Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed.

SV channels dominate the vacuolar Ca2+ release during intracellular signaling.

Vacuoles have long been suggested to mediate a rise in the cytosolic free Ca(2+) during environmental signal transduction. This study addresses the issue of the control of vacuolar calcium release by some of the known signaling molecules such as IP(3), cADPR, ABA, ATP, cAMP, cGMP, H(2)O(2) and CaM. Over 30 concentrations and/or combinations of these signaling compounds were studied in a series of electrophysiological experiments involving non-invasive ion flux measurements (the MIFE) and patch-clamp techniques. Our results suggest that calcium, calmodulin and nucleotides cause calcium release via SV channels.

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.

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.

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.

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.

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(+).