Halophyte species implement a "salt-including" strategy, sequestering significant amounts of Na + to cell vacuoles. This requires a reduction of passive Na + leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na + -permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na + accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na + exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na + sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na + , this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K + than for Na + , and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mM NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2-and 7-fold lower in older compared with young leaves in control-and saltgrown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na + leak, thus enabling efficient sequestration of Na + to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species.The increasing problem of global land salinization (Flowers, 2004;Rengasamy, 2006) and its associated multibillion dollar losses in agricultural production require a better understanding of the key physiological mechanisms that confer salinity tolerance in crops. One effective way of gaining such knowledge comes from studying halophytes (Glenn et al., 1999;Flowers and Colmer, 2008;Shabala and Mackay, 2011).One of the prominent features of halophytes is their ability to efficiently sequester cytosolically toxic Na + to the cell vacuole. The classic view is that this sequestration is achieved by tonoplast Na + /H + antiporters (Barkla et al., 1995;Flowers and Colmer, 2008), a process energized by both vacuolar H + pumps: ATPase (Ayala et al., 1996;Vera-Estrella et al., 1999;Wang et al., 2001) and pyrophosphatase (Parks et al., 2002;Vera-Estrella et al., 2005;Guo et al., 2006;Krebs et al., 2010). However, recent studies have added more complexity to the relationship between Na + /H + antiporters and vacuolar Na + sequestration, assigning a role to the transporter in the regulation of K + and H + homeostasis (for review, see Rodríguez-Rosales et al., 2009;Jiang et al., 2010; Bassil et al., 2011 (Yamaguchi et al., 2005). Consequently, other transporters, in addition to and different from NHX, are likely to be involved in vacuolar Na + sequestration. In addition...

Halophytes species can be used as a highly convenient model system to reveal key ionic and molecular mechanisms that confer salinity tolerance in plants. Earlier, we reported that quinoa (Chenopodium quinoa Willd.), a facultative C3 halophyte species, can efficiently control the activity of slow (SV) and fast (FV) tonoplast channels to match specific growth conditions by ensuring that most of accumulated Na+ is safely locked in the vacuole (Bonales-Alatorre et al. (2013) Plant Physiology). This work extends these finding by comparing the properties of tonoplast FV and SV channels in two quinoa genotypes contrasting in their salinity tolerance. The work is complemented by studies of the kinetics of net ion fluxes across the plasma membrane of quinoa leaf mesophyll tissue. Our results suggest that multiple mechanisms contribute towards genotypic differences in salinity tolerance in quinoa. These include: (i) a higher rate of Na+ exclusion from leaf mesophyll; (ii) maintenance of low cytosolic Na+ levels; (iii) better K+ retention in the leaf mesophyll; (iv) a high rate of H+ pumping, which increases the ability of mesophyll cells to restore their membrane potential; and (v) the ability to reduce the activity of SV and FV channels under saline conditions. These mechanisms appear to be highly orchestrated, thus enabling the remarkable overall salinity tolerance of quinoa species.

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 μM) 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 (Kbg) 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 Kbg and Kv1.3 channels to the transient and posttransient STS-induced K+ efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking Kbg current, and the Kv1.3-specific blocker margatoxin. Channel-mediated K+ efflux provoked a substantial cellular shrinkage and affected the activation of caspases.

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.

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²⁺ and Mg²⁺. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³⁺ (10 μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 μM) and SKF 96365 (250 μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²⁺ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.

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.

Efforts to breed salt tolerant crops could benefit from investigating previously unexplored traits. One of them is a tissue succulency. In this work, we have undertaken an electrophysiological and biochemical comparison of properties of mesophyll and storage parenchyma leaf tissues of a succulent halophyte species Carpobrotus rosii ("pigface"). We show that storage parenchyma cells of C. rossii act as Na sink and possessed both higher Na sequestration (298 vs. 215 mM NaCl in mesophyll) and better K retention ability. The latter traits was determined by the higher rate of H -ATPase operation and higher nonenzymatic antioxidant activity in this tissue. Na uptake in both tissues was insensitive to either Gd or elevated Ca ruling out involvement of nonselective cation channels as a major path for Na entry. Patch-clamp experiments have revealed that Caprobrotus plants were capable to downregulate activity of fast vacuolar channels when exposed to saline environment; this ability was higher in the storage parenchyma cells compared with mesophyll. Also, storage parenchyma cells have constitutively lower number of open slow vacuolar channels, whereas in mesophyll, this suppression was inducible by salt. Taken together, these results provide a mechanistic basis for efficient Na sequestration in the succulent leaf tissues.

a b s t r a c tActivity 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.