Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii

Glucose‐6‐phosphate dehydrogenase (G6PDH) is important for the activation of plant resistance to environmental stresses, and ion homeostasis is the physiological foundation for living cells. In this study, we investigated G6PDH roles in modulating ion homeostasis under salt stress in Carex moorcroftii callus. G6PDH activity increased to its maximum in 100 mM NaCl treatment and decreased with further increased NaCl concentrations. K⁺/Na⁺ ratio in 100 mM NaCl treatment did not exhibit significant difference compared with the control; however, in 300 mM NaCl treatment, it decreased. Low‐concentration NaCl (100 mM) stimulated plasma membrane (PM) H⁺‐ATPase and NADPH oxidase activities as well as Na⁺/H⁺ antiporter protein expression, whereas high‐concentration NaCl (300 mM) decreased their activity and expression. When G6PDH activity and expression were reduced by glycerol treatments, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio dramatically decreased. Simultaneously, NaCl‐induced hydrogen peroxide (H₂O₂) accumulation was abolished. Exogenous application of H₂O₂ increased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein expression and K⁺/Na⁺ ratio in the control and glycerol treatments. Diphenylene iodonium (DPI), the NADPH oxidase inhibitor, which counteracted NaCl‐induced H₂O₂ accumulation, decreased G6PDH, PM H⁺‐ATPase and NADPH oxidase activities, Na⁺/H⁺ antiporter protein level and K⁺/Na⁺ ratio. Western blot result showed that G6PDH expression was stimulated by NaCl and H₂O₂, and blocked by DPI. Taken together, G6PDH is involved in H₂O₂ accumulation under salt stress. H₂O₂, as a signal, upregulated PM H⁺‐ATPase activity and Na⁺/H⁺ antiporter protein level, which subsequently resulted in the enhanced K⁺/Na⁺ ratio. G6PDH played a central role in the process.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Cytochrome c oxidase: the mechanistic significance of structural H+ in energy transduction

Changes in the bulk-phase concentration of O₂ and H⁺ associated with the reduction of O₂ to water are simultaneously determined in reactions catalyzed by fully reduced cytochrome c oxidase both isolated and embedded in liposomes. Consistent with the polyphasic kinetics of electron transfer through the oxidase, the time course of O₂ consumption and H⁺ translocation exhibit the following novel characteristics: (1) The uptake of scalar protons (H_m ⁺), the ejection of vectorial protons (H⁺ _v), and the consumption of O₂, all proceed in a kinetically polyphasic process. (2) During the first phase of the reaction the rates of O₂ uptake and H⁺ transfer are extremely fast and compatible with the rates of electron flow through the oxidase. (3) The K_m of the oxidase for O₂ is close to 75 M, the same for O₂ consumption and scalar H⁺ uptake. The V_max of O₂ reduction to water in reactions catalyzed by the isolated enzyme is, at least, 0.5 × 10⁴ s^–1. (4) The extent of vectorial H⁺ ejection by cytochrome c oxidase embedded in liposomes is an exponential function dependent on both enzyme concentration and extent of O₂ consumption. (5) The H⁺/O stoichiometry of H⁺ ejection is a variable that may reach a maximum value of 4.0 only when the enzyme undergoes net oxidation at extremely high enzyme/O₂ molar ratios. It is postulated that the generation of useful energy at the level of cytochrome c oxidase depends not only on the number of molecules of O₂ reduced to water but also on the extent and state of reduction and/or protonation of the enzyme.     

Effects of deuterium oxide on growth, proton extrusion, potassium influx, and in vitro plasma membrane activities in maize root segments

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by ²H₂O in the incubation medium. Short-term exposure (30 min) to ²H₂O slightly reduced O₂ uptake and significantly increased ATP levels. ²H₂O inhibited H⁺ extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K⁺ (about 30 and 53%, respectively at 50% [v/v] ²H₂O). Experiments on plasma membrane vesicles showed that H⁺-pumping and ATPase activities were greatly inhibited by ²H₂O (about 35% at 50% [v/v] ²H₂O); NADH-ferricyanide reductase and 1,3-β-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by ²H₂O. Therefore, the effect of short incubation time and low concentration of ²H₂O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H⁺ -ATPase. K⁺ uptake was inhibited by ²H₂O only when K⁺ was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (E_m) was slightly hyperpolarized by ²H₂O at low K⁺, but was not affected at the higher K⁺ concentrations. These results suggest a relationship between H⁺ extrusion and K⁺ uptake at low K⁺ external concentration.     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

The C-terminal cytoplasmic portion of the NhaP2 cation–proton antiporter from Vibrio cholerae affects its activity and substrate affinity

In this work, we report the phenotypic and biochemical effects of deleting the C-terminal cytoplasmic portion of the NhaP2 cation/proton antiporter from Vibrio cholerae. While the deletion changed neither the expression nor targeting of the Vc-NhaP2 in an antiporter-less Escherichia coli strain, it resulted in a changed sensitivity of the host to sodium ions at neutral pH, indicating an altered Na⁺ transport through the truncated variant. When assayed in inside-out sub-bacterial vesicles, the truncation was found to result in greatly reduced K⁺/H⁺ and Na⁺/H⁺ antiport activity at all pH values tested and a greater than fivefold decrease in the affinity for K⁺ (measured as the apparent K _m) at pH 7.5. Being expressed in trans in a strain of V. cholerae bearing a chromosomal nhaP2 deletion, the truncated nhaP2 gene was able to complement its inability to grow in potassium-rich medium at pH 6.0. Thus the residual K⁺/H⁺ antiport activity associated with the truncated Vc-NhaP2 was still sufficient to protect cells from an over-accumulation of K⁺ ions in the cytoplasm. The presented data suggest that while the cytoplasmic portion of Vc-NhaP2 is not involved in ion translocation directly, it is necessary for optimal activity and substrate binding of the Vc-NhaP2 antiporter.     

Hormone action on transmembrane electron and h transport

A possible involvement of two different systems in proton translocation was investigated by simultaneous measurement of transmembrane electron flow and proton secretion in a pH-stat combined with a redoxstat. The pH gradient between cytoplasm and apoplast is probably maintained by an H⁺ -pumping ATPase and by a second proton extrusion system, which seems to be linked to a redox chain with NAD(P)H as electron donor. Indole acetic acid inhibits both e⁻ and H⁺ efflux, but only if the `electron draw' from the outside is not too high. The electron draw depends on the hexacyanoferrate level at the plasmalemma surface and on the Ca²⁺ concentration. The inhibiting effect of auxin on e⁻ and H⁺ efflux in the presence of hexacyanoferrate can be only detected at low levels of bivalent cations and of the artificial electron acceptor. The inhibition of e⁻ and H⁺ efflux by auxin requires high oxygen levels. The influence of auxin on both e⁻ and H⁺ transfer disappears below 2 kilopascals O₂, a level which does not influence respiration. Ethanol and fusicoccin do not increase the e⁻ flux, probably because the electron transfer from the plasma membrane to HCF III is the limiting step. If electron transfer is reduced by IAA pretreatment, ethanol increases e⁻ flux. Fusicoccin decreases e⁻ and increases H⁺ efflux if the rates have been lowered previously by indole acetic acid pretreatment. This effect depends on high oxygen levels and is reversible by lowering oxygen pressure. Auxin and Ca²⁺ change e⁻ flow and H⁺ ejection in a 1:1 ratio.     

Possible Linkage between NADH-Oxidation and Proton Secretion in Zea mays L. Roots

A possible involvement of two different systems in proton translocationand the correlation of this factor to growth rates were measuredsimultaneously by means of a pH stat and an optical system.Ferricyanide, which can accept electrons at the plasmalemma,led to an immediate increase of net H⁺ -efflux but also decreasedroot growth rate. The reduced form, ferrocyanide, inhibitednet H⁺ -effluxwithout changing the growth rate. Thus, corn rootgrowth was not determined by proton secretion exclusively. Vanadatestrongly inhibited net H⁺ -efflux by the roots but did not preventthe stimulating effect of fcrricyanide. Moreover, the extentof enhancement of net H⁺ -effluxby ferricyanide was exactlythe same in vanadate pretreated as in untreated roots. Alcoholswere used to try to increase the intracellular NADH level throughthe action of the cytoplasmic alcohol dehydrogenase presentin the roots and coleoptiles. Alcohols, known to be substratesfor alcohol dehydrogenase such as propan- 1-ol, ethanol andbutan-l -ol increased net H⁺ -effluximmediately but methanoland secondary alcohols which are not substrates had no effecton proton secretion. The K_m values of alcohol dehydrogenasefor the alcohols correspond only partly to their effects onproton secretion. However, the specificlty observed suggeststhat increased H⁺ -efflux arose from reduction of endogenousNAD by ADH and consequent increased membrane NADH-oxidasc activitytrans locating protons and electrons out of the cells. Decreased oxygen concentrations slowed proton secretion at valuesfar higher than are necessary to saturate cytochrome c oxidase.The results of these experiments suggest two distinct systemscontributing to proton efflux. Key words: ADH, proton transport, redox chain     

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O₂ dependence of net H⁺ efflux of maize coleoptiles has been investigated. Below 100 M O₂, H⁺ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 M O₂. Old coleoptiles show the same decrease in net H⁺ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO₂ production to the change in net proton efflux is about 1:1 at 40–80 M O₂ but not at 10 M O₂. An influx can be observed at 10 M O₂ in young as well as in old coleoptiles if the H⁺ concentration is held at values below pH 6.5. Lower O₂ concentrations lead to an increase of net H⁺ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO₂ production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O₂ concentration which is within the same range as the high K_m of the alternative path.Abbreviation FC fusicoccin     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Mycochromone decreases ATP-driven proton translocation in plant plasma membrane- and tonoplast-enriched vesicles

Abstract Mycochromone, a metabolite produced by Mycosphaerella rosigena, inhibits the ATP-dependent proton translocation and the ATP-generated electrical potential in pea stem tonoplast-enriched vesicles, without affecting the H⁺/K⁺ exchange induced by nigericin or an artificially imposed proton gradient. The inhibition is dependent on the time of pre-incubation and mycochromone concentration. In addition, mycochromone inhibits the ATP-dependent proton translocation in radish plasma membrane-enriched vesicles, though it does not alter ATPase activity (evaluated by hydrolysis of ATP) in either type of plant vesicle. Mycochromone seems to act on the H⁺ channels for proton translocation of the H⁺-pumping ATPase localized on plasmalemma and tonoplast, without affecting the catalytic site of ATP hydrolysis.     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

Effect of tentoxin on K+transport in winter wheat seedlings of different K+-status

Klotz, M. G. and Erdei, L. 1988. Effect of tentoxin on K⁺ transport in winter wheat seedlings of different K⁺-status. The influence of the phytoeffective mycotoxin, tentoxin, [cyclo-(L-leucyl-N-methyltrans-dehydronhenyl-alanyl-glycyl-N-methyl-L-alanyl)] (in K⁺ uptake and on translocation of K⁺ from roots to shoot was studied in 14-day-old winter wheat plants (Triticum aestivum L. cv. Martonvásári-8) grown at different levels of K⁺ supply. For comparison, the effects of 2,4-dinilrophcnol and valinomycin were also investigated. In I-h experiments I pM tentoxin reduced K⁺ influx in the routs over the external K⁺ concentration range 0.1 to 1 m^M (low-K⁺ plants), whereas stimulation was observed al lower and higher K⁺ concentrations. On the other hand, in plants grown at 0.3 m^M K⁺, tentoxin stimulated the translocation of K⁺ from roots to shoots in 5-h experiments. Valinomycin affected K⁺ transport only al high K⁺-status (slight stimulation). In low-K⁺ plants 2,4-dinitrophenol (DNP) caused drastic inhibition of K⁺ uptake, but in high-K⁺ plants uptake was only slightly inhibited and translocation slightly stimulated, It is concluded that the opposite effects of tentoxin on K⁺ uptake and translocation agree1 with the directions of the H⁺-ATPases pumping H⁺ towards the apoplast and located at the cortex plasmalemma and the xylem parenchyma plasma-membrane, respectively. These effects should probably be attributed to the interaction between tentoxin and the K⁺-carrier protein rather than to a direct influence of tentoxin on H⁺-ATPase.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Plasma membrane K+/H+-ATPase From leishmania donovani

Leishmania donovani has an active ^K+/H⁺ exchange system on the surface membrane. Modulation of external K⁺ concentration resulted in a corresponding change in internal pH (pH_i) suggesting a link between proton and potassium transport. Although a Na⁺/H⁺ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K⁺/H⁺ exchange. Reduction of cellular ATP with NaN₃ and KCN inhibits K⁺/H⁺ exchange showing thereby that the process is energy dependent. The K⁺/H⁺ exchange is sensitive to inhibitors of the gastric K⁺/H⁺-ATPase. It is concluded that the H⁺-ATPase previously reported on the plasma membrane of L. donovani is in fact a K⁺/H⁺-ATPase. © 1994 wiley-Liss, Inc.     

Inhibition of fusicoccin-stimulated K+/H+ transport in root tips from maize seedlings pretreated with Chlorsulfuron

Abstract Fusicoccin (FC)-stimulated K⁺ (₈₆Rb) uptake and proton extrusion of maize (Zea mays) root apical segments were inhibited by pretreatment of 4-day-old seedlings with the herbicide Chlorsulfuron. In the range of Chlorsulfuron concentrations 0.01-10 mmol m^?3, the percentage of inhibition was 15% at 0.01 mmol m^?3 and progressively increased with Chlorsulfuron concentration up to 60% at 10 mmol m^?3. At the maximum concentration tested (10 mmol m^?3), the inhibition was evident after 1.5 h of pre-treatment. The binding of FC to microsomal fractions of root segments from Chlorsulfuron-pretreated seedlings was inhibited by 30%. It is suggested that Chlorsulfuron causes an alteration at the plasmalemma level involving the FC binding sites. The ineffectiveness of Chlorsulfuron in inhibiting FC-stimulaled K⁺ uptake when administered to excised segments, while inhibiting the enzyme acetolactate synthase, pointed out by Ray (1984) as the site of action of Chlorsulfuron in pea plants, suggests that the observed inhibition of K⁺ uptake and H⁺ extrusion is not induced by Chlorsulfuron inhibition of this enzyme. An alternative site of action of Chlorsulfuron is hypothesized in maize plants.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied.Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux.The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K⁺ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the Δμ_H component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K⁺ and Na⁺.     

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     

Fluxes of h and k in corn roots : characterization and stoichiometries using ion-selective microelectrodes

We report here on an experimental system that utilizes ion-selective microelectrodes to measure the electrochemical potential gradients for H⁺ and K⁺ ions within the unstirred layer near the root surface of both intact 4-day-old corn seedlings and corn root segments. Analysis of the steady state H⁺ and K⁺ electrochemical potential gradients provided a simultaneous measure of the fluxes crossing a localized region of the root surface. Net K⁺ influx values obtained by this method were compared with unidirectional K⁺ (⁸⁶Rb⁺) influx kinetic data; at any particular K⁺ concentration, similar values were obtained by either technique. The ionspecific microelectrode system was then used to investigate the association between net H⁺ efflux and net K⁺ influx. Although the computed H⁺:K⁺ stoichiometry is dependent upon the choice of diffusion coefficients, the values obtained were extremely variable, and net K⁺ influx rarely appeared to be charge-balanced by H⁺ efflux. In contrast to earlier studies, we found the cortical membrane potential to be highly K⁺ sensitive within the micromolar K⁺ concentration range. Simultaneous measurements of membrane potential and K⁺ influx, as a function of K⁺ concentration, revealed similar K_m values for the depolarization of the potential (K_m 6-9 micromolar K⁺) and net K⁺ influx (K_m 4-7 micromolar K⁺). These data suggest that K⁺ may enter corn roots via a K⁺-H⁺ cotransport system rather than a K⁺/H⁺ antiporter.     

New Drugs for the Na+/H+ Exchanger. Influence of Na+ Concentration and Determination of Inhibition Constants with a Microphysiometer

The NHE-1 isoform of the Na⁺/H⁺ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na⁺/H⁺ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na⁺/H⁺ antiporter. The Na⁺/H⁺ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K _M = 30 ± 4 mm for Na⁺. Addition of either one of the four inhibitors decreased the acidification rate. The IC₅₀ values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC₅₀ values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na⁺/H⁺ antiporter and allows a quantitative kinetic analysis of the proton excretion rate. Received: 3 September 1998/Revised: 20 November 1998