Slow Vacuolar Channel Research Articles

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.

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.

Inhibition of vacuolar ion channels by polyamines.

In this work, direct effects of cytosolic polyamines on the two principle vacuolar ion channels were studied by means of patch-clamp technique. Fast and slow activating vacuolar channels were analyzed on membrane patches isolated from vacuoles of the red beet taproot. The potency of the fast and of the slow vacuolar channel blockage by polyamines decreased with a decrease of the polycation charge, spermine4+ > spermidine3+ > putrescine2+. In contrast to the inhibition of the fast vacuolar channel, the blockage of the slow vacuolar channel by polyamines displayed a pronounced voltage-dependence. Hence, in the presence of high concentration of polyamines the slow vacuolar channel was converted into a strong inward rectifier as evidenced by its unitary current-voltage characteristic. The blockage of the slow vacuolar channel by polyamines was relieved at a large depolarization, in line with the permeation of polyamines through this channel. The voltage-dependence of blockage was analyzed in terms of the conventional model, assuming a single binding site for polyamines within the channel pore. Taking advantage of a simple linear structure of naturally occurring polyamines, conclusions on a possible architecture of the slow vacuolar channel pore were drawn. The role of common polyamines in regulation of vacuolar ion transport was discussed.

Redox agents regulate ion channel activity in vacuoles from higher plant cells

The ability of redox agents to modulate certain characteristics of voltage- and calcium-activated channels has been recently investigated in a variety of animal cells. We report here the first evidence that redox agents regulate the activation of ion channels in the tonoplast of higher plants. Using the patch-clamp technique, we have demonstrated that, in tonoplasts from the leaves of the marine seagrass Posidonia oceanica and the root of the sugar beet, a variety of sulphydryl reducing agents, added at the cytoplasmic side of the vacuole, reversibly favoured the activation of the voltage-dependent slow vacuolar (SV) channel. Antioxidants, like dithiothreitol (DTT) and the reduced form of glutathione, gave a reversible increase of the voltage-activated current and faster kinetics of channel activation. Other reducing agents, such as ascorbic acid, also increased the SV currents, although to a lesser extent in comparison with DTT and glutathione, while the oxidising agent chloramine-T irreversibly abolished the activity of the channel. Single channel experiments demonstrated that DTT reversibly increased the open probability of the channel, leaving the conductance unaltered. The regulation of channel activation by glutathione may correlate ion transport with other crucial mechanisms that in plants control turgor regulation, response to oxidative stresses, detoxification and resistance to heavy metals.

Signal transduction and ion channels in guard cells.

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity. At the plasmalemma, loss of K+ requires depolarization of the membrane potential into the range at which the outward K+ channel is open. ABA-induced activation of a non-specific cation channel, permeable to Ca2+, may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are activated by Ca2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K+ current at any given membrane potential; this activation is Ca(2+)-independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C. Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca(2+)-activated, have been identified which are capable of K+ efflux; these are the voltage-independent VK channel specific to K+, and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K+ and Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN

Phosphate and slow vacuolar channels in Beta vulgaris

Movement of phosphate through slow vacuolar (SV) ion channels and the effects of phosphate on SV currents were investigated using vacuoles from Beta vulgaris L. When the vacuoles contained 50 mM phosphate, the addition of phosphate to the bath shifted the apparent reversal potential for whole vacuole currents to more positive values, suggesting an outward rectifying current due to movement of phosphate ions out of the vacuole. However absolute values for reversal potentials obtained from the current-voltage curves and tail currents for whole vacuoles suggested that the membranes were relatively impermeable to phosphate. Single-channel data showed that the vacuole preparations contained more than one species of ion channel and therefore the whole-vacuole data will not give definitive information about individual species of ion channels. One of the channels had a single channel reversal potential that indicated a permeability of H2PO4- ions relative to Cl- of 7. The probability of this outwardly rectifying channel being open had a marked dependence on voltage and, in these experiments, it was effectively closed for potentials negative of +20 mV. The single-channel conductance was 19.4 ± 3.1 pS with 50 mM KH2PO4 in the vacuole and 10 mM total phosphate concentration in the bath. A channel with these characteristics has not been reported previously. In addition to the data identifying a phosphate channel, it was found that the presence of phosphate in the bath solution slowed the rate of activation of the SV currents. This effect was partially reversed when phosphate was removed from the bath.

Reversible protein phosphorylation regulates the activity of the slow‐vacuolar ion channel

Protein storage vacuoles (PSVs) within barley (Hordeum vulgare) aleurone cells contain abundant K, Ca, Mg and P reserves. These minerals are transported from the PSV and are used to support growth of the embryo. In this study, the regulation of transport through slow‐vacuolar (SV) ion channels in the tonoplast of barley aleurone PSVs was examined using the patch—clamp technique. Okadaic acid (OA), an inhibitor of protein phosphatase types 1 and 2A, reduced whole‐vacuole SV currents by 60%. This inhibition by OA was overcome by exogenous calcineurin. Adding ATP (200 µM) to the bath solution as a substrate for kinase activity decreased SV channel activity by 70%. This reduction in activity was prevented by the kinase inhibitor H‐7. From these data, it is concluded that protein phosphorylation can inhibit SV channel activity, and that both the protein kinase and protein phosphatase involved in this regulation are present at the PSV tonoplast. Whole‐vacuolar SV currents were significantly higher when 2 mM ATP was used to bathe PSVs than with 200 µM ATP. Calmodulin‐like domain protein kinase (CDPK) at either ATP concentration increased SV channel activity by ∼ 150%, implying that protein phosphorylation can also stimulate SV channel activity. When PSVs were treated with the ATP analog AMP‐PNP, SV channel activity was not reduced. Hence, ATP hydrolysis is not essential for sustained SV channel activity. A model in which SV channel activity is regulated by protein phosphorylation at two sites is presented.

Ion channels in the vacuoles of the seagrass Posidonia oceanica

Voltage-dependent ionic channels were investigated by the patch-clamp technique in the vacuolar membrane from the leaves of the seagrass Posidonia oceanica. Vacuoles extruded from the meristematic white part of the leaves displayed rectifying slow currents which activated in several seconds at positive potentials and deactivated at negative voltages within a few hundreds of ms. Like the Slow Vacuolar (SV) channel already identified in the tonoplast of terrestrial plants, the SV voltage-dependent channel of Posidonia leaves was activated by micromolar concentrations of Ca 2+ and was equally permeable to K + and Na +. The single-channel conductance of the Posidonia SV-type channel was 106±12 pS (in symmetric 400 mM K +). In the same ionic solutions, another channel, occasionally observed in vacuoles from the green part of the leaves, displayed a single-channel conductance of 47±4 pS. To our knowledge, this is the first electrophysiological characterization of ion transport pathways in Posidonia, a marine plant of crucial importance for the ecology of the Mediterranean sea.

Signalling in guard cells and regulation of ion channel activity

A review is presented of the properties of ion channels in plasmalemma and tonoplast of stomatal guard cells, their regulation, with particular reference to Ca(2+) and protein phosphorylation/dephosphorylation, and of the evidence for ABA-induced changes in specific ion channels, with an attempt to identify the signalling chains involved in each such change. A key question is whether a local increase in Ca(2+), close to cell membranes and capable of triggering Ca(2+)-dependent changes in a variety of ion channels, is a universal feature of the ABA-reponse. If this is so, then there exist Ca(2+)-coupled mechanisms for most of the observed changes, including inhibition of the inward K(+) channel and activation of the slow anion channel in the plasmalemma, and activation of two channels in the tonoplast, the K(+)-selective (VK) channel and the slow vacuolar (SV) channel, initiating efflux of both anions and cations from the vacuole. The detailed signalling chains are not complete, and the role of protein phosphorylation/dephosphorylation is not clearly defined, nor linked to ABA. Control of the outward K(+) channel is Ca(2+)-independent; its activation by ABA may be mediated by cytoplasmic alkalinization, but the role of protein dephosphorylation in the signalling chain has still to be clarified. If Ca(2+) is not available as second messenger, then the signalling chains involved have hardly begun to be understood. Detailed comparison of the efflux transients in different conditions provides evidence that ABA changes the 'set-point' of a stretch-activated channel, initiating loss of vacuolar K(+). The inclusion of Ba(2+) in the bathing solution has effects similar to those of reduced ABA concentration, a delay in initiating the vacuolar transient, and a slower rise to a reduced peak height. It is suggested that this could be the result of inhibition of the process of Ca(2+) release from internal stores, by blocking a charge-balancing K(+) flux.

Ionic channels of the sugar beet tonoplast are regulated by a multi-ion single-file permeation mechanism.

Ionic channels of the sugar beet tonoplast were studied using the patch-clamp technique. At micromolar concentrations of cytosolic calcium, several (at least four) distinct single-channel current levels were routinely identified. On the basis of channel voltage dependence, kinetic properties and conductance of single openings, the largest channel (103 +/- 2 pS in symmetric 150 mm KCl) corresponds to the slow vacuolar (SV) channel already identified by Hedrich and Neher (1987). The majority of the whole-vacuole current was ascribed to this time-dependent slow-activating channel elicited by positive vacuolar potentials. The channel of intermediate amplitude (41 +/- 1 pS in 150 mM KCl) did not show any voltage dependence and delay in the activation upon the application of voltage steps to both positive and negative transmembrane potentials. Owing to its voltage independence this channel was denominated FV1. The opening probability of the SV-type channel increased by increasing the cytoplasmic calcium concentration, while the activity of the FV1 channel did not increase appreciably by changing the calcium concentration in the range from 6 microM to 1 mM. All the channels identified showed a linear current-voltage characteristic in the range +/-100 mV and at least the three most conductive ones displayed potassium selectivity properties. Substitution of potassium with tetramethylammonium (TMA) on the cytosolic side demonstrated that both the SV and FV1 channels are impermeable to TMA influx into the vacuole and support the potassium selectivity properties of these two channels. Moreover, the single channel conductances of all the channels identified increased as a function of the potassium concentration and reached a maximum conductivity at [K+] approximately 0.5 M. This behavior can be explained by a multi-ion occupancy single-file permeation mechanism.

Ca2+-Calmodulin Modulates Ion Channel Activity in Storage Protein Vacuoles of Barley Aleurone Cells.

Many plant ion channels have been identified, but little is known about how these transporters are regulated. We have investigated the regulation of a slow vacuolar (SV) ion channel in the tonoplast of barley aleurone storage protein vacuoles (SPV) using the patch-clamp technique. SPV were isolated from barley aleurone protoplasts incubated with CaCl2 in the presence or absence of gibberellic acid (GA) or abscisic acid (ABA). A slowly activating, voltage-dependent ion channel was identified in the SPV membrane. Mean channel conductance was 26 pS when 100 mM KCl was on both sides of the membrane, and reversal potential measurements indicated that most of the current was carried by K+. Treatment of protoplasts with GA3 increased whole-vacuole current density compared to SPV isolated from ABA- or CaCl2-treated cells. The opening of the SV channel was sensitive to cytosolic free Ca2+ concentration ([Ca2+]i) between 600 nM and 100 [mu]M, with higher [Ca2+]i resulting in a greater probability of channel opening. SV channel activity was reduced greater than 90% by the calmodulin (CaM) inhibitors W7 and trifluoperazine, suggesting that Ca2+ activates endogenous CaM tightly associated with the membrane. Exogenous CaM partially reversed the inhibitory effects of W7 on SV channel opening. CaM also sensitized the SV channel to Ca2+. In the presence of ~3.5 [mu]M CaM, specific current increased by approximately threefold at 2.5 [mu]M Ca2+ and by more than 13-fold at 10 [mu]M Ca2+. Since [Ca2+]i and the level of CaM increase in barley aleurone cells following exposure to GA, we suggest that Ca2+ and CaM act as signal transduction elements mediating hormone-induced changes in ion channel activity.