Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana.

There is increasing evidence of the important roles of glutamate receptors (GLRs) in plant development and in adaptation to stresses. However, the studies of these putative ion channels, both in planta and in Xenopus oocytes, may have been limited by our lack of knowledge of possible GLR heteromer formation in plants. We have developed a modification of the single-cell sampling technique to investigate GLR co-expression, and thus potential heteromer formation, in single cells of Arabidopsis thaliana leaves. Micro-EXpression amplification (MEX) has allowed us to amplify gene transcripts from a single cell, enabling expression of up to 100 gene transcripts to be assayed. We measured, on average, the transcripts of five to six different AtGLRs in a single cell. However, no consistent patterns of co-expression or cell-type-specific expression were detected, except that cells sampled from the same plant showed similar expression profiles. The only discernible feature was the detection of AtGLR3.7 in every cell examined, an observation supported by GUS staining patterns in plants stably expressing promoter::uidA fusions. In addition, we found AtGLR3.7 expression in oocytes induces a Ba2+-, Ca2+- and Na+-permeable plasma membrane conductance.

Partitioning of nutrient transport processes in roots.

Roots have a range of cell types that each contribute to the acquisition of nutrients and their subsequent transfer to the xylem. The activities of these cells must be co-ordinated to ensure that delivery of nutrients to the shoot occurs at a rate that matches the demands of growth. The partitioning of transport processes between different cell types is thus essential for roots to function effectively. This partitioning is considered at the level of proteins, organelles and cells in relation to the accepted concepts of how nutrients are taken up by roots and delivered to the xylem. Using K+ as an example, the evidence underpinning current concepts is examined, gaps in understanding identified and the contribution of some new approaches assessed.

Where do all the ions go? The cellular basis of differential ion accumulation in leaf cells.

Leaf cells accumulate solutes differently depending on their cell type. The accumulation profiles of inorganic ions have been well documented for the mesophyll and epidermis, particularly in cereals. These cell types accumulate ions such as phosphate and calcium to strikingly different extents. Understanding the processes that control ion accumulation could reveal how plants respond to either a limiting supply of important micro- and macronutrient ions or to potentially toxic loads of salts or heavy metal ions. Research has recently begun to reveal the processes that underlie this remarkable sorting of nutrient ions within the leaf.

Differential ion accumulation and ion fluxes in the mesophyll and epidermis of barley.

In barley (Hordeum vulgare L.) leaves, differential ion accumulation commonly results in inorganic phosphate (Pi) being confined to the mesophyll and Ca(2+) to the epidermis, with preferential epidermal accumulation of Cl(-), Na(+), and some other ions. The pattern was confirmed in this study for major inorganic anions and cations by analysis of barley leaf protoplasts. The work focused on the extent to which differences in plasma membrane ion transport processes underlie these observations. Ion transport across the plasma membrane of barley epidermal and mesophyll protoplasts was investigated electrophysiologically (by microelectrode impalement and patch clamping) and radiometrically. Data from both approaches suggested that similar types of ion-selective channels and membrane transporters, which catalyze the transport of Ca(2+), K(+), Na(+), and Pi, exist in the plasma membrane of the two cell types. In general, the simple presence or absence of ion transporters could not explain cell-type-specific differences in ion accumulation. However, patch-clamp data suggested that differential regulation of instantaneously activating ion channels in the plasma membrane could explain the preferential accumulation of Na(+) in the epidermis.

Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase.

The bisphosphonates (general structure PO3-R-PO3) competitively inhibit soluble and membrane-bound inorganic pyrophosphatases (PPases) with differing degrees of specificity. Aminomethylenebisphosphonate (AMBP; HC(PO3)2NH2) is a potent, specific inhibitor of the PPase of higher plant vacuoles (V-PPase). To explore the possibility of constructing photoactivatable probes from bisphosphonates to label the active site of V-PPase we analysed the effects of different analogues on the hydrolytic and proton pumping activity of the enzyme. Bisphosphonates with a range of structures inhibited competitively and the effects on PPi hydrolysis correlated with the effects on proton pumping. Low-molecular-mass bisphosphonates containing hydrophilic groups (alpha-NH2 or OH) were the most effective, suggesting that the catalytic site is in a restricted polar pocket. Bisphosphonates containing a benzene ring were less active but the introduction of a nitrogen atom into the ring increased activity. Compounds of the general formula NH2(CH2)nC(PO3)2OH were more inhibitory than compounds of the H(CH2)nC(PO3)2NH2, NH2(CH2)nC(PO3)2NH2 or OH(CH2)nC(PO3)2NH2 series, with activity decreasing as n increased. A nitrogen atom in the carbon chain increased activity but activity was decreased by the presence of an oxygen atom. An analogue with a ring attached via a four-carbon chain, which included an amide linkage and a hydroxy group on the alpha-carbon atom, inhibited competitively (Ki=62.0 microM), suggesting that it may be possible to design bisphosphonate inhibitors which contain a photoactivatable azido group for photoaffinity labelling of V-PPase active site.

Tris Is a Competitive Inhibitor of K+ Activation of the Vacuolar H+-Pumping Pyrophosphatase.

The effects of a range of commonly used pH buffers on the hydrolytic activity of the plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase) from mung bean (Vigna radiata L.) hypocotyls were tested. All of the buffers inhibited K+ stimulation of the V-PPase, and the degree of inhibition was dependent on the concentrations of both the buffer and K+. The effects were dependent on the organic cation used in the buffers, and those tested inhibited in the order: Tris > Bis-Tris-propane > Bicine = Tricine > imidazole. Detailed studies revealed that a model in which Tris affects both the Km and Vmax for K+ stimulation provided an accurate description of the observed kinetics. The ability of different cations to stimulate the V-PPase was measured with a noncompeting buffer (5 mM imidazole-HCl) and the order of effectiveness was K+ = Rb+ > NH4+ >> Cs+ > Na+ > Li+, with the Km for K+ stimulation being about 1 to 2 mM. Published experiments performed in the presence of Tris were re-evaluated and all could be fitted to mixed inhibition kinetics, with kinetic parameters similar to those measured for the mung bean V-PPase. It is concluded that the variations in the published Km for K+ stimulation of the V-PPase are probably due to the effects of pH buffer cations and that the real value for this parameter is in the low millimolar range. The implications of this for regulation of the V-PPase by K+ in vivo and for the role of the enzyme in K+ transport into the vacuole are discussed.

Pathways for the permeation of Na+ and Cl- into protoplasts derived from the cortex of wheat roots.

Sodium permeation into cortex cells of wheat roots was examined under conditions of high external NaCI and low Ca(2+). Two types of K(+) inward rectifier were observed in some cells. The time-dependent K(+) inward rectifier was Ca(2+)-sensitive, increasing in magnitude as external Ca(2+) was decreased from 10 mM to 0.1 mM, but did not show significant permeability to Na(+). However, the spiky inward rectifier showed significant Na+ permeation at Ca(2+) concentrations of 1 and 10 mM. In cells that initially did not show K(+) inward rectifier channels, fast and sometimes slowly activating whole-cell inward currents were induced at membrane potentials negative of zero with high external Na(+) and low Ca(2+) concentrations. With 1 mM Ca(2+) in the external solution, large inward currents were carried by Rb(+), Cs(+), K(+), Li(+), and Na(+). The permeability sequence shows that K(+), Rb(+) and Cs(+) are all more permeant than Na(+), which is about equally as permeant as Li(+). When some K(+) was present with high concentrations of Na(+) the inward currents were larger than with K(+) or Na(+) alone. About 60% of the inward current was reversibly blocked when the external Ca(2+) activity was increased from 0.03 mM to 2.7 mM (half inhibition at 0.31 mM Ca(2+) activity). Changes in the characteristics of the current noise indicated that increased Ca(2+) reduced the apparent single channel amplitude. In outside-out patches inward currents were observed at membrane potentials more positive than the equilibrium potentials for K(+) and Cl(-) when the external Na(+) concentration was high. These channels were difficult to analyse but three analysis methods yielded similar conductances of about 30 pS.

Effects of Prolonged Washing on Primary and Secondary Transport Processes at the Plasma Membrane in Red Beet Storage Tissue.

Changing patterns of enzyme activity and solute transport in response to washing were investigated in red beet (Beta vulgaris L.) storage tissue. Washing had a pronounced effect on the plasma membrane (PM) H+-ATPase with an increase in both hydrolytic and proton-pumping activities. Immunoblotting indicated that this may be due, in part, to a higher amount of this enzyme in the PM of washed tissue. Activities of the tonoplast (V)H+-ATPase and pyrophosphatase fluctuated during a 4-d washing period, but overall showed no marked change in activity. In tissue discs sucrose (Suc), glucose (Glc), and fructose uptakes increased significantly in response to washing. Cycloheximide, cordycepin, and tunicamycin inhibited both Glc- and Suc-inducible uptake. Monensin also strongly inhibited inducible Glc uptake, but the effect on Suc was less marked. N-Ethylmaleimide inhibited both Suc and Glc uptake, with its effects being more pronounced in fresh tissue. Other protein-modifying reagents showed no significant difference in their level of inhibition between fresh and washed tissue. Transport studies, carried out using energized PM vesicles from fresh and washed tissue, indicated that there was no rise in Suc and Glc uptake rates in response to washing. Results with a range of inhibitors indicated that there was no marked change in transporter sensitivity in vesicles isolated from fresh and washed tissue. The results indicate that the well-described enhancement of solute transport in washed storage tissue may be due to an increased PM H+-ATPase activity rather than to changes in PM carrier activity or to changes in metabolism such as invertase activity.

Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity. Study of plasma membrane H(+)-ATPase, vacuolar H(+)-PPase, alkaline and acid phosphatases.

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inhibition. Secondly, since previous data suggested that beticolins could also be active against other Mg2(+)-dependent enzymes, we tested beticolin-1 on the vacuolar H(+)-PPase, which requires Mg2+ as co-substrate, and on the alkaline and acid phosphatases, which do not use Mg2+ as co-substrate. Only vacuolar H(+)-PPase is sensitive to beticolin-1, which suggests that beticolins are specific to enzymes that use a complex of Mg2+ as the substrate. The same Mg2H2B2 complex which is responsible of the plasma membrane H(+)-ATPase inhibition appears to be also involved in the inhibition of the vacuolar H(+)-PPase.

Potassium homeostasis in vacuolate plant cells.

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understanding the way this nutrient affects plant growth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (aK) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measurements revealed that vacuolar aK declined linearly with decreases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have been used in thermodynamic calculations to predict the need for, and likely mechanisms of, active K+ transport into the vacuole and cytosol. The direction of active K+ transport at the vacuolar membrane changes with tissue K+ status.

The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H+-Pumping Inorganic Pyrophosphatase (Studies Using Ligand Protection from Covalent Inhibitors).

Inhibitors preferentially and covalently reactive with cysteine, arginine, histidine, and carboxyl-containing residues were inhibitory to the plant vacuolar H+-transporting inorganic pyrophosphatase (H+-PPase) from Vigna radiata (mung bean) and Beta vulgaris (red beet), but hydrophobic compounds and those reactive with tyrosine and lysine were less effective. Inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, and N-ethylmaleimide was decreased in the presence of Mg2+ or mixtures of Mg2+ and inorganic pyrophosphate (PPi) but not by PPi alone. None of these ligands affected inhibition by reagents reactive with histidine. The Mg2+ dependence of protection from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide inhibition followed first-order kinetics and yielded a Km for free Mg2+ of 20 to 23 [mu]M. Protection from inhibition by N-ethylmaleimide and phenylglyoxal varied as a function of Mg2PPi concentration, suggesting that this is the substrate for the H+-PPase. Protection by Mg2PPi followed Michaelis-Menten kinetics with a Km of approximately 2 [mu]M. These results are consistent with the predictions of a kinetic model for the H+-PPase (R.A. Leigh, A.J. Pope, I.R. Jennings, D. Sanders [1992] Plant Physiol 100: 1698-1750), which identified free Mg2+ as an allosteric activator (Km = 25 [mu]M) and Mg2PPi as the substrate (Km = 2.5-5 [mu]M).

Cells of the Upper and Lower Epidermis of Barley (Hordeum vulgare L.) Leaves Exhibit Distinct Patterns of Vacuolar Solutes.

Vacuolar saps were extracted from individual, anatomically uniform cells of the upper (adaxial) and lower (abaxial) epidermis of the third leaf of barley (Hordeum vulgare L.) using a modified pressure probe. Saps (volume 80-200 pL) were sampled at various times between 3 d before and 7 d after full-leaf expansion and were analyzed for their osmolality and their concentrations of NO3-, malate, CI-, K+, and Ca2+. The osmolalities of upper and lower epidermis both increased with time but were similar to each other. In young leaves, K+ and Ca2+ were evenly distributed between the two epidermal layers, but as the leaf aged, the upper epidermis accumulated high (40-100 mM) Ca2+, whereas cells of the lower epidermis accumulated K+ instead. Nitrate concentration was 100 to 150 mM higher in the upper than in the lower epidermis, whereas CI- was 50 to 120 mM higher in the lower epidermis. These differences did not depend on the leaf developmental stage. The uneven distribution of epidermal NO3- and CI- was maintainedover a wide range of epidermal sap concentrations of these ions and was not affected by NO3- or CI- starvation or by an increase in the light intensity from 120 to 400 [mu]mol m-2 s-1. However, the latter did cause a decrease in epidermal NO3- and the appearance and accumulation of epidermal malate, particularly in the upper epidermis. The physiological implications of the results for solute storage in leaves and for the pathways of ion distribution to the epidermis are discussed.

The H(+)-pumping inorganic pyrophosphatase of the vacuolar membrane of higher plants.

The vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase) of plants is a member of a new class of energized ion translocases. The development of our understanding of this enzyme is briefly reviewed, including the evidence for its physiological role in H(+)-pumping and K+ transport into the vacuole, the identity of the polypeptides components, the cloning and sequencing of a cDNA encoding the catalytic subunit, and the partitioning of function between cytosolic and membrane domains of the protein. Lack of information about the identity of the substrate, activators and inhibitors of the H(+)-PPase has been a major barrier to the latter work. The various analyses that have been done of the kinetics of the enzyme predict different sets of activators and inhibitors, but work with residue-specific covalent inhibitors is now being done to resolve this. The results suggest that Mg2PPi is the substrate and that Mg2+ is an activator, but whether other PPi complexes inhibit the enzyme is still to be established.

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors.

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H(+)-PPase) from oat (Avena sativa L.) roots to changes in Mg(2+) and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H(+)-PPase is Mg(2)PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg(2+) and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H(+)-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H(+)-PPase. It is demonstrated that models incorporating Mg(2)PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H(+)-PPase. Calculations of the likely concentrations of Mg(2)PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H(+)-PPase would be about 70% saturated in vivo.

Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis.

The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca(2+) and a number of other divalent cations (Co(2+), Mn(2+), Zn(2+)). By contrast, the H(+)-translocating ATPase (H(+)-ATPase) located on the same membrane is insensitive to Ca(2+). Here we examine the mechanism and feasibility of regulation of the vacuolar H(+)-PPase by cytosolic free Ca(2+) under the conditions thought to prevail in vivo with respect to Mg(2+), inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca(2+) or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg(2)PPi) at one site, inhibitory binding of Mg(2)PPi to a lower-affinity second site, binding of activator (Mg(2+)) at a third site, and direct binding of Ca(2+) or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca(2+) or CaPPi are explicable only if Ca(2+), rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca(2+) on the substrate concentration dependence of H(+)-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H(+)-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca(2+) might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.

Functional expression of a plant plasma membrane transporter in Xenopus oocytes.

A full-length cDNA clone for the H+/hexose co-transporter (STP1) from Arabidopsis thaliana has been transcribed in vitro and the mRNA injected into Xenopus oocytes. Under optimized conditions, oocytes injected with the STP1 mRNA accumulated 3-O-[methyl-14C]glucose at rates of more than a 1000-fold greater than water-injected control oocytes. A hexose-elicited depolarization of the oocyte membrane potential was demonstrated, and uptake was shown to be stimulated by low external pH, confirming the activity of a H+/hexose co-transport system. This is the first example of the functional expression of a plant membrane transporter in oocytes.

Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling.

Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m(-3) nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m(-3) (n=19) and 41.8 ± 2.6 mol · m(-3) (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m(-3) (n = 4) and 72.8 ± 8.4 mol · m(-3) (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m(-3) (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m(-3) (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m(-3), while the vacuole accumulated nitrate to 30-50 mol · m(-3). The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.

Characterization of Cl- transport in vacuolar membrane vesicles using a Cl(-)-sensitive fluorescent probe: reaction kinetic models for voltage- and concentration-dependence of Cl- flux.

The effects of Cl- concentration and membrane potential (delta psi) on Cl- influx in isolated vesicles of vacuolar membrane (tonoplast) from red beet (Beta vulgaris L.) storage tissue have been characterized using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). The initial rate of Cl- transport into the vesicles was enhanced both by the imposition of a positive delta psi and by increases in extravesicular Cl- concentration. The kinetic mechanism underlying these responses was investigated by examining the accuracy with which the data could be described by several transport models. A model based on constant field theory yielded a poor description of the data, but satisfactory fits were generated by pseudo-two-state reaction kinetic models based on classical carrier schemes. Fits were equally good when it was assumed that charge translocation accompanied Cl- entry, or when charge was carried by the unloaded transport system, as long as only a single charge is translocated in each carrier cycle. Expansion of the models to three states enabled description of the Cl- concentration dependence of transport by changes in a single, voltage insensitive rate constant which is tentatively identified with Cl- binding at the external surface of the membrane. The derived value of the dissociation constant between Cl- and the transport system is estimated at between 30 and 52 mM.

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe : Effects of other anions, membrane potential, and transport inhibitors.

The characteristics of Cl(-) transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl(-)-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K(+) and valinomycin, increased the initial rate of Cl(-) transport, demonstrating that Cl(-) could be electrically driven into the vesicles. Chloride influx was unaffected by SO 4 (2-) , but was competitively blocked by NO 3 (-) , indicating that both Cl(-) and NO 3 (-) may be transported by the same porter. In some preparations, increases in free-Ca(2+) concentration from 10(-8) to 10(-5) mol·dm(-3) caused a significant decrease in Cl(-) influx, which may indicate that cytosolic Ca(2+) concentration has a role in controlling Cl(-) fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl(-) transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.