Polyamines interact with hydroxyl radicals in activating Ca(2+) and K(+) transport across the root epidermal plasma membranes.

Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca(2+) dynamics by activating a range of nonselective Ca(2+)-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K(+) and Ca(2+) fluxes induced by hydroxyl radicals (OH(•)) in pea (Pisum sativum) roots. OH(•), but not hydrogen peroxide, activated a rapid Ca(2+) efflux and a more slowly developing net Ca(2+) influx concurrent with a net K(+) efflux. In isolated protoplasts, OH(•) evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K(+) efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca(2+). Active OH(•)-induced Ca(2+) efflux in roots was suppressed by the PM Ca(2+) pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH(•)-induced ionic currents in root protoplasts and K(+) efflux and Ca(2+) influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH(•)-induced cation fluxes. The net OH(•)-induced Ca(2+) efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH(•)-induced net K(+) efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca(2+) homeostasis by modulating both Ca(2+) influx and efflux transport systems at the root cell PM.

Calcium efflux systems in stress signaling and adaptation in plants.

Transient cytosolic calcium ([Ca(2+)](cyt)) elevation is an ubiquitous denominator of the signaling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca(2+)](cyt) elevations vary in magnitude, frequency, and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium "signature" that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca(2+) influx mechanisms to shaping [Ca(2+)](cyt) signatures, restoration of the basal [Ca(2+)](cyt) levels is impossible without both cytosolic Ca(2+) buffering and efficient Ca(2+) efflux mechanisms removing excess Ca(2+) from cytosol, to reload Ca(2+) stores and to terminate Ca(2+) signaling. This is the topic of the current review. The molecular identity of two major types of Ca(2+) efflux systems, Ca(2+)-ATPase pumps and Ca(2+)/H(+) exchangers, is described, and their regulatory modes are analyzed in detail. The spatial and temporal organization of calcium signaling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca(2+) efflux systems in plant responses to a range of abiotic and biotic factors is summarized. Contribution of Ca(2+)-ATPase pumps and Ca(2+)/H(+) exchangers in shaping [Ca(2+)](cyt) signatures is then modeled by using a four-component model (plasma- and endo-membrane-based Ca(2+)-permeable channels and efflux systems) taking into account the cytosolic Ca(2+) buffering. It is concluded that physiologically relevant variations in the activity of Ca(2+)-ATPase pumps and Ca(2+)/H(+) exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca(2+)](cyt) signatures in response to environmental stimuli, emphasizing the crucial role these active efflux systems play in plant adaptive responses to environment.

Infection by Trypanosoma cruzi enhances anion conductance in rat neonatal ventricular cardiomyocytes.

Recent studies on malaria-infected erythrocytes have shown increased anion channel activity in the host cell membrane, increasing the exchange of solutes between the cytoplasm and exterior. In the present work, we addressed the question of whether another intracellular protozoan parasite, Trypanosoma cruzi, alters membrane transport systems in the host cardiac cell. Neonatal rat cardiomyocytes were cultured and infected with T. cruzi in vitro. Ion currents were measured by patch-clamp technique in the whole-cell configuration. Two small-magnitude instantaneous anion currents, outward- and inward-rectifying, were recorded in all noninfected cardiomyocytes. In addition, ~10% of cardiomyocytes expressed a large anion-preferable, time-dependent current activated at positive membrane potentials. Hypotonic (230 mOsm) treatment resulted in the disappearance of the time-dependent current but provoked a dramatic increase of the instantaneous outward-rectifying one. Both instantaneous currents were suppressed by intracellular Mg(2+). T. cruzi infection did not provoke new anion currents in the host cells but caused an increase of the density of intrinsic swelling-activated outward current, up to twice in heavily infected cells. The occurrence of a time-dependent current dramatically increased in infected cells in the presence of Mg(2+) in the intracellular solution, from ~10 to ~80%, without a significant change of the current density. Our findings represent one further, besides the known Plasmodium falciparum, example of an intracellular parasite which upregulates the anionic currents expressed in the host cell.

K(bg) and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes.

Microelectrode ion flux estimation (MIFE) and patch-clamp techniques were combined for noninvasive K(+) flux measurements and recording of activities of the dominant K(+) channels in the early phases of apoptosis in Jurkat cells. Staurosporine (STS, 1 microM) evoked rapid (peaking around 15 min) transient K(+) efflux, which then gradually decreased. This transient K(+) efflux occurred concurrently with the transient increase of the K(+) background (K(bg)) TWIK-related spinal cord K(+) channel-like current density, followed by a drastic decrease and concomitant membrane depolarization. The Kv1.3 current density remained almost constant. Kv1.3 activation was not altered by STS, whereas the inactivation was shifted to more positive potentials. Contribution of K(bg) and Kv1.3 channels to the transient and posttransient STS-induced K(+) efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking K(bg) current, and the Kv1.3-specific blocker margatoxin. Channel-mediated K(+) efflux provoked a substantial cellular shrinkage and affected the activation of caspases.

TRESK-like potassium channels in leukemic T cells.

In this study, we present patch-clamp characterization of the background potassium current in human lymphoma (Jurkat cells), generated by voltage-independent 16 pS channels with a high ( approximately 100-fold) K+/Na+ selectivity. Depending on the background K+ channels density, from few per cell up to approximately 1 open channel per microm2, resting membrane potential was in the range of -40 to -83 mV, approaching E (K) = -88 mV. The background K+ channels were insensitive to margotoxin (3 nM), apamine (3 nM), and clotrimazole (1 microM), high-affinity blockers of the lymphocyte Kv1.3, SKCa2, and IKCa1 channels. The current depended weakly on external pH. Arachidonic acid (20 microM) and Hg2+ (0.3-10 microM) suppressed background K+ current in Jurkat cells by 75-90%. Background K+ current was weakly sensitive to TEA+ (IC50 = 14 mM), and was efficiently suppressed by externally applied bupivacaine (IC50 = 5 microM), quinine (IC50 = 16 microM), and Ba2+ (2 mM). Our data, in particular strong inhibition by mercuric ions, suggest that background K+ currents expressed in Jurkat cells are mediated by TWIK-related spinal cord K+ (TRESK) channels belonging to the double-pore domain K+ channel family. The presence of human TRESK in the membrane protein fraction was confirmed by Western blot analysis.

Patch clamp characterization of a non-selective cation channel of ER membranes purified from Beta vulgaris taproots.

The ER fraction from red beet taproot was purified on sucrose gradient and giant liposomes, suitable for patch clamping, were formed by dehydration-rehydration of the lipid film. Single-channel recordings on excised and attached patches revealed a large conductance (165 pS) cation (P(Cl-)/P(K+) < 0.03) channel with equal conductance and relative permeability for Na+ and K+. This non-selective cation channel was also highly permeable for Ca2+. We failed to detect any single-channel currents activated by a direct application of d-myo-inositol 1,4,5 trisphosphate, despite the fact that the ER membranes were native.

Na-K transport in roots under salt stress.

Salinity causes billion dollar losses in annual crop production. So far, the main avenue in breeding crops for salt tolerance has been to reduce Na(+) uptake and transport from roots to shoots. Recently we have demonstrated that retention of cytosolic K(+) could be considered as another key factor in conferring salt tolerance in plants. A subsequent study has shown that Na(+)-induced K(+) efflux in barley root epidermis occurs primarily via outward rectifying K(+) channels (KORC). Surprisingly, expression of KORC was similar in salt- tolerant and sensitive genotypes. However, the former were able to better oppose Na(+)-induced depolarization via enhanced activity of plasma membrane H(+)-ATPase (thus minimizing K(+) leak from the cytosol). In addition to highly K(+)-selective KORC channels, activities of several types of non-selective cation channels were detected at depolarizing potentials. Here we show that the expression of one of them, NORC, was significantly lower in salt-tolerant genotypes. As NORC is capable of mediating K(+) efflux coupled to Na(+) influx, we suggest that the restriction of its activity could be beneficial for plants under salt stress.

Root plasma membrane transporters controlling K+/Na+ homeostasis in salt-stressed barley.

Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H(+) pump activity; (3) better ability of root cells to pump Na(+) from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca(2+). At the same time, no significant difference was found between contrasting cultivars in their unidirectional (22)Na(+) influx or in the density and voltage dependence of depolarization-activated outward-rectifying K(+) channels. Overall, our results are consistent with the idea of the cytosolic K(+)-to-Na(+) ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.

Methyl-beta-cyclodextrin reversibly alters the gating of lipid rafts-associated Kv1.3 channels in Jurkat T lymphocytes.

The voltage-dependent Kv1.3 potassium channels mediate a variety of physiological functions in human T lymphocytes. These channels, along with their multiple regulatory components, are localized in cholesterol-enriched microdomains of plasma membrane (lipid rafts). In this study, patch-clamp technique was applied to explore the impact of the lipid-raft integrity on the Kv1.3 channel functional characteristics. T lymphoma Jurkat cells were treated for 1 h with cholesterol-binding oligosaccharide methyl-beta-cyclodextrin (MbetaCD) in 1 or 2 mM concentration, resulting in depletion of cholesterol by 63 +/- 5 or 75 +/- 4%, respectively. Treatment with 2 mM MbetaCD did not affect the cells viability but retarded the cell proliferation. The latter treatment caused a depolarizing shift of the Kv1.3 channel activation and inactivation by 11 and 6 mV, respectively, and more than twofold decrease in the steady-state activity at depolarizing potentials. Altogether, these changes underlie the depolarization of membrane potential, recorded in a current-clamp mode. The effects of MbetaCD were concentration- and time-dependent and reversible. Both development and recovery of the MbetaCD effects were completed within 1-2 h. Therefore, cholesterol depletion causes significant alterations in the Kv1.3 channel function, whereas T cells possess a potential to reverse these changes.

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.

Potassium-selective channel in the red beet vacuolar membrane.

In higher plants the vacuolar K(+)-selective (VK) channel was identified solely in guard cells. This patch-clamp study describes a 40 pS homologue of the VK channel in Beta vulgaris taproot vacuoles. This voltage-independent channel is activated by submicromolar Ca(2+), and is ideally selective for K(+) over Cl(-) and Na(+).

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 sigma = 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 approximately 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.