Abscisic acid effects on intracellular pH and ion fluxes in barley leaf segments

Changes in intracellular pH and in H⁺, K⁺ and Cl^? fluxes were evaluated in different experimental conditions in leaf segments of barley (Hordeum vulgare cv. Georgie) incubated in the dark, at pH 5.5, in the presence or absence of abscisic acid (ABA), and a comparison was made between the effects of ABA and those of erythrosin B (EB), a plasmalemma H⁺-pump inhibitor. In all conditions tested, ABA induced a cell sap acidification, an alkalinization of the external medium, a decrease in K⁺ intracellular contents, and an increase in the contents of Cl^?. The ABA-induced decrease in K⁺ content was chiefly due to the inhibition of K⁺ influx. On the contrary, ABA did not influence the uptake of Cl^?, but inhibited Cl^? efflux, the inhibition satisfactorily accounting for the larger Cl^? content observed in the presence of the hormone. The intracellular acidification and the decrease in apparent outward net transport of H⁺ observed with ABA were seemingly not associated with the activity of the proton pump, the transmembrane electrical potential difference, or K⁺ transport. On the contrary, a correlation was evident with the changes in Cl^? content. These results and, in particular, the similarity between the effects of ABA and those induced by 4,4 -diisothiocyano-2,2-disulfonic acid stilbene (DIDS), a Cl^? channel-blocking agent, suggest that the ABA-induced changes in intracellular pH and in H⁺ transport might depend on the capability of ABA to inhibit Cl^? efflux, more than on a primary inhibition of the H⁺ pump, and propose an important role for ABA in regulating the Cl^? channels.     

Changes in Chloride Fluxes and Cytosolic pH Induced by Abscisic Acid in Elodea densa Leaves

The effects of ABA on intracellular pH, net H⁺ extrusion, Cl^? fluxes and E_m values were studied in Elodea densa leaves, and the possible relationships between the ABA-induced changes of cytosolic pH and of Cl^? and H⁺ fluxes were investigated. Cytosolic and vacuolar pH were calculated by the weak acid and weak base distribution method. The data show that, also in this material (a water plant without stomata), ABA induces a decrease in both net H⁺ extrusion and intracellular pH, and strongly inhibits Cl^? efflux. No significant effect of ABA is detectable on E_m values, either at short or long intervals in the presence or absence of K⁺. Cl^? efflux is apparently independent of the activity of the plasmalemma H⁺ pump and of the E_m values. Conversely, it strongly depends on the value of cytosolic pH, a larger efflux occurring for the lower pH values both in the presence and in the absence of ABA. These results indicate that the ABA-induced cytosolic acidification cannot be the cause but, possibly, a consequence of the decrease in Cl^? efflux, and are consistent with the hypothesis of a primary role of ABA in regulating Cl^? efflux, presumably by directly affecting a class of Cl^?-permeable channels.     

Ion fluxes and pH changes induced by trans-plasmalemma electron transfer and fusicoccin inLemna gibba L. (strain G1)

During the reduction of extracellular [Fe(CN)₆]^3– at the plasmalemma of intact, K⁺-starvedLemna gibba L. fronds, the external medium was acidified and K⁺ released, in the absence of inhibitors with rates of 10 e^–/8.5 H⁺/1.5 K⁺ (mol·(g FW)^–1·^–1). In K⁺ plants the larger K⁺ efflux caused a lag phase in extracellular acidification and a change in rates to 10 e^–/6 H⁺/4 K⁺ and in the presence of CN^–+salicylhydroxamic acid at pH 5 to 5.2 e^–/0 H⁺/6.6 K⁺. The e^– transfer was accompanied by a membrane depolarization of up to 100 mV and a cytosolic acidification of about 0.6 pH units, but only in K⁺ plants, where the extracellular acidification was smaller. These results indicate that a stimulation of the plasmalemma H⁺-ATPase may be triggered either by a cytosolic acidification or by a strong membrane depolarization. It is concluded that the redox system catalyses only uncoupled e^– transfer without H⁺ transfer across the plasmalemma. The obligatory, but secondary charge compensation is partially achieved by the rapid K⁺ release upon membrane depolarization and partially by the activity of the plasma membrane H⁺-ATPase, but not by an e^–/anion exchange. The extracellular acidification during [Fe(CN)₆]^3– reduction is generated by the conversion of a strong trivalent into a strong tetravalent anion. This acidification is caused by changes in the concentration ratio of strong cations to strong anions. Efflux of K⁺ and not the production of organic acids or NAD(P)H oxidation is the chemical cause of the measurable cytosolic acidification. Extracellular acidification was inversely correlated with intracellular acidification. Similarly, fusicoccin-induced pH changes were correlated with changes in the strong-ion concentration difference. Extracellular ± FC-dependent acidification and intracellular alkalinization of up to 0.6 pH units were strongly dependent on K⁺ fluxes. The ferricyanide-triggered trans-plasmalemma electron-transfer system is an example of how measurable pH changes are the consequence and not the cause of charge-transfer-induced changes in strong-ion fluxes.Abbreviations DCCD dicyclohexylcarbodiimide - E_m electrical membrane potential difference - FC fusicoccin - pH_c cytosolic pH - FW fresh weight - PM plasmalemma - SHAM salicylhydroxamic acid - SID strong-ion concentration difference This work was supported by the Deutsche Forschungsgemeinschaft. We gratefully acknowledge the Alexander von Humboldt award donated to J.G. We thank Professor Ulrich Lüttge (TH Darmstadt, FRG) for his kind support and Annett Ehrhardt and Dr. Karl Fischer (TH Darmstadt, FRG) for their valuable help with Cl^– and CO₂ experiments. Special thanks are due to Professor Erasmo Marrè (Università di Milano, Italy) for continuous discussions and also to Professor Alessandro Ballio (Università di Roma, Italy) for their kind gifts of fusicoccin.     

The effect of CO2 on potassium transport by Chlorella fusca

Abstract. The effect of CO₂ on net K⁺ uptake by Chlorella fusca grown on high CO₂ levels was examined by passing 1.5% CO₂ through algal suspensions gassed previously with air or CO₂-free air Addition of CO₂ in the light caused a large net uptake of K⁺ (initial velocity 4.2–9.2 mmol s^?1 m^?3 cells) which decreased the concentration of K⁺ in the supernatant from 0.1–0.2 mol m^?3 to 3–10 mmol m^?3. In the dark and in the presence of 30 mmol m^?3 DCMU, no effects were found. Measurement or the unidirectional K⁺ fluxes by using ⁸⁶Rb⁺ as a label showed that in the presence of 1.5% CO₂, influx of K⁺ was increased by a factor of 2–4 while efflux was inhibited completely. CO₂ hyperpolarized the membrane potential (determined through TPP⁺ uptake) from –120mV to –130 mV which could not explain the more than 15,000-fold K⁺ accumulations. In the light, CO₂ lowered the intracellular pH (determined with DMO) by 0.5 units. In the dark and in the presence of DCMU only, a small acidification of 0.1 units was found. During the first 15 min after addition of CO₂ the malate content of the cells increased from 0.7 to 1.5 mol m^?3 packed cells. On the basis of these and earlier results, CO₂-induced net K⁺ uptake is interpreted as a stimulation of an electroneutral ATP-dependent K⁺/H⁺ exchange at the plasmalemma. This exchange acts as a ‘pHstat’ by reducing the intracellular acidification caused by production of acidic assimilation products.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Early Effects of Penconazole on H+ and K+ Transport,Electrolyte Leakage and Transmembrane Electrical Potential in Egeria densa Leaves

The early effects of penconazole (PCZ) at relatively high concentration (10^?4 to 5 × 10^?4 M) on changes in pH and in titratable acidity of the medium, transmembrane electrical potential difference (Em), electrolyte leakage and cell morphology were investigated in Egeria densa leaves. At the lowest (10^?4 M) concentration and in the presence of a very low (10 μM) K⁺ concentration, triazole induced an early, moderate hyperpolarization of Em, associated with a decrease of net K⁺ uptake, suggesting some increase in the passive permeability to K⁺. This Em hyperpolarization was no longer detectable at high (2 mM) K⁺_out concentration. At high PCZ concentrations (3 × 10^?4 M and 5 × 10^?4 M) the early hyperpolarization detectable in the presence of a low K⁺_out concentration became transient, and was followed by a marked depolarization. PCZ, at these concentrations, suppressed acidification of the medium, stimulated electrolyte leakage and, in the mesophyll cells, induced some shrinking of the cytoplasm and its disconnection from the cell walls. These results are interpreted as due to an early effect of this triazole leading to the disorganization of the plasma membrane.     

Transmembrane potential-mediated coupling between H+ pump operation and K+ fluxes in Elodea densa leaves hyperpolarized by fusicoccin, light or acid load

In isolated Elodea densa leaves, the relationships between H⁺ extrusion (-ΔH⁺), K⁺ fluxes and membrane potential (E_m) were investigated for two different conditions of activation of the ATP-dependent H⁺ pump. The ‘basal condition’ (darkness, no pump activator present) was characterized by low values of-ΔH⁺ and K⁺ uptake (ΔK⁺), wide variability of the ?ΔH⁺/ΔK⁺ ratio, relatively low membrane polarization and E_m values more positive than EK for external K⁺ concentrations (|K⁺]_o of up to 2mol m^?3. A net K⁺ uptake was seen already at [K⁺]_o below 1 mol m^?3, suggesting that K⁺ influx in this condition was a thermodynamically uphill process involving an active mechanism. When the H⁺ pump was stimulated by fusicoccin (FC), by cytosol acidification, or by light (the ‘high polarization condition’), K⁺ influx largely dominated K⁺ and C^? efflux, and the ?ΔH⁺/ΔK⁺ ratio approached unity. In the range 50 mmol m^?3?5 mol m^?3 [K⁺]₀, E_m was consistently more negative than E_K. The curve of K⁺ influx at [K⁺]₀ ranging from 50 to 5000mmol m^?3 fitted a monophasic, hyperbolic curve, with an apparent half saturation value = 0–2 mol m^?3. Increasing |K⁺]₀ progressively depolarized E_m, counteracting the strong hyperpolarizing effect of FC. The effects of K⁺ in depolarizing E_m were well correlated with the effects on both K⁺ influx and ?ΔH⁺, suggesting a cause-effect chain: K⁺₀ influx → depolarization → activation of H⁺ extrusion. Cs⁺ competitively inhibited K⁺ influx much more strongly in the ‘high polarization’ than in the ‘basal’ condition (50% inhibition at [Cs⁺]/[K⁺]₀ ratios of 1:14 and 1:2, respectively) thus confirming the involvement of different K⁺ uptake systems in the two conditions. These results suggest that in E. densa leaves two distinct modes of interactions rule the relationships between H⁺ pump, membrane polarization and K⁺ transport. At low membrane polarization, corresponding to a low state of activation of the PM H⁺-ATP^ase and to E_m values more positive than E_K, K⁺ influx would mainly     

Plasmalemma redox activity and h extrusion: I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm acidification

Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K⁺ to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H⁺ extrusion no longer requires the presence of K⁺ in the medium. The (ferricyanide reduced)/(H⁺ extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H⁺ extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K⁺ release to the medium. The ratio (ferricyanide reduced)/(H⁺ extruded + K⁺ released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump, stimulated by both intracellular acidification and depolarization. K⁺ efflux would depend on potential depolarization.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

The ionic relations of Porphyra purpurea(Roth) C.Ag. (Rhodophyta, Bangiales)

Abstract Radioisotope equilibration techniques have been used to determine the intracellular concentration of K⁺, Na⁺ and Cl_?, together with the unidirectional ion fluxes across the plasmalemma of Porphyra purpurea. Influx and efflux of ⁴²K⁺, ²⁴Na⁺ and ³⁶C1^? are biphasic, the rapid, initial uptake and loss of tracer from individual thalli being attributable to desorption from extracellular regions. Cellular fluxes are slower and monophasic, cells discriminating in favour of K⁺ and Cl^? and against Na⁺. A comparison between the equilibrium potential of individual ion species and the measured membrane potential demonstrates that there is an active component of K⁺ and Cl^? influx and Na⁺ efflux. ‘Active’ uptake and ‘passive’ loss of K⁺ and Cl^? are reduced when plants are kept in darkness, suggesting that a fraction of the transport of K⁺ and Cl^? may be due to ‘exchange diffusion’ (K⁺/K⁺ and Cl^?/Cl^?antiport).     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K⁺ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K⁺. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K⁺ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K⁺ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line. Received: 4 October 1999/Revised: 21 December 1999     

Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl‐prolyl cis‐trans isomerase of the FK506‐binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over‐expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H⁺‐ATPase) is indirect: over‐expression of ROF2 activates K⁺ uptake, causing depolarization of the plasma membrane, which activates the electrogenic H⁺ pump. The depolarization of ROF2 over‐expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Na+/H+ antiporter activity of the SOS1 gene: lifetime imaging analysis and electrophysiological studies on Arabidopsis seedlings

Based on sequence analysis, the salt overly sensitive (SOS1) gene has been suggested to function as a Na⁺/H⁺ antiporter located at the plasma membrane of plant cells, being expressed mostly in the meristem zone of the root and in the parenchyma cells surrounding the vascular tissue of the stem. In this study, we compared net H⁺ and Ca²⁺ fluxes and intracellular pH and [Ca²⁺]_cyt in the root meristem zone of Arabidopsis wild‐type (WT) and sos mutants before and after salt stress. In addition, we studied the effect of pretreatment with amiloride (an inhibitor of Na⁺/H⁺ antiporters) on net ion fluxes, intracellular pH and intracellular Ca²⁺ activity ([Ca²⁺]_cyt) in WT plants and sos1 mutants before and after salt stress. Net ion fluxes were measured using microelectrode ion flux estimation (MIFE) and intracellular pH and [Ca²⁺]_cyt using fluorescence lifetime imaging microscopy (FLIM) techniques. During the first 15 min after NaCl application, sos1 mutants showed net H⁺ efflux and intracellular alkalinization in the meristem zone, whereas sos2 and sos3 mutants and WT showed net H⁺ influx and slight intracellular acidification in the meristem zone. Treatment with amiloride led to intracellular acidification and lower net H⁺ flux in WT plants and to a decrease in intracellular Ca²⁺ in WT and sos1 plants. WT plants pretreated with amiloride did not show positive net H⁺ flux and intracellular acidification. After NaCl application, internal pH shifted to higher values in WT and sos1 plants. However, absolute values of H⁺ fluxes were higher and internal pH values were lower in WT plants pretreated with amiloride compared with sos1 mutants. Therefore, the SOS1 transporter is involved in H⁺ influx into the meristem zone of Arabidopsis roots, or it may function as a Na⁺/H⁺ antiporter. Amiloride affects SOS1 and other Na⁺/H⁺ antiporters in plant cells because of its ability to decrease the H⁺ gradient across the plasma membrane.     

Cation flux in the ehrlich ascites tumor cell evidence for Na-for-Na and K-for-K exchange diffusion

In a previous study, evidence was presented for an external Na⁺-dependent, ouabain-insensitive component of Na⁺ efflux and an external K⁺-dependent component of K⁺ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na⁺ and K⁺ they represent Na⁺-for-Na⁺ and K-⁺for-K⁺ exchange mechanisms. Using ⁸⁶Rb to monitor K⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb⁺ influx and a component of Rb⁺ efflux, both representing approx. 30% of the total fux. Inhibition of Rb⁺ efflux is greatly reduced by removal of extracellular K⁺. Furosemide does not alter steady-state levels of intracellular K⁺ and it does not prevent cells depleted of K⁺ by incubation in the cold from regaining K⁺ upon warming. Using ²²Na to monitor Na⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na⁺ efflux which represents approx. 22% of total Na⁺ efflux. Furosemide does not alter steady-state levels of intracellular Na⁺ and does not prevent removal of intracellular Na⁺ upon warming from cells loaded with Na⁺ by preincubation in the cold. The ability of furosemide to affect unidirectional Na⁺ and K⁺ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of α-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na⁺-dependent amino acid transport system A.     

Effect of potassium deficiency on the plasma membrane H+-ATPase of the wood ray parenchyma in poplar

The effect of K⁺ deficiency on the plasma membrane (PM) H⁺‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K⁺ supply. Immunological assays using different antibodies were applied to test if K⁺ deficiency affects the amount of immunodetectable PM H⁺‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H⁺‐ATPases under conditions of low K⁺ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H⁺‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K⁺ supply. Measurements of extracellular H⁺ concentrations using non‐invasive, H⁺‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H⁺ in plants grown with low K⁺ supply. The present results indicate an up‐regulation of specific isoforms of the PM H⁺‐ATPase in VACs under K⁺‐deficient conditions and suggest a key role for these PM H⁺‐ATPases in unloading K⁺ from the xylem stream.     

Spontaneous inactivation of the Ca-sensitive K channels of human red cells at high intracellular Ca activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a (dCa²⁺/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about −65 mV was observed. At a membrane potential of about −70 mV and an intracellular concentration of about 2·10⁻⁴M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.