Vacuolar Channel Research Articles

On the Interaction of Neomycin with the Slow Vacuolar Channel ofArabidopsis thaliana

This study investigates the interaction of the aminoglycoside antibiotic neomycin with the slow vacuolar (SV) channel in vacuoles from Arabidopsis thaliana mesophyll cells. Patch-clamp experiments in the excised patch configuration revealed a complex pattern of neomycin effects on the channel: applied at concentrations in the submicromolar to millimolar range neomycin (a) blocked macroscopic SV currents in a voltage- and concentration-dependent manner, (b) slowed down activation and deactivation kinetics of the channel, and most interestingly, (c) at concentrations above 10 μM, neomycin shifted the SV activation threshold towards negative membrane potentials, causing a two-phasic activation at high concentrations. Single channel experiments showed that neomycin causes these macroscopic effects by combining a decrease of the single channel conductance with a concomitant increase of the channel's open probability. Our results clearly demonstrate that the SV channel can be activated at physiologically relevant tonoplast potentials in the presence of an organic effector molecule. We therefore propose the existence of a cellular equivalent regulating the activity of the SV channel in vivo.

Different properties of SV channels in root vacuoles from near isogenic Al-tolerant and Al-sensitive wheat cultivars

Patch-clamp experiments revealed that near isogenic ET8 (Al-tolerant) and ES8 (Al-sensitive) wheat cultivars differed significantly in slow vacuolar channel properties. Under control conditions, whole vacuole currents displayed faster deactivation in ES8. Application of 1.4 μM vacuolar Al 3+ caused a 20 mV increase in the activation threshold and slowed activation kinetics in ET8 but not in ES8. Channel density was about 30% higher in ES8 than ET8, and was not altered by 24 h aluminium pre-treatment. However, the activation threshold was reduced in Al-pre-treated ES8. Overall, our data suggests that Alt1 locus may control more than the plasma membrane malate channel in wheat.

A Perspective on the Slow Vacuolar Channel in Vacuoles from Higher Plant Cells

A Perspective on the Slow Vacuolar Channel in Vacuoles from Higher Plant Cells

The barley two-pore K +-channel HvKCO1 interacts with 14-3-3 proteins in an isoform specific manner

Members of the highly conserved family of 14-3-3 proteins play an important role in various cellular processes. Recent studies, using electrophysiological techniques, show that the 14-3-3 proteins also regulate plasma membrane and vacuolar K + conducting channels. The molecular mechanism behind the regulatory effect of 14-3-3 proteins on K + channels remains to be shown. One vacuolar channel down-regulated by 14-3-3 proteins is the slow-activating vacuolar (SV) channel. In Arabidopsis, the protein coded by the KCO1 gene was recently shown to be present in the vacuolar membrane and identified as a component of the SV channel. These two observations raised the question whether the KCO1 protein does interact with 14-3-3 proteins. Therefore, we isolated the barley HvKCO1 gene and the encoded protein indeed contains a canonical 14-3-3 interaction motif, which is conserved in all other KCO1 orthologues from other plant species. Using surface plasmon resonance (SPR) we determined in real-time the affinity between the phospho-peptide derived from the putative KCO1 14-3-3 interaction motif and three barley 14-3-3 proteins. The 14-3-3A protein showed the highest affinity, whereas the binding of all three isoforms was dependent on the presence of either Ca 2+ or Mg 2+. Interestingly, the barley SV current was strongly reduced by 14-3-3B and C protein, but not by 14-3-3A. This difference between the SPR and patch-clamp data will be discussed, along with the role for Ca 2+ in activation of the SV channel by direct interaction and inactivation of the channel by facilitating the binding of 14-3-3 to the channel.

Regulation of the Slow Vacuolar Channel by Luminal Potassium: Role of Surface Charge

Voltage-dependent activation of slow vacuolar (SV) channels has been studied on isolated patches from red beet (Beta vulgaris L.) vacuoles. Isoosmotic variation of vacuolar K(+) from 10 to 400 mM in Ca(2+)-free solutions at the vacuolar side shifted the SV channel activation threshold to more positive voltages. The effect of K(+) could be mimicked by additions of choline or N-methyl D-glucamine and could be explained by unspecific screening of the negative surface charge. Fitting the dependence of voltage shift on K(+) concentration to the Gouy-Chapman model yields a surface charge density of 0.36 +/- 0.05 e(-)/nm(2). Negative surface potential also tended to increase the local concentration of permeable ions (K(+)), resulting in anomalously high single-channel conductance, approximately 200 pS in 10 mM KCl. An increase of ionic strength due to addition of impermeable cations greatly reduced the unitary conductance. Large positive shift of the SV channel voltage dependence, caused by physiological (0.5 mM) free vacuolar Ca(2+), was partly ameliorated by increasing luminal K(+). We interpreted these results as follows: K(+)induced a reduction of surface potential, hence i) causing a positive shift of the voltage dependence and ii) a dilution of Ca(2+) in the membrane vicinity, thus reducing the inhibitory effect of vacuolar Ca(2+) and causing a negative shift of the SV channel voltage dependence, with a sum of the two shifts being negative.

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.

K+ currents through SV‐type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.

Redox-dependent modulation of the carrot SV channel by cytosolic pH

Currents mediated by a slow vacuolar (SV) channel were recorded and characterized in vacuoles from cultured carrot cells. The carrot channel shows the typical functional characteristics reported for channels of the SV category previously identified in other plants, i.e., slow voltage-dependent activation kinetics, current activation favoured by cytosolic calcium and permeability to different monovalent cations. The carrot channel is strongly activated by cytosolic reducing agents (such as dithiothreitol, DTT, and glutathione, GSH) and has a peculiar dependence on cytosolic pH, which, in turn, is affected by the concentration of cytosolic reducing agents. Specifically, in 1 mM DTT or GSH the channel displayed a maximum conductance at neutral pH. The normalized conductance did not depend significantly on DTT concentration at acidic pH, while at alkaline pH the attenuation of the normalized conductance declines with increasing DTT concentration. Our results suggest two pH-titratable groups within the carrot SV channel, one of these depending on cysteine residues exposed to the cytosolic side of the vacuole.

Mechanism of luminal Ca2+ and Mg2+ action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca(2+)-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channel's closed conformations. Vacuolar Ca2+ and Mg2+ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a 'permeable blocker'. Vacuolar Ca(2+)-with a much higher affinity than Mg(2+)-slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca(2+)-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channel's closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca2+ at the vacuolar side. The specificity for Ca2+ compared to Mg2+ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca2+ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.

Slow vacuolar channels of non-embryogenic and embryogenic cultures of winter wheat

Slowly activating vacuolar channels (SV), were examined in embryogenic and non-embryogenic cultures of winter wheat using a patch-clamp technique. Four different types of cultures were examined: embryogenic and non-embryogenic calli from embryos, embryogenic and non-embryogenic calli from inflorescences. In a cell-attached mode single SV channel events were recorded. Unitary conductance of single SV channels was between 37 pS and 48 pS and did not significantly depend on the kind of the culture, although it was a tendency that SV channels of embryogenic calli possessed lower unitary conductance than those of non-embryogenic. 2,4-D caused significant lowering of unitary conductance from 48±6 pS in the control culture of embryogenic embryos to 28±6 pS in vacuoles treated. The SV channel density was estimated as 0.34 µm−2.

The gating kinetics of the slow vacuolar channel. A novel mechanism for SV channel functioning?

Although there is consensus that the slow vacuolar or SV channel is a Ca2+ release channel, the underlying mechanism of operation is still controversial. The main reason is that the voltage sensitivity of SV gating seems to exclude activation at hyperpolarized (physiological) membrane potentials. Inspired by a study of Gambale et al. (1993) and supported by simulation studies presented here, we interpreted SV activation and deactivation kinetics in terms of a cyclic state diagram originally applied to animal cation-selective channels. A cyclic state diagram allows two pathways of activation operating in opposite directions. One pathway represents the frequently observed slow activation at moderate depolarization (< 130 mV). With the open state (O) next to the closed state initially occupied (C1), direct transitions from C1 to O can account for the fast activation observed at higher depolarized potentials (> 130 mV). We hypothesize that similar state transitions directly to O may also occur during hyperpolarization. The implication of this proposed mechanism is that SV accomplishes its physiological role during hyperpolarization-evoked deactivation. Despite their rare occurrence and possibly short duration, these opening events may last long enough to substantially raise the local cytosolic free Ca2+ level at the channel mouth by as much as 600 nM/ms. Because under in vivo conditions the Ca2- flux is inwardly directed, the mechanism presented here revives the notion that the SV channel can be subject to calcium-induced calcium release.

The transient receptor potential channel on the yeast vacuole is mechanosensitive.

Ca2+ is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca2+ conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg = 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca2+ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca2+ concentration and is apparently independent of its known Ca2+ activation, which we now propose to be an amplification mechanism (Ca2+-induced Ca2+ release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.

Regulation of the Fast Vacuolar Channel by Cytosolic and Vacuolar Potassium

At resting cytosolic Ca 2+, passive K + conductance of a higher plant tonoplast is likely dominated by fast vacuolar (FV) channels. This patch-clamp study describes K +-sensing behavior of FV channels in Beta vulgaris taproot vacuoles. Variation of K + between 10 and 400 mM had little effect on the FV channel conductance, but a pronounced one on the open probability. Shift of the voltage dependence by cytosolic K + could be explained by screening of the negative surface charge with a density σ = 0.25 e −/nm 2. Vacuolar K + had a specific effect on the FV channel gating at negative potentials without significant effect on closed-open transitions at positive ones. Due to K + effects at either membrane side, the potential at which the FV channel has minimal activity was always situated at ∼50 mV below the potassium equilibrium potential, E K +. At tonoplast potentials below or equal to E K +, the FV channel open probability was almost independent on the cytosolic K + but varied in a proportion to the vacuolar K +. Therefore, the release of K + from the vacuole via FV channels could be controlled by the vacuolar K + in a feedback manner; the more K + is lost the lower will be the transport rate.

Research at channel level on the effect of LaCl3 on Radish (Raphanus satirus L.) vacuolar membrane

We recorded slow vacuolar (SV-type) channel currents of Radish vacuoles successfully for the first time by using the whole-vacuolar patch-clamp recording mode. SV-type currents would increase and threshold potentials of activation would shift towards more negative values with the increase of concentrations of cytosolic Ca2+. When 2.5 mmol/L LaCl3 and 4 mmol/L EGTA were added to bath solutions, SV-type currents were suppressed remarkably. Then adding LaCl3 with different concentrations to pipette solutions, we found that LaCl3 with higher concentrations (>4 × 10−7 mol/L) had a strong inhibitory effect on SV-type currents, while LaCl3 with lower concentrations (⩽4 × 10−7 mol/L) promoted channel currents. This promoting effect provides an important basis at channel level for researching further the effects of rare earth on physiological activities of plants and the production-increase effects of rare earth fertilizers on crops.

Ion transport and metal sensitivity of vacuolar channels from the roots of the aquatic plant Eichhornia crassipes

AbstractUsing the patch‐clamp technique, we investigated the transport properties of vacuolar ion channels from the roots of water hyacinth, Eichhornia crassipes (Mart. Solms, Pontederiacae). Eichhornia crassipes vacuoles displayed large voltage‐dependent rectifying slow‐vacuolar (SV) currents, which activated in a few seconds at positive potentials and deactivated at negative voltages in a few hundreds of millseconds. Similarly to SV channel previously identified in the tonoplast of terrestrial plants, SV currents in E. crassipes were activated by micromolar concentrations of Ca2+ and current slightly increased (25%) on addition (10 mm) of the reducing agent dithiothreitol (DTT). Eichhornia crassipes SV channels were equally permeable to K+ and Na+. The permeability sequence derived from current values is: K+ ≈ Na+ &gt; Rb+ &gt; NH4+ ≈ Cs+ &gt;&gt; TEA+. Excised membrane patches displayed single channel transitions typical of SV‐type single channel openings with a conductance of (83·0 ± 5·6) pS; a smaller channel with a conductance of (31·0 ± 2·7) pS was also identified. Metals such as Ni2+ and Zn2+ decreased the vacuolar current in a reversible manner. However, although Zn2+ inhibition is comparable to that induced by the same metal in vacuoles from the main root of sugar beet (Beta vulgaris L.), the inhibition of the SV currents by Ni2+ is not as substantial in E. crassipes as in sugar beet. To our knowledge, this is the first electrophysiological characterization of ionic transport in E. crassipes, a pervasive troublesome aquatic weed, which has exceptional absorption properties of several water contaminants such as heavy metals, pesticides and phenols.

Anion modulation of the slowly activating vacuolar channel.

This study addresses the permeability and modulation by anions of the slowly-activating vacuolar (SV) channel in the tonoplast of Mesembryanthemum crystallinum. Although we conclude that the M. crystallinum SV channel is impermeable to anions, the anion species at the cytosolic side of the membrane clearly affects the channel. We discuss two types of modulation, both consistent with a single binding site for anions at the cytosolic face of the channel. The first effect is related to the conducting properties of SV. When the site is occupied, the bound anion exerts an effect on the Ca2+ and Na+ fluxes through the channel. Because the Stokes-diameter of both ion species is different, both fluxes are not influenced to the same extent. This differential effect on the permeation of both ion species is reflected in a permeability ratio Ca2+ over Na+ (P(Ca)/P(Na)) that depends on the identity of the anion species. The second type of modulation deals with the activation of the channel by anions. Previous studies revealed that a low level of cytosolic Cl- inactivates SV (Pantoja, Dainty & Blumwald, 1992a). We critically evaluated reported effects of anions on the whole-vacuole SV current. Given that cytosolic Ca2+ and Cl- both modulate SV, we conclude that both levels of regulation show some kind of interaction. High cytosolic Ca2+ can overrule the inactivation caused by low cytosolic Cl-.

Effects of cytoplasmic Mg2+ on slowly activating channels in isolated vacuoles of Beta vulgaris.

The slow vacuolar (SV) channel can mediate a large part of the ionic current in plant tonoplasts, but its actual physiological role is still unclear. We demonstrate that in vacuoles from the taproots of sugar beet (Beta vulgaris L.), besides Ca2+, cytoplasmic Mg2+ also plays an important role in promoting the activation of the SV channel. An increase in Mg2+ concentration decreases the time constants of channel activation and deactivation, and determines a consistent shift, towards negative voltages, of the conductance characteristic; as an example, when the free concentration of Mg2+ was increased from the micromolar range up to 10 mM the activation shifted by about -60 mV. The experimental results obtained, which are based on a fast perfusion procedure allowing us to change the solution bathing the vacuole in a few milliseconds, suggest that magnesium-binding is a faster process than the voltage-activation gating of the channel, which constitutes the rate-limiting step controlling channel opening. Interestingly, the activation of the channel mediated by Mg2+ depends on the cooperative binding of at least three magnesium ions. We verified that cytoplasmic magnesium favours the activation of SV channels in the presence of nanomolar cytoplasmic calcium concentrations. A critical discussion on the Calcium Induced Calcium Release (CICR) mechanism proposed for the SV channel is presented.

Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins

The conductance of the vacuolar membrane at elevated cytosolic Ca 2+ levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca 2+ concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca 2+-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.

Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwortConocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolyzed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves ofArabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slowly activating vacuolar channel inC. conicum displayed the characteristic features of higher-plant slowly activating vacuolar channels.

Magnesium Sensitizes Slow Vacuolar Channels to Physiological Cytosolic Calcium and Inhibits Fast Vacuolar Channels in Fava Bean Guard Cell Vacuoles.

Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca(2+), calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg(2+) levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg(2+) inhibited fast vacuolar (FV) currents with a K(i) of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg(2+) at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg(2+), cytosolic Ca(2+) at less than 10 µM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg(2+) was present, submicromolar concentrations of cytosolic Ca(2+) activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg(2+)-Ca(2+) effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg(2+) concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca(2+) with an affinity in the submicromolar range and a cytosolic low-affinity Mg(2+)-Ca(2+) binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca(2+) and is inhibitory. In conclusion, cytosolic Mg(2+) sensitizes SV channels to physiological cytosolic Ca(2+) elevations. Furthermore, we propose that cytosolic and vacuolar Mg(2+) concentrations ensure that FV channels do not function as a continuous vacuolar K(+) leak, which would prohibit stomatal opening.