respect to the composition of phloem sap reported for other plant species.     

Accumulation and radial transport of ions from potassium salts by cucumber roots

Accumulation of K(+) is insensitive to the anion supplied with it at a solution concentration below 1 mm. Rates of K(+) transport to the xylem from the same solutions are, however, dependent upon the anion present and decrease in the order NO(3) (-) > Cl(-) > SO(4) (2-). Parallel effects on rates of exudation and anion transport result from kind and concentration of anion supplied and time of exposure to the solution. When high K salt concentrations are used, only linear relationships are found between solution concentrations and transport rates. However, ion concentration in the exudate increases more than external solution concentration, while exudation rate is unaffected. It is suggested that some of the ions transported are from compartments within the cells. At high solution concentrations KNO(3) results in more exudation and in higher ion concentration in the exudate than is found with KCl.     

Transport of sodium into the xylem exudate of tobacco

When tobacco (Nicotiana tabacum L. var. Virginia Gold) plants were pretreated with Na (²²Na) several days before detopping, from 2.3 to 4.9% of Na previously accumulated in roots appeared in the xylem exudate in 7 days after detopping. Na from the external medium, however, was readily transported to the exudate. Moreover, the amount of the pretreatment Na that was transported to the exudate was not influenced by the presence of Na in the external medium. When Na was present in the external medium after detopping, about 4% (with an NaNO₃ post treatment) to 10% (with an NaCl post treatment) of the Na transported to the xylem in the 7 days following detopping originated in the vacuoles. Nitrate salts of K or Na in the external medium after detopping resulted in transport of large quantities of the respective cation to the exudate, but not in increased transport of the pretreatment Na. A much larger percentage of the K that was accumulated after detopping than of the Na similarly accumulated was transferred to the xylem exudate.     

The effects of valinomycin on respiration and volume changes in plant mitochondria

The effects of valinomycin on the respiration and volume changeshave been studied with isolated mitochondria from bean hypocotyl(Phaseolus vulgaris L.) and cauliflower bud (Brassica oleraceaL.). In the presence of 10 mM K salts of chloride, acetate,phosphate, and sulfate respiration is stimulated by valinomycinconcomitant with osmotic swelling. When swelling declines respirationwith organic acid substrates also declines. In the presenceof the K salts of acetate and PO₄ but not Cl^– the terminationof respiration leads to contraction. The contraction in K-PO₄is inhibited by addition to the external medium of between 65to 100 mM K-PO₄. The results are interpreted to suggest thatvalinomycin in the presence of KCl facilitated the movementof K down an electrical gradient, with the Cl anion followingand osmotic swelling resulting. However, in a medium containingacetate or PO₄ the anions are actively transported against anelectrical gradient at the expense of metabolic energy. Valinomycinfacilitates the influx of K⁺ with the actively transported anionand swelling follows. When respiration terminates the activelytransported anions move passively back down their electrochemicalgradient and osmotic contraction follows. ¹ Present address: Department of Biology, Fort Lewis College,Durango, Colorado 81301, U.S.A. (Received July 21, 1972; )     

Effect of lithium salts on lactate dehydrogenase,adenylate kinase,and 1-phosphofructokinase activities

Inhibitions of 30?nM rabbit muscle 1-phosphofructokinase (PFK-1) by lithium, potassium, and sodium salts showed inhibition or not depending upon the anion present. Generally, potassium salts were more potent inhibitors than sodium salts; the extent of inhibition by lithium salts also varied with the anion. Li₂CO₃ was a relatively potent inhibitor of PFK-1 but LiCl and lithium acetate were not. Our results suggest that extents of inhibition by monovalent salts were due to both cations and anions, and the latter needs to be considered before inhibition can be credited to the cation. An explanation for monovalent salt inhibitions is proffered involving interactions of both cations and anions at negative and positive sites of PFK-1 that affect enzyme activity. Our studies suggest that lithium cations per se are not inhibitors: the inhibitors are the lithium salts, and we suggest that in vitro studies involving the effects of monovalent salts on enzymes should involve more than one anion.     

Origins of the Electrical Potential Difference between the Xylem Sap of Maize Roots and the External Solution

The effects of 2,4-dinitrophenol, of changes in the temperatureand concentration of the ambient solution and of variationsin salt status on the electrical potential difference betweenthe xylem exudate of maize roots and the ambient solution havebeen examined. The results are discussed in the light of someof the factors which could give rise to a potential differencebetween the sap and the solution. The rapid response of thepotential difference to dinitrophenol and to changes in temperaturesuggest that, at least in part, it arises directly from metabolicprocesses. Rapid changes in the potential difference broughtabout by addition of salts may be attributed to differentialrates of movement of anions and cations in the initial uptakeprocess. Over longer periods the potential difference appearsto be dependent on the concentration, but not the compositionof the ambient solution, and on the salt status of the roots.The salt status influences the relative rates at which anionsand cations are transported to the xylem sap, and a correlationhas been found between the potential difference and the ratioof the rates of movement of chloride or sulphate to potassiumto the sap. The implications of these findings on the elucidationof the pathways whereby ions are transported to the sap arediscussed.     

Interaction of anions with iron-transferrin-chelate complexes.

Preliminary evidence suggested that phosphate or borote destabilize iron-ovotransferrin-nitrilotriacetate complexes in the absence of added bicarbonate. The iron-ovotransferrin-EDTA complex was prepared in the absence of bicarbonate, and a number of anions, including phosphate, sulfate, and citrate, were found to perturb the visible absorbance (lambdamax = 490 nm) of this complex. Other anions, such as chloride, nitrate, and perchlorate, had little or no effect on the spectrum. Also, when bicarbonate was added to a solution of the iron-transferrin-EDTA complex (A515 = 0.45), within 2 min, the visible absorbance had decreased to A515 = 0.13. Slowly a new peak appeared (lambdamax = 470 nm), evidently the iron-transferrin-CO3 complex. When these spectral changes were monitored in detail, the lack of an isosbestic point indicated the existence of one or more intermediates in the conversion of iron-transferrin-EDTA complex to the iron-transferrin-CO3 complex. Experiments using ternary complexes containing either 59Fe or [14C]EDTA show that both iron and EDTA nearly completely dissociate from the protein (most likely concomitantly within 2 min after bicarbonate is added. These observations are best explained by a paradigm which includes anion binding to the apoprotein. It is clear that there is an intimate relationship between anions and the binding of iron chelates by transferrin.     

Effects of anions on a soluble ATPase from mitochondria of pea cotyledons

We investigated the nature of the stimulation by salts of theactivity of a solubilized ATPase from pea cotyledon mitochondria.Our experiments demonstrated that salt stimulations were causedor regulated by anions. Stimulatory anions included oxyanionssuch as bicarbonate, but anions such as chloride and bromidewere also affective, as were anions of organic acids. Sincethe specific polypeptide inhibitor of ATPase was shown to beabsent from our preparation, the stimulations of the enzymeby anions were not caused by destruction or dissociation ofthe inhibitor. In most respects the anion stimulations werevery similar to those reported with F₁-ATPases from yeast andmammalian tissues. The lack of oxyanion specificity requiresthat postulated roles for oxyanions be re-examined. (Received May 8, 1978; )     

Cation-Anion Balance during Potassium and Sodium Absorption by Barley Roots

Steady-state rates of potassium ion and sodium ion absorption by excised barley roots accompanied by various anions were compared with the rates of anion absorption and the concomitant H⁺ and base release by the roots. The cation absorption rates were found to be independent of the identities, concentrations, and rates of absorption of the anions of the external solution, including bicarbonate. Absorption of the anion of the salt plus bicarbonate could not account for the cation absorption. H⁺ is released during cation absorption and base during anion absorption. The magnitude by which one or the other predominates depends on the relative rates of anion and cation absorption under various conditions of pH, cation and anion concentration, and inhibitor concentrations. The conclusion is that potassium and sodium ions are absorbed independently of the anions of the absorption solution in exchange for H⁺, while anions are exchanged for a base. The H⁺ release reflects a specificity between K⁺ and Na⁺ absorption such that it appears to be H⁺ exchanged in the specific rate-limiting reactions of the cation absorption.     

Nitrate increases in soil water following conversion of chaparral to grass

An Arizona watershed converted from chaparral to grass, released high concentrations of nitrate to stream water. The nitrate originated from the rooting zone of the decomposing shrubs. High nitrate concentrations (44–373 ppm) were found in soil solutions from 1.5-, 3.0-, and 4.6-m depths on the converted watershed as compared with low nitrate concentrations (0.2–6.2 ppm) found in an adjacent undisturbed area. Soil solution nitrate concentrations at the 0.3-m depth were generally low, especially in the untreated area. High nitrate concentrations were balanced mainly by relative decreases in bicarbonate anions in the soil solutions and in the stream water. Multiple stepwise regression analyses showed improvement in the regression of bicarbonate on nitrate when chloride and sulfate anions were entered as variables.     

Allantoin and Allantoic Acid in Tissues and Stem Exudate from Field-grown Soybean Plants

Samples of stem exudate and plant tissue collected from field-grown soybean (Glycine max [L.] Merr.) plants were analyzed for allantoin and allantoic acid. Nitrogen in nitrate plus amino acids exceeded ureide N concentration in stem exudate prior to flowering. During all of reproductive development (from about 40 days after planting until maturity), ureide N concentration was two to six times greater than amino acid plus nitrate N concentration. Allantoin and allantoic acid, not asparagine, are the principal forms of nitrogen transported from nodulated roots to shoots of the soybean plant. During pod and seed development ureide N comprised as high as 2.3, 37.7, and 15.8% of total N in leaf blades, stems + petioles, and fruits, respectively. The concentration of ureide in stems and fruits declined to nearly zero at maturity.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

COMPOSITION OF SIEVE TUBE SAP FROM MACROCYSTIS PYRIFERA (PHAEOPHYTA) WITH EMPHASIS ON THE INORGANIC CONSTITUENTS1

A quantitative accounting of the solids in the sieve tube sap from Macrocystis pyrifera (L.) e. Ag. was performed. The major organic compounds (mannitol, amino acids, and protein) and inorganic cations (K⁺, Na⁺, Mg²⁺ and Ca²⁺) were present near previously reported levels. The anions, until now unreported (except for iodide), included chloride as the major inorganic ion, bromide, phosphate, nitrate, and bicarbonate as the major inorganic carbon species. Sulfate and ammonium were below the detection limits of 1.5 ppm and 2 ppm, respectively. The elements B and As were also present although their speciation was not determined.     

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

Histochemical Differentiation of Chloride from Other Ions Precipitated by Silver Nitrate in Freeze-Substitution Fixation

The method is based on substitution fixation at —25° C of quickly frozen tissue with a 90% alcohol solution saturated with silver nitrate. The silver salts are photochemically reduced in the histological preparations. At this low temperature very little staining of the protein structure of the tissue takes place. Silver ions adsorbed by the tissue can be removed by treatment with a sodium nitrate solution. About 2/3 of the brown material in the histological preparations of cerebral cortex was due to the chloride in the tissue, 1/6 to the phosphate, 1/10 to an unidentified (probably organic) anion, and 1/20 to bicarbonate. When the alcoholic silver nitrate solution used for the fixation is acidified, or the sections are treated with nitric acid, the colored material consists of reduced silver chloride only. A comparison of the light absorption in histological preparations of cortex treated with neutral and with acid solutions supported the conclusion that about 2/3 of the colored material in the tissue is reduced silver chloride.     

Carbonic anhydrase inhibitors. Inhibition of the newly isolated murine isozyme XIII with anions

The inhibition of the newly discovered cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme XIII of murine origin (mCA XIII) has been investigated with a series of anions, such as the physiological ones (bicarbonate, chloride), or the metal complexing anions (cyanate, cyanide, azide, hydrogen sulfide, etc), nitrate, nitrite, sulfate, sulfamate, sulfamide as well as with phenylboronic and phenylarsonic acids. The best mCA XIII inhibitors were cyanate, thiocyanate, cyanide and sulfamide, with K(I)-s in the range of 0.25microM-0.74 mM, whereas fluoride, iodide, azide, carbonate and hydrogen sulfide were less effective (K(I)-s in the range of 3.0-5.5mM). The least effective inhibitors were sulfate, chloride and bicarbonate (K(I)-s in the range of 138-267 mM). The affinity of mCA XIII for anions is very different from that of the other cytosolic isozymes (hCA I and II) or the mitochondrial isozyme hCA V. This resistance to inhibition by the physiological anions bicarbonate and chloride suggests an evolutionary adaptation of CA XIII to the presence of high concentrations of such anions (e.g., in the reproductive tract of both female and male), and the possible participation of this isozyme (similarly to CA II, CA IV and CA V) in metabolons with proteins involved in the anion exchange and transport, such as the anion exchangers (AE1-3) or the sodium bicarbonate co-transporter (NBC1 and NBC3) proteins, which remain to be identified.     

CHARACTERISTICS OF SALTS SECRETED BY TAMARIX APHYLLA

The composition and concentration of salts secreted by the salt glands of Tamarix aphylla L. grown under controlled nutrient conditions were determined. Eight ions, Na, K, Mg, Ca, Cl, NO₃, HCO₃, and SO₄, constituted 99 % + of the dry weight of salts secreted by plants grown on half-strength Hoagland's solution. The divalent cations Mg and Ca accounted for most of the cations; HCO₃ comprised about 60 % of the anions. The micronutrients B, Mn, Cu, Zn, and Mo were present in enriched concentrations in the secretion. The composition of the secretions was highly dependent on the composition of the root environment. The predominating cation in the saline culture solutions was also the predominant cation secreted. The accompanying anion in the culture solution influences the cation composition of the secreted salt. The concentration of the salt gland secretion averaged 0.5n , a 50-fold increase in concentration over the nutrient solution in which the plants were grown.     

Active transport of ions across sunflower roots

Summary The electrical potential difference across exuding roots of Helianthus annuus in two strengths of complete culture solution was measured. The determination of the concentration of the major nutrient ions in the outside solution and the xylem sap enabled the Nernst potential for each ion to be calculated. A comparison of the measured and calculated potentials indicated that the anions NO₃, SO₄, H₂PO₄ and HPO₄ were actively transported into the sap against the electrochemical potential gradient. The cations Ca and Mg, on the other hand, appeared to move passively into the sap. The behaviour of potassium depended on its concentration in the medium. With a relatively low external concentration (0.75 mM) it appeared to be actively tansported into the sap, whilst at higher outside concentrations (7.5 mM) it was apparently moving passively into the xylem down the electrochemical potential gradient. The possibility of potassium being pumped out of the sap with relatively high external concentrations is discussed.