Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Efficiency of Potassium Utilization by Barley Varieties: The Role of Subcellular Compartmentation

Memon, A. R., Saccomani, M. and Glass, A. D. M. 1985. Efficiencyof potassium utilization by barley varieties: The role of subcellularcompartmentation.?J. exp. Bot. 36: 1860–1876. The subcellulardistributions of K⁺ in roots of three barley (Hordeum vulgareL.) varieties, grown at 10 and 100 mmol m^–3 external K⁺([K⁺]_o) were estimated by compartmental analyses. In general,increased [K⁺]_o caused a 2–3 fold increase in vacuolar[K⁺], but cytoplasmic [K⁺] increased only slightly. Nevertheless,the three varieties, which had been selected for study on thebasis of their different rates of K⁺ utilization, showed distinctdifferences in the allocation of K⁺ between cytoplasm and vacuole.At 10 mmol m^–3 [K⁺]_o var. Betzes exhibited typical K⁺deficiency symptoms while var. Fergus and var. Compana did not,even though Betzes had higher [K⁺] in shoots and roots. Theinefficient utilization of K⁺ in this variety appears to beassociated with a failure to mobilize vacuolar K⁺ into the cytoplasmiccompartment (the ratio of vacuolar: cytoplasmic K⁺ contentsfor Betzes was 4.1 compared to 2.7 and 2.5, respectively, forFergus and Compana). Fergus and Betzes, which demonstrate pronouncedgrowth responses to increased [K⁺]₀ between 10 and 100 mmolm^–3, showed significant increases of cytoplasmic [K⁺]in this range of [K⁺]_o. By contrast, cytoplasmic [K⁺] in Compana,a variety whose growth is not stimulated by increased [K⁺]₀(from 10 to 100 mmol m^–3) showed virtually no increase.It is suggested that the efficiency of K⁺ utilization and thegrowth response to [K⁺]₀ in these varieties are functions ofthe subcellular distribution of this ion between cytoplasm andvacuole. Key words: Barley varieties, K⁺ subcellular compartmentation, utilization efficiency     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

Amino Acid Sequence Similarities between the Vacuolar Proton-Pumping Inorganic Pyrophosphatase and the c-Subunit of F0F1-ATPases

Comparison of the Arabidopsis thaliana vacuolar proton-pumpinginorganic pyrophosphatase with three F₀F₁-ATPase c-subunitsrevealed a strong similarity between a stretch containing aminoacids 227–245 of the H⁺-PPase and a transmembrane a-helixof the c-subunits which contains the glutamate which binds N,N'-dicyclohexylcarbodiimide. (Received November 16, 1992; Accepted December 22, 1992)     

Gastric type H+,K+-ATPase in the cochlear lateral wall is critically involved in formation of the endocochlear potential

Cochlear endolymph has a highly positive potential of approximately +80 mV known as the endocochlear potential (EP). The EP is essential for hearing and is maintained by K⁺ circulation from perilymph to endolymph through the cochlear lateral wall. Various K⁺ transport apparatuses such as the Na⁺,K⁺-ATPase, the Na⁺-K⁺-2Cl^– cotransporter, and the K⁺ channels Kir4.1 and KCNQ1/KCNE1 are expressed in the lateral wall and are known to play indispensable roles in cochlear K⁺ circulation. The gastric type of the H⁺,K⁺-ATPase was also shown to be expressed in the cochlear lateral wall (Lecain E, Robert JC, Thomas A, and Tran Ba Huy P. Hear Res 149: 147–154, 2000), but its functional role has not been well studied. In this study we examined the precise localization of H⁺,K⁺-ATPase in the cochlea and its involvement in formation of EP. RT-PCR analysis showed that the cochlea expressed mRNAs of gastric ₁-, but not colonic ₂-, and -subunits of H⁺,K⁺-ATPase. Immunolabeling of an antibody specific to the ₁ subunit was detected in type II, IV, and V fibrocytes distributed in the spiral ligament of the lateral wall and in the spiral limbus. Strong immunoreactivity was also found in the stria vascularis. Immunoelectron microscopic examination exhibited that the H⁺,K⁺-ATPase was localized exclusively at the basolateral site of strial marginal cells. Application of Sch-28080, a specific inhibitor of gastric H⁺,K⁺-ATPase, to the spiral ligament as well as to the stria vascularis caused prominent reduction of EP. These results may imply that the H⁺,K⁺-ATPase in the cochlear lateral wall is crucial for K⁺ circulation and thus plays a critical role in generation of EP. hydrogen, potassium-adenosine triphosphatase; stria vascularis; spiral ligament     

Responses by Coleoptiles of Intact Rice Seedlings to Anoxia: K+ Net Uptake from the External Solution and Translocation from the Caryopses

This study evaluated the effects of anoxia on K⁺ uptake andtranslocation in 3–4-d-old, intact, rice seedlings (Oryzasativa L. cv. Calrose). Rates of net K⁺ uptake from the mediumover 24 h by coleoptiles of anoxic seedlings were inhibitedby 83–91 %, when compared with rates in aerated seedlings.Similar uptake rates, and degree of inhibition due to anoxia,were found for Rb⁺ when supplied over 1·5–2 h,starting 22 h after imposing anoxia. The Rb⁺ uptake indicatedthat intact coleoptiles take up ions directly from the externalsolution. Monovalent cation (K⁺ and Rb⁺) net uptake from thesolution was inhibited by anoxia to the same degree for thecoleoptiles of intact seedlings and for coleoptiles excised,‘aged’, and supplied with exogenous glucose. Transportof endogenous K⁺ from caryopses to coleoptiles was inhibitedless by anoxia than net K⁺ uptake from the solution, the inhibitionbeing 55 % rather than 87 %. Despite these inhibitions,osmotic pressures of sap (_sap) expressed from coleoptiles ofseedlings exposed to 48 h of anoxia, with or without exogenousK⁺, were 0·66 ± 0·03 MPa; however,the contributions of K⁺ to _sap were 23 and 16 %, respectively.After 24 h of anoxia, the K⁺ concentrations in the basal10 mm of the coleoptiles of seedlings with or without exogenousK⁺, were similar to those in aerated seedlings with exogenousK⁺. In contrast, K⁺ concentrations had decreased in aeratedseedlings without exogenous K⁺, presumably due to ‘dilution’by growth; fresh weight gains of the coleoptile being 3·6-to 4·7-fold greater in aerated than in anoxic seedlings.Deposition rates of K⁺ along the axes of the coleoptiles werecalculated for the anoxic seedlings only, for which we assessedthe elongation zone to be only the basal 4 mm. K⁺ depositionin the basal 6 mm was similar for seedlings with or withoutexogenous K⁺, at 0·6–0·87 µmolg^–1 f. wt h^–1. Deposition rates in zones above6 mm from the base were greater for seedlings with, thanwithout, exogenous K⁺; the latter were sometimes negative. Weconclude that for the coleoptiles of rice seedlings, anoxiainhibits net K⁺ uptake from the external solution to a muchlarger extent than K⁺ translocation from the caryopses. Furthermore,K⁺ concentrations in the elongation zone of the coleoptilesof anoxic seedlings were maintained to a remarkable degree,contributing to maintenance of _sap in cells of these elongatingtissues.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway

The influences of the gastric H⁺/K⁺ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the - and -subunits of human H⁺/K⁺-ATPase (H⁺/K⁺-, cells). The pH values of trans-Golgi network (pH_TGN) and recycling endosomes (pH_RE) were measured by transfecting H⁺/K⁺-, cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H⁺/K⁺-ATPase was present in the plasma membrane, TGN, and RE. The pH_TGN was similar in both H⁺/K⁺-, cells (pH_TGN 6.36) and vector-transfected ("mock") cells (pH_TGN 6.34); pH_RE was also similar in H⁺/K⁺-, (pH_RE 6.40) and mock cells (pH_RE 6.37). SCH28080 (inhibits H⁺/K⁺-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H⁺ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pH_TGN of 7.0–7.5, close to pH_cytosol. Similar results were observed in RE. Thus H⁺/K⁺-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H⁺ v-ATPase. Mathematical modeling predicted a large number of H⁺ v-ATPases (8,000) active in the TGN to balance a large, passive H⁺ leak (with P_H 10^–3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H⁺/K⁺-ATPases (estimated to be 4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane. pHluorin; H⁺ v-ATPase; trans-Golgi network; organelle pH; H⁺ permeability     

Differential stimulation by Cl- of H+ transport and ATPase activity in purified plasma membrane vesicles from maize (Zea mays L.) roots

Maize (Zea mays L.) root plasma membranes purified by the aqueouspolymer two-phase technique have previously been shown to bevery low in tonoplast H⁺ -ATPase and H⁺ -PPase activities. Westernblots of a similar preparation showed that, compared to a microsomalfraction, there was practically no reaction with antibodiesto the tonoplast enzymes, but a strong reaction with an antibodyto the plasma membrane H⁺ -ATPase. Freeze/thaw treatment ofthe plasma membrane vesicles increased the proportion with aninsideout orientation to about 40%. This preparation was usedto demonstrate that substitution of KCl for K₂S0₄ resulted ina 14-fold stimulation of H⁺ transport, but an increase in ATPaseactivity of less than 10%. In contrast to its effect on tonoplastvesicles, Cl^– had only a small effect on the membranepotential of plasma membrane vesicles, assayed by oxonol V fluorescencequench recovery. To account for the apparent variability inthe H⁺/ATP coupling ratio, it may be necessary to devise a modelthat takes into consideration the possibility of non-linearbehaviour with respect to the membrane potential of the protonleak and/or of slip in the ATPase. Key words: ATPase, plasma membrane, anion stimulation, proton transport     

Salinity-induced Malate Accumulation in Chara

Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m^–3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na⁺ or Li⁺salts. Duringion absorption from various salts of K⁺ and Rb⁺ vacuolar cationconcentration exceeded Cl^– concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m₃^–,with accumulation occurring at rates of up to 0.45 mol m^–3h^–1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO₃^– concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H⁺efflux at a rate of about40 nmol m^–2 s^–1from the cell. The results presentedargue against cytoplasmic Cl^–, HCO₃^– or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity     

Internal Factors Regulating Nitrate and Chloride Influx in Plant Cells

The primary factor determining the observed decrease in activeC1^– influx during salt accumulation in carrot and barleyroot cells has been shown to be the concentration of C1^–+ NO₃^– in the vacuole. The relationship between C1^– influx and the vacuolar concentrationsof various substances was examined after the tissues had accumulatedions from various salt solutions. After accumulating K⁺ malate,C1^– influx was not reduced, but after accumulating C1^–or NO₃^– salts, C1^– influx was reduced by up to 90per cent. Considering all treatments, C1^– influx was notcorrelated with the vacuolar concentration of K⁺, Na⁺, (K⁺+Na⁺),reducing sugars, malate, C1^–, or NO₃^–, nor withthe cellular osmotic pressure. The correlation coefficient betweenCl^– influx and log (C1^– + NO₃^– concentrationin the vacuole) was highly significant, and accounted for allthe variation in C1^– influx in this experiment. Net NO₃^– influx is similarly reduced by a high C1^–concentration in the vacuole. External Cl^– and NO₃^–have quantitatively different, apparently competitive, effectson C1^– influx. These differ from the apparently negative-feedbackeffects of C1^– and NO₃^– in the vacuole, which arequantitatively similar. Decreasing the internal hydrostatic pressure by raising theexternal osmotic pressure increased active K⁺ influx in Valoniaventricosa, but had no effect on C1^– or K⁺ influx in carrotor maize root cells. Cl^– influx is not related to thereducing sugar concentration during ageing drifts in excisedcarrot root tissue. Acetazolamide did not inhibit C1^– influx to carrot tissue. The implications of this type of negative feedback regulation,and the relationship between C1^– and NO₃^– transportare discussed.     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Light-induced Activation of Electrogenic H+ Extrusion and K+ Uptake in Elodea densa Depends on Photosynthesis and is Mediated by the Plasma membrane H+ ATPase

In Elodea densa leaves light strongly stimulates electrogenic,K ⁺-dependent, vanadate- and erythrosin B-sensitive H⁺ extrusionand hyperpolarizes the transmembrane electrical potential. Theseeffects of light are suppressed by treatment with DCMU, an inhibitorof photosynthesis, which has no effect on H⁺ extrusion in thedark. Light-induced H⁺ extrusion requires the presence of K⁺in the medium and is associated with increased K⁺ uptake andalkalinization of the cell sap. Light-induced H⁺ extrusion increaseswith increased CO₂ concentration. At constant CO₂ concentration(10⁴ parts 10^–6) the rate of H⁺ extrusion is stronglyenhanced by an increased light intensity up to 30 W m^–2.Different wavelengths, between 400 and 730 nm, induce a significantstimulation of both proton secretion and transmembrane potentialhyperpolarization. The stimulating effects of light on H⁺ extrusion, K⁺ uptakeand cell sap pH are very similar to those induced in the darkby fusicoccin, a toxin known to stimulate strongly ATP-driven,vanadate- and erythrosin B-sensitive H⁺ transport. In the light,the effects of fusicoccin are only partially additive to thoseof light, thus suggesting that the two factors influence thesame system. The identification of this system with the plasmamembrane H⁺-ATPase is indicated by the observed inhibition ofthe effects of either light or fusicoccin by the H⁺-ATPase inhibitorsvanadate and erythrosin B. These data indicate that the activation of electrogenic H⁺ extrusionand of K⁺ uptake by light is mediated by some products of photosynthesis.The mechanism and the possible physiological implications ofthis phenomenon are discussed. Key words: Photosynthesis, H⁺ pump, K⁺ uptake, Elodea densa     

The Ionic Relations of Acetabularia mediterranea

The concentrations of K⁺, Na⁺, and Cl^– in the cytoplasmand the vacuole of Acetabularia mediterranea have been measured,as have the vacuolar concentrations of SO₄^–– andoxalate. The electrical potential difference between externalsolution, and vacuole and cytoplasm has been measured. The resultsindicate that Cl^– and SO₄^–– are probably transportedactively into the cell, and that active transport of Na⁺ isoutwards. The results for K⁺ are equivocal. The fluxes of K⁺,Na⁺, Cl^–, and S0₄^–– into the cell and theeffluxes of Na⁺ and Cl^– have been determined. The Cl^–fluxes are extremely large. In all cases the plasmalemma isthe rate-limiting membrane for ion movement. A technique isdescribed for the preparation of large, completely viable cellfragments containing only cytoplasm, with no vacuole.     

Efficiency of K+ Utilization by Barley Varieties: Activation of Pyruvate Kinase

Barley (Hordeum vulgare L.) varieties differed in their raponseto [K⁺]₀, in terms of their utilization efficiencies (UE = freshweight. concentration of [K⁺]₁^–1). At low [K⁺]₀, Compana,an efficient-non-responder demonstrated superior utilizationof absorbed K⁺. On the other hand, at high [K⁺]₀, Fergus (anefficient responder) and BT 334 (an inefficient responder) hadhigher UE values for K⁺ than Compana which performed poorlyat this [K⁺]₀. Kinetic parameters for K⁺ activation of the enzyme pyruvatekinase from 12 barley varieties, representing a range of UEvalues, were determined. Varieties showed substantial differencesin their V_max values (P<0·01). Compana, an efficientvariety, had the highest V_max (31 µmol g^–1 freshwt. h^–1) which was about 50% higher than that of Mingo,an inefficient variety. By contrast, K_m values for the enzymeswere not significantly different among varieties The mean valuesfor all varieties (3·9±0·15 mol m^–3K⁺) is far below the estimated cytoplasmic [K⁺] (100-200 molm^–3). It is, therefore, unlikely that differences in theutilization of K⁺ by these varieties can be explained on thebasis of differential requirements for (K⁺) activation of theseenzymes. Alternative possibilities for differences in the utilizationof K⁺ are discussed. Key words: K⁺ utilization efficiency, Pyruvate kinase, Barley varieties     

The spontaneous growth response of maize coleoptile segments and the change in tissue 8

Decapitated segments from maize (Zea mays L.) coleoptiles orientedvertically in an upright position show a strong spontaneousgrowth response (SGR) 3 h after decapitation. The latent periodof the SGR is markedly reduced when these segments are orientedin an inverted position. Coleoptile segments with intact tipsexhibit a weak and transient SGR in the vertical upright orientation.However, in the inverted orientation, these segments show atypical SGR. The data are inconsistent with the current hypothesisthat the SGR is caused by a time-dependent increase in tissuesensitivity to auxin. The parallel increase in membrane potentialdifference and growth rate during the time-course of the SGRindicates a possible role for PM H⁺-ATPase in the establishmentof the SGR in maize coleoptile segments. Key words: Auxin, spontaneous growth response, membrane potential, plasma membrane H⁺-ATPase, Zea mays L.     

Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells

The functional properties of the Saccharomyces cerevisiae bicarbonate transporter homolog Bor1p (YNL275wp) were characterized by measuring boron (H₃BO₃), Na⁺, and Cl^– fluxes. Neither Na⁺ nor Cl^– appears to be a transported substrate for Bor1p. Uphill efflux of boron mediated by Bor1p was demonstrated directly by loading cells with boron and resuspending in a low-boron medium. Cells with intact BOR1, but not the deletant strain, transport boron outward until the intracellular concentration is sevenfold lower than that in the medium. Boron efflux through Bor1p is a saturable function of intracellular boron (apparent K_m 1–2 mM). The extracellular pH dependences of boron distribution and efflux indicate that uphill efflux is driven by the inward H⁺ gradient. Addition of 30 mM HCO₃^– does not affect boron extrusion by Bor1p, indicating that HCO₃^– does not participate in Bor1p function. Functional Bor1p is present in cells grown in medium with no added boron, and overnight growth in 10 mM H₃BO₃ causes only a small increase in the levels of functional Bor1p and in BOR1 mRNA. The fact that Bor1p is expressed when there is no need for boron extrusion and is not strongly induced in the presence of growth-inhibitory boron concentrations is surprising if the main physiological function of yeast Bor1p is boron efflux. A possible role in vacuolar dynamics for Bor1p was recently reported by Decker and Wickner (10). Under the conditions used presently, there appears to be mildly abnormal vacuolar morphology in the deletant strain. boron; SLC4; YNL275w     

Potassium-Ammonium Uptake Interactions in Tobacco Seedlings

Short-term (< 12 h) uptake experiments were conducted with6–7-week-old tobacco (Nicotiana tabacum L. cv. Ky 14)seedlings to determine absorption interactions between K⁺ andNH₄⁺. At equal solution concentrations (0.5 mol m^–3) netK⁺ uptake was inhibited 30–35% by NH₄⁺ and NH₄⁺ uptakewas decreased 9–24%. Removal of NH₄⁺ resulted in completerecovery in K⁺ uptake rate, but NH⁴⁺ uptake rate did not recoverwhen K⁺ was removed. In both cases, inhibition of the uptakerate of one cation saturated as the concentration of the othercation was increased up to 0.5 mol m^–3. The relative effectof K⁺-NH₄⁺ interactions was not altered when Cl- was replacedwith SO₄^2–, but the magnitudes of the uptake rates wereless in the absence of Cl-. The V_max for NH₄⁺ uptake was reducedfrom 128 to 105 µmol g^–1 dry wt. h^–1 in thepresence of 0.5 mol m^–3 K⁺ and the K_m for NH₄⁺ doubledfrom 12 to 27 mmol m^–3 in the presence of K⁺. The resultsof these K⁺-NH₄⁺ experiments are interpreted as mixed-noncompetitiveinteractions. However, an enhanced efflux of K⁺ coupled to NH₄⁺influx via an antiporter cannot be ruled out as contributingto the decrease in net K⁺ uptake. Key words: Nicotiana tabacum, K⁺, NH₄⁺, Uptake interactions     

Uptake of Imidazole and its Effects on the Intracellular pH and Ionic Relations of Chara corallina

Ammonia (pK_a 9.25) and methylamine (pK_a, 10.65) increase cytoplasmicpH and stimulate Cl^– influx in Chara corallina, theseeffects being associated with influx of the amine cations ona specific porter. The weak base imidazole (pK_a 6.96) has similareffects but diffuses passively into the cell both as an unionizedbase and as a cation. When the external pH is greater than 6.0influx of the unionized species predominates. Imidazole accumulates to high concentrations in the vacuole,where it is protonated. Cytoplasmic pH and vacuolar pH riseby only 0.2–0.3 units, suggesting a large balancing protoninflux across the plasma membrane. Balance of electric chargeis partially maintained by net efflux of K⁺ and net influx ofCl^–. Calculation of vacuolar concentrations of imidazole(from (¹⁴C] imidazole uptake, assuming that there is no metabolism)plus K⁺ and Na⁺ indicates an excess of cations over inorganicanions (Cl^–). However, although the osmotic potentialof the cells increases, also indicating increased solute concentrations,the increase is less than that predicted by the calculated ionicconcentrations. This discrepancy remains to be resolved. Becausethe osmotic potential also increases when imidazole is absorbedfrom Cl^–-free solutions it is likely that maintenanceof charge-balance can also involve synthesis and vacuolar storageof organic or amino acids. Key words: Imidazole, potassium, intracellular pH, membrane transport, Chara