Electrogenic plasma membrane H<Superscript>+</Superscript>-ATPase activity using voltage sensitive dyes

Fast responding voltage sensitive dyes, RH421 and di-4-ASPBS, were used to study the electrogenic properties of plant plasma membrane proton pumps on sealed plasma membrane vesicles extracted by two-phase partitioning from Beta vulgaris and Avena sativa cv Swan root material. Fluorescence spectroscopy in the presence of the dye RH421 (10.8 nM) was sufficiently sensitive to detect electrogenic activity of the extracted plant vesicles. The dye detection system could detect inhibition of electrogenic activity of vesicles by vanadate (75 μM) and stimulation by nigericin (0.5 μM). The newly developed dye di-4-ASPBS was less sensitive to detecting the electrogenic proton pump activity. This study represents an important innovation in plant biophysics as this class of fast responding voltage sensitive dyes have never to our knowledge been used to study electrogenic proton pump activity derived from plant membranes and represents a novel approach for carrying out such studies.     

Exogenous IAA and ABA stimulate germination of petunia male gametophyte by activating Ca<Superscript>2+</Superscript>-dependent K<Superscript>+</Superscript>-channels and by modulating the activity of plasmalemma H<Superscript>+</Superscript>-ATPase and actin cytoskeleton

To date, the molecular mechanisms underlying the osmoregulation of pollen grains (PGs) related to the maintenance of their water status and allowing pollen tubes (PTs) to regulate concentrations in them of osmolytes and transmembrane water transport remain to be not so far characterized. In the present work, the data on the participation of IAA and ABA in the osmoregulation of germinating in vitro petunia male gametophyte were obtained. It has been established that the growth-stimulating effect of these phytohormones is due to their action on intracellular pH (pH_c), the membrane potential of plasmalemma (PM), the activity of PM H⁺-ATPase, K⁺-channels in the same membrane and organization of actin cytoskeleton (AC). Two possible targets of the action of these compounds are revealed. These are represented by (1) PM H⁺-ATPase, electrogenic proton pump responsible for polarization of this membrane, and (2) Ca²⁺-dependent K⁺-channels. The findings of the present work suggest that the hormone-induced pH_c shift is involved in cascade of the events including the functioning of pH-dependent K⁺-channels. It was shown that the hormoneinduced hyperpolarization of the PM is a result of stimulation of electrogenic activity of PM H⁺-ATPase and the hormonal effects are mediated by transient elevation in the level of free Ca²⁺ in the cytosol and generation of reactive oxygen species (ROS). The results on the role of K⁺ ions in the control of water-driving forces for transmembrane water transport allowed us to formulate the hypothesis that IAA and ABA stimulate germination of PGs and growth of PTs by activating K⁺-channels. In addition, the studies performed showed that the AC of male gametophyte is sensitive to the action of exogenous phytohormones, with to more extent to the action of IAA. As judged by the action of latrunculin B (LB) the AC may serve as the determinant of the level of endogenous phytohormones that most likely participate in the regulation of the polar growth of PTs impacting on the pool of F-actin in their apical and subapical zones.     

Improvement of alcoholic fermentation by calcium ions under enological conditions involves the increment of plasma membrane H<Superscript>+</Superscript>-ATPase activity

The effect of Ca²⁺ on alcoholic fermentation and plasma membrane H⁺-ATPase activity of wine yeast under enological conditions were investigated in this study. The results showed that fermentation rate, cell growth and ethanol production were improved by 0.5 and 1.5 mM Ca²⁺ supplementation, which correlated well with the increment of ATPase activity and protein levels. Considering the important role of ATPase in the tolerance of yeast to ethanol, the improvement could be, at least partially, attributed to the increment of ATPase activity. No activation of ATPase by Ca²⁺ was observed in the early phase of fermentation and the increment of activity was only observed when ethanol concentration exceeded 6.5%. Therefore, the enhancement of ATPase activity by Ca²⁺ was ascribed to alleviating the inhibition of ATPase activity by ethanol through protection of membrane structure. Our results suggest that, besides maintenance of cell membrane structure, the increment of plasma membrane ATPase activity was also responsible for the improvement of alcoholic fermentation by Ca²⁺ supplementation.     

Cell physiological aspects of the plasma membrane electrogenic H<Superscript>+</Superscript> pump

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H⁺) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H⁺ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H⁺ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H⁺ efflux supports the notion that the ion carried by the electrogenic ion pump is H⁺. The role of the H⁺ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H⁺ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the K_m value for the ATP of H⁺-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H⁺-ATPase in guard cells is presented. However, there are cases where H⁺-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H⁺-ATPase activity. Finally, modulation of H⁺-pumping or H⁺-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H⁺ pump.     

Role of the plasma membrane H<Superscript>+</Superscript>-ATPase in auxin-induced elongation growth: historical and new aspects

This article will cover historical and recent aspects of reactions and mechanisms involved in the auxin-induced signalling cascade that terminates in the dramatic elongation growth of cells and plant organs. Massive evidence has accumulated that the final target of auxin action is the plasma membrane H⁺-ATPase, which excretes H⁺ ions into the cell wall compartment and, in an antiport, takes up K⁺ ions through an inwardly rectifying K⁺ channel. The auxin-enhanced H⁺ pumping lowers the cell wall pH, activates pH-sensitive enzymes and proteins within the wall, and initiates cell-wall loosening and extension growth. These processes, induced by auxin or by the "super-auxin" fusicoccin, can be blocked instantly and specifically by a voltage inhibition of the H⁺-ATPase due to removal of K⁺ ions or the addition of K⁺-channel blockers. Vice versa, H⁺ pumping and growth are immediately switched on by addition of K⁺ ions. Furthermore, the treatment of segments either with auxin or with fusicoccin (which activates the H⁺-ATPase irreversibly) or with acid buffers (from outside) causes an identical transformation and degradation pattern of cell wall constituents during cell-wall loosening and growth. These and other results described below are in agreement with the acid-growth theory of elongation growth. However, objections to this theory are also discussed.     

Suppression of the plasma membrane H<Superscript>+</Superscript>-conductance on the background of high H<Superscript>+</Superscript>-pump activity in dithiothreitol-treated <Emphasis Type="Italic">Chara</Emphasis> cells

Photosynthesizing cells of characean algae exposed to light are able to produce pH bands corresponding to alternate areas with dominant H⁺-pump activity and high H⁺-conductance of the cell membrane. The action potential generation temporally arrests the counter-directed H⁺ fluxes, which gives rise to opposite pH shifts in different cell regions and represents a suitable indicator for activities of the plasma membrane H⁺-transporting systems. Measurements of pH near the cell surface by means of microelectrodes and microspectrophotometry in the presence of pH-indicating dye thymol blue have shown that the treatment of cells with dithiothreitol (SH-group reducing agent) suppresses pH changes induced by the action potential generation in the alkaline cell areas and considerably increases the concurrent pH changes in the acid regions. Measurements of plasma membrane resistance in the alkaline zones revealed that dithiothreitol inhibits the light-dependent conductance of the resting cell and diminishes the conductance inactivation caused by the action potential generation. The data suggest that the reduction of accessible disulfide bonds results in the decrease of H⁺-conductance, whereas the activity of plasma membrane H⁺-pump remains unimpaired or is even enhanced.     

Properties of plasma membrane H<Superscript>+</Superscript>-ATPase in salt-treated <Emphasis Type="Italic">Populus euphratica</Emphasis> callus

The plasma membrane (PM) vesicles from Populus euphratica (P. euphratica) callus were isolated to investigate the properties of the PM H⁺-ATPase. An enrichment of sealed and oriented right-side-out PM vesicles was demonstrated by measurement of the purity and orientation of membrane vesicles in the upper phase fraction. Analysis of pH optimum, temperature effects and kinetic properties showed that the properties of the PM H⁺-ATPase from woody plant P. euphratica callus were consistent with those from herbaceous species. Application of various thiol reagents to the reaction revealed that reduced thiol groups were essential to maintain the PM H⁺-ATPase activity. In addition, there was increased H⁺-ATPase activity in the PM vesicles when callus was exposed to NaCl. Western blotting analysis demonstrated an enhancement of H⁺-ATPase content in NaCl-treated P. euphratica callus compared with the control.     

Close-Up and Genomic Views of the Yeast Vacuolar H<Superscript>+</Superscript>-ATPase

The yeast V-ATPase has emerged as an excellent model for other eukaryotic V-ATPases. In this review, recent biochemical and genomic studies of the yeast V-ATPase are described, with a focus on: 1) the role of V₁ subunit H in coupling ATP hydrolysis and proton pumping and 2) identification of the full set of yeast haploid deletion mutants that exhibit the pH and calcium-sensitive growth characteristic of loss of V-ATPase activity. The combination of “close-up” biochemical views of V-ATPase structure and mechanism and “geomic” views of its functional reach promises to provide new insights into the physiological of V-ATPases.     

H<Superscript>+</Superscript> translocation by weak acid uncouplers is independent of H<Superscript>+</Superscript> electrochemical gradient

According to the common view, weak acid uncouplers increase proton conductance of biological (and phospholipid bilayer) membranes, thus effecting H⁺ fluxes driven by their electrochemical gradients. Under certain conditions, however, uncouplers can induce unexpected effects opposite to the dissipation of H⁺ gradients. Results are presented here demonstrating CCCP-induced proton influx into Saccharomyces cerevisiae cytosol driven by the electrochemical potentials of CCCP and its CCCP^? anions, independent of electrochemical H⁺-gradient. Another view of week acid uncouplers’ action is proposed that is logically consistent with these observations.     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Fumonisin B<Subscript>1</Subscript>, a sphingoid toxin,is a potent inhibitor of the plasma membrane H<Superscript>+</Superscript>-ATPase

Fumonisin B₁ (FB₁) is an amphipathic toxin produced by the pathogenic fungus Fusarium verticillioides which causes stem, root and ear rot in maize (Zea mays L.). In this work, we studied the action of FB₁ on the plasma membrane H⁺-ATPase (EC 3.6.1.34) from germinating maize embryos, and on the fluidity and lipid peroxidation of these membranes. In maize embryos the toxin at 40 M inhibited root elongation by 50% and at 30 M decreased medium acidification by about 80%. Irrespective of the presence and absence of FB₁, the H⁺-ATPase in plasma membrane vesicles exhibited non-hyperbolic saturation kinetics by ATPH-Mg, with Hill number of 0.67. Initial velocity studies revealed that FB₁ is a total uncompetitive inhibitor of this enzyme with an inhibition constant value of 17.5±1 M. Thus FB₁ decreased V_max and increased the apparent affinity of the enzyme for ATP-Mg to the same extent. Although FB₁ increased the fluidity at the hydrophobic region of the membrane, no correlation was found with its effect on enzyme activity, since both effects showed different FB₁-concentration dependence. Peroxidation of membrane lipids was not affected by the toxin. Our results suggest that, under in vivo conditions, the plasma membrane H⁺-ATPase is a potentially important target of the toxin, as it is inhibited not only by FB₁ but also by its structural analogs, the sphingoid intermediates, which accumulate upon the inhibition of sphinganine N-acyltransferase by this toxin.     

An Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na⁺/H⁺ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (Δnhx1), TaNHX2 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na⁺/H⁺ antiporter, and caused partial recovery of growth of Δnhx1 in NaCl and LiC1 media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane