Slow Vacuolar Channel Research Articles

Slow vacuolar channels in vacuoles from winter and spring varieties of rape ( Brassica napus)

Currents passing through slowly activating vacuolar channels (SV) in isolated vacuoles from winter (Górczański) and spring (Młochowski) varieties of rape (Brassica napus) were examined using the patch-clamp technique. Eight-week-long vernalization at 5/2 degrees C (day/night) was applied to obtain the generative stage of winter rape. SV channels of vacuoles isolated from vegetative (rosette) and generative leaves of both varieties were examined in order to investigate a possible role of these ion channels in rape flowering. Single SV channel conductance measured in a vacuole-attached configuration (natural cell sap) ranged from 60 to 83 pS. Lower values were observed in the generative leaves of both varieties. Unitary conductance measured in excised cytoplasm-out membrane patches did not differ significantly among the experimental variants, with the exception of spring generative vacuoles, where it was significantly lower. There was also no difference in SV current densities measured in the whole-vacuole configuration. Gibberellic acid (GA(3)) (2mg/l) caused lowering of macroscopic SV currents by 20%, and had no significant effect on the single channel conductance. We conclude that SV channels play a role in rape vernalization and flowering owing to their multifactor regulation abilities rather than structural changes.

Characteristics of Anion Channels in the Tonoplast of the Liverwort Conocephalum conicum

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses

The putative two-pore Ca(2+) channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K(+)-selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca(2+)-activated slow vacuolar (SV) currents, which were increased in AtTPC1-over-expressing Arabidopsis compared to the wild-type. A Ca(2+)-insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1-2 knockout plants. The kinetics of ABA- and CO(2)-induced stomatal closure were similar in wild-type and tpc1-2 knockout plants, excluding a role of SV channels in guard-cell signalling in response to these physiological stimuli. ABA-, K(+)-, and Ca(2+)-dependent root growth phenotypes were not changed in tpc1-2 compared to wild-type plants. Given the permeability of SV channels to mono- and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca(2+) entry into the cytosol. Ca(2+) responses as measured in aequorin-expressing wild-type, tpc1-2 knockout and TPC1-over-expressing plants disprove a contribution of TPC1 to any of the stimulus-induced Ca(2+) signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca(2+) concentration and biotic factors (elf18, flg22). In good agreement, stimulus- and Ca(2+)-dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization-activated Ca(2+)-permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca(2+) homeostasis.

Vacuolar calcium channels

The central vacuole is the largest Ca2+ store in a mature plant cell. Ca2+ release from this store contributes to Ca2+-mediated intracellular signalling in a variety of physiological responses. However, the routes for vacuolar Ca2+ release are not well characterized. To date, at least two voltage-dependent and two ligand-gated Ca2+-permeable channels have been reported in plant vacuoles. However, the so-called VVCa (vacuolar voltage-gated Ca2+) channel most probably is not a separate channel but is identical to another voltage-dependent channel-the so-called SV (slow vacuolar) channel. Studies in the last few years have added a new dimension to our knowledge of SV channel-mediated ion transport and the mechanisms of its regulation by multiple natural factors. Recently, the SV channel was identified as the product of the TPC1 gene in Arabidopsis. In contrast, the TPC1 channel from other species was thought to be localized in the plasma membrane. A re-evaluation of this work under the assumption that the TPC1 channel is generally a vacuolar channel provides interesting insights into the physiological function of the TPC1/SV channel. Considerably less is known about vacuolar Ca2+ channels that are supposed to be activated by inositol 1,4,5-trisphosphate or cADP ribose. The major problems are controversial reports about functional characteristics, and a remarkable lack of homologues of animal ligand-gated Ca2+ channels in higher plants. To help understand Ca2+-mediated intracellular signalling in plant cells, a critical update of existing experimental evidence for vacuolar Ca2+ channels is presented.

Effects of Cu2+ on characteristic of SV currents

Applying the patch-clamp technique to vacuoles from Radish we studied the effects of Cu 2+ on Slow Vacuolar (SV) current’s characteristic. Our results show that Cu 2+ in bath solution at higher concentration inhibits SV currents and the percentage of inhibition increases with increasing concentration and changes with different voltage. When at lower concentration, Cu 2+ significantly promotes the SV currents and the promotion ratio decrease with increasing voltage. At the same time, the time constants of activation become lesser after adding Cu 2+ . These results show that there may be some Cu 2+ binding sites on SV channels and binding to which can change SV current’s characteristic.

The effect of dihydroquercetin on slow vacuolar channels

The effect of dihydroquercetin on slow vacuolar channels

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

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.

Effects of BaCl2 on slow vacuolar ion channels on radish by patch-clamp

The effects of BaCl 2 on slow vacuolar (SV) currents of radish are studied by using the whole-vacuolar patch-clamp recording mode. The Ca 2+ -dependent SV channel can be activated by cytosolic Ca 2+ . When 1 mmol/L BaCl 2 is added into pipette solution, SV currents are suppressed remarkably. Then adding BaCl 2 of different concentrations into the bath solution, SV currents reflect different effects. The results show that BaCl 2 with a lower concentration (＜ 3 mmol/L) promotes the channel currents and the currents are saturated when BaCl 2 concentrations are between 1 mmol/L and 1 mmol/L, but BaCl 2 with higher concentration (≥ 3 mmol/L) inhibits SV currents.

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.

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.