A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.     

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to?cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast.     

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to␣cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast. Received: 17 August 1998 / Accepted: 7 December 1998     

Mechanical stress-evoked but angiotensin II-independent activation of angiotensin II type 1 receptor induces cardiac hypertrophy through calcineurin pathway

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT₁) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca²⁺-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT₁ receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT₁ receptor and siRNA of calcineurin. Mechanical stresses for 2 weeks in vivo and for 24 h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG^−/−) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG^−/− mice. Furthermore, an AT₁ receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT₁ receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT₁ receptor induces cardiac hypertrophy through calcineurin pathway.     

Chloride Channel Function in the Yeast TRK-Potassium Transporters

The TRK proteins—Trk1p and Trk2p— are the main agents responsible for “active” accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration—qualitatively as expected for H⁺ coupling to K⁺ uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K⁺ or H⁺, but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K⁺-dependent and can be enhanced by K⁺ starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K_0.5 at 46 μM [K⁺]_out and 6.8 mM [K⁺]_out, and saturating fluxes of ∼5 mM/min and ∼10 mM/min (referred to intracellular water). These numbers are compatible with the normal K⁺-transport properties of Trk1p and Trk2p, respectively.This revised version was published online in August 2005 with a corrected cover date.     

A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5

A chimeric CaHAK1–LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K⁺ uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K⁺ uptake shown by K⁺-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K⁺ uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K⁺ uptake was not correlated with the root K⁺ content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K⁺ starvation or growth in the presence of NH₄ ⁺, but which do not decrease the K⁺ content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K⁺ starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K⁺ transporter.     

The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter

Protein kinases dedicated to the phosphorylation of SR proteins have been implicated in the processing and nuclear export of mRNAs. Here we demonstrate in Saccharomyces cerevisiae their participation in cation homeostasis. A null mutant of the single yeast SR protein kinase Sky1p is viable but exhibits increased tolerance to diverse toxic cations such as Na⁺, Li⁺, spermine, tetramethylammonium, hygromycin B and Mn²⁺. This pleiotropic phenotype correlates with reduced accumulation of cations, suggesting a decrease in membrane electrical potential. Genetic analysis and Rb⁺ uptake measurements indicate that Sky1p modulates Trk1,2, the high-affinity K⁺ uptake system of yeast and a major determinant of membrane potential.     

Arabidopsis thaliana vacuolar TPK channels form functional K+ uptake pathways in Escherichia coli

Very few vacuolar two pore potassium channels (TPKs) have been functionally characterized. In this paper we have used complementation of K⁺ uptake deficient Escherichia coli mutant LB2003 to analyze the functional properties of Arabidopsis thaliana TPK family members. The four isoforms of AtTPKs were cloned and expressed in LB2003 E. coli background.The expression of channels in bacteria was analyzed by RT-PCR. Our results show that AtTPK1, AtTPK2 and AtTPK5 are restoring the LB2003 growth on low K⁺ media. The analysis of potassium uptake exhibited elevated level of K⁺ uptake in the same three types of AtTPKs transformants.     

Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: A possible link between the nervous system and atherosclerosis development

Objective

The parasympathetic nervous system regulates inflammation in peripheral tissues through a pathway termed the “cholinergic anti-inflammatory reflex” (CAIR). Mice deficient in the alpha 7 nicotinic acetylcholine receptor (α7^−/−) have an impaired CAIR due to decreased signaling through this pathway. The purpose of this study was to determine if the increased inflammation in α7^−/− mice is associated with enhanced serum and macrophage atherogenicity.

Methods

We measured serum markers of inflammation and oxidative stress, and macrophage atherogenicity in mouse peritoneal macrophages harvested from α7^−/− mice on the background of C57BL/6 mice, as well as on the background of the atherosclerotic Apolipoprotein E-deficient (ApoE^−/−) mice.

Results

α7-Deficiency had no significant effects on serum cholesterol, or on markers of serum oxidative stress (TBARS and paraoxonase1 activities). However, α7-deficiency significantly increased serum CRP and IL-6 (p < 0.05) levels in atherosclerotic mice, confirming an anti-inflammatory role for the α7 receptor. Macrophage cholesterol mass was increased by 25% in both normal and atherosclerotic mice in the absence of the α7 receptor (p < 0.05). This was accompanied by conditional increases in oxidized LDL uptake and in macrophage total peroxide levels. Furthermore, α7-deficiency reduced macrophage paraoxonase2 mRNA and activity by 50-100% in normal and atherosclerotic mice (p < 0.05 for each), indicating a reduction in macrophage anti-oxidant capacity in the α7^−/− mice.

Conclusion

The above results suggest an anti-atherogenic role for the macrophage α7nAchr, through a mechanism that involves attenuated macrophage oxidative stress and decreased uptake of oxidized LDL.     

Complexity of potassium acquisition: How much flows through channels?

The involvement of potassium (K⁺)-selective, Shaker-type channels, particularly AKT1, in primary K⁺ acquisition in roots of higher plants has long been of interest, particularly in the context of low-affinity K⁺ uptake, at high K⁺ concentrations, as well as uptake from low-K⁺ media under ammonium (NH₄⁺) stress. We recently demonstrated that K⁺ channels cannot mediate K⁺ acquisition in roots of intact barley (Hordeum vulgare L.) seedlings at low (22.5 µM) external K⁺ concentrations ([K⁺]_ext) and in the presence of high (10 mM) external NH₄⁺, while the model species Arabidopsis thaliana L. utilizes channels under comparable conditions. However, when external NH₄⁺ was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K⁺ influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K⁺ influx, illustrating a hitherto unexplored magnitude and rapidity of K⁺-uptake capacity and plasticity. Here, we expand on our previous work by offering further characterization of channel-mediated K⁺ fluxes in intact barley, with particular focus on anion effects, root respiration and pharmacological sensitivity and highlight key additions to the current model of K⁺ acquisition.     

Adaptation of plants to salt stress: the role of the ion transporters

Adaptation to high salinity is achieved by cellular ion homeostasis which involves regulation of toxic sodium ion (Na⁺) and Chloride ion (Cl⁻) uptake, preventing the transport of these ions to the aerial parts of the plants and vacuolar sequestration of these toxic ions. Ion transporters have long been known to play roles in maintaining ion homeostasis. Na⁺ enters the cell through various voltage dependent selective and non-selective ion channels. High Na⁺ concentration in the plasma membrane is balanced either by uptake of potassium ion (K⁺) by various potassium importing channels, by salt exclusion mechanism or by sequestration of Na⁺ in the vacuoles. Therefore, the role of high-affinity potassium transporter, the salt overly sensitive pathway, the most well-defined Na⁺ exclusion pathway that exports Na⁺ from cell into xylem and tonoplast localized cation transporters that compartmentalizes Na⁺ in vacuoles need to be studied in detail and applied to make the plant adaptable to saline soil. Knowledge on the regulation of expression of these transporters by the hormones, microRNAs and other non-coding RNAs can be utilized to manipulate the ion transport. Here, we reviewed paradigm of the ion transporters in salt stress signalling pathways from the recent and past studies aiding transformation of basic knowledge into biotechnological applications to generate engineered salt stress tolerant crops.

     

Nitrogen uptake responses of Gracilaria vermiculophylla (Ohmi) Papenfuss under combined and single addition of nitrate and ammonium

The ammonium (NH₄⁺) and nitrate (NO₃⁻) uptake responses of tetrasporophyte cultures from a Portuguese population of Gracilaria vermiculophylla were studied. Thalli were incubated at 5 nitrogen (N) levels, including single (50 μM of NH₄⁺ or NO₃⁻) and combined addition of each of the N sources. For the combined additions, the experimental conditions attempted to simulate 2 environments with high N availability (450 μM NO₃⁻ + 150 μM NH₄⁺; 250 μM NO₃⁻ + 50 μM NH₄⁺) and the mean N concentrations occurring at the estuarine environment of this population (30 μM NO₃⁻ + 5 μM NH₄⁺). The uptake kinetics of NH₄⁺ and NO₃⁻ were determined during a 4 h time-course experiment with N deprived algae. The experiment was continued up to 48 h, with media exchanges every 4 h. The uptake rates and efficiency of the two N sources were calculated for each time interval. For the first 4 h, G. vermiculophylla exhibited non-saturated uptake for both N sources even for the highest concentrations used. The uptake rates and efficiency calculated for that period (V_0-4 h), respectively, increased and decreased with increasing substrate concentration. NO₃⁻ uptake rates were superior, ranging from 1.06 ± 0.1 to 9.65 ± 1.2 μM g(dw)⁻¹ h⁻¹, with efficiencies of 19% to 53%. NH₄⁺ uptake rates were lower (0.32 ± 0.0 to 5.75 ± 0.08 μM g(dw)⁻¹ h⁻¹) but G. vermiculophylla removed 63% of the initial 150 μM and 100% at all other conditions. Uptake performance of both N sources decreased throughout the duration of the experiment and with N tissue accumulation. Both N sources were taken up during dark periods though with better results for NH₄⁺. Gracilaria vermiculophylla was unable to take up NO₃⁻ at the highest concentration but compensated with a constant 27% NH₄⁺ uptake through light and dark periods. N tissue accumulation was maximal at the highest N concentration (3.9 ± 0.25% dw) and superior under NH₄⁺ (3.57 ± 0.2% dw) vs NO3 (3.06 ± 0.1% dw) enrichment. The successful proliferation of G. vermiculophylla in estuarine environments and its potential utilization as the biofilter component of Integrated Multi-Trophic Aquaculture (IMTA) are discussed.     

Expressing the yeast HAL1 gene in tomato increases fruit yield and enhances K+/Na+ selectivity under salt stress

The yeast HAL1 gene facilitates K⁺/Na⁺ selectivity and salt tolerance of cells. Ectopic expression of HAL1 in transgenic tomato (Lycopersicon esculentum Mill.) plants minimized the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K⁺ contents in both leaf calli and leaves in the presence of salt, which indicates that HAL1 functions in plants using a similar mechanism to that in yeast, namely by facilitating K⁺/Na⁺ selectivity under salt stress.     

K homeostasis and central pattern generation in the metathoracic ganglion of the locust

Stress-induced arrest of ventilatory motor pattern generation is tightly correlated with an abrupt increase in extracellular potassium concentration ([K⁺]_o) within the metathoracic neuropil of the locust, Locusta migratoria. Na⁺/K⁺-ATPase inhibition with ouabain elicits repetitive surges of [K⁺]_o that coincide with arrest and recovery of motor activity. Here we show that ouabain induces repetitive [K⁺]_o events in a concentration-dependent manner. 10⁻⁵ M, 10⁻⁴ M, and 10⁻³ M ouabain was bath-applied in semi-intact locust preparations. 10⁻⁴ M and 10⁻³ M ouabain reliably induced repetitive [K⁺]_o events whereas 10⁻⁵ M ouabain had no significant effect. In comparison to 10⁻⁴ M ouabain, 10⁻³ M ouabain increased the number and hastened the time to onset of repetitive [K⁺]_o waves, prolonged [K⁺]_o event duration, increased resting [K⁺]_o, and diminished the absolute value of [K⁺]_o waves. Recovery of motor patterning following [K⁺]_o events was less likely in 10⁻³ M ouabain. In addition, we show that K⁺ channel inhibition using TEA suppressed the onset and decreased the amplitude of ouabain-induced repetitive [K⁺]_o waves. Our results demonstrate that ventilatory circuit function in the locust CNS is dependent on the balance between mechanisms of [K⁺] accumulation and [K⁺] clearance. We suggest that with an imbalance in favour of accumulation the system tends towards a bistable state with transitions mediated by positive feedback involving voltage-dependent K⁺ channels.     

Potassium Translocation into the Root Xylem

Abstract: Potassium is the most abundant cation in cells of higher plants and plays vital roles in plant growth and develop ment. Since the soil is the only source of potassium, plant roots are well adapted to exploit the soil for potassium and supply it to the leaves. Transport across the root can be divided into three stages: uptake into the root symplast, transport across the symplast and release into the xylem. Uptake kinetics of potassium have been studied extensively in the past and sug gested the presence of high and low affinity systems. Molecular and electrophysiological techniques have now confirmed the existence of discrete transporters encoded by a number of genes. Surprisingly, detailed characterisation of the transpor ters using reverse genetics and heterologous expression shows that a number of the transporters (AKT and AtKUP family) func tion both in the low (μM) and high (mM) K⁺ range. Electrophy siological studies indicate that K⁺ uptake by roots is coupled to H⁺, to drive uptake from micromolar K⁺. However, thus far only Na⁺ coupled K⁺ transport has been demonstrated (HKT1). Ion channels play a major role in the exchange of potassium be tween the symplast and the xylem. An outward rectifying chan nel (KORC) mediates potassium release. Cloning of the gene en coding this channel (SKOR) shows that it belongs to the Shaker super-family. Both electrophysiological and genetic studies demonstrate that K⁺ release through this channel is controlled by the stress hormone abscisic acid. Interestingly, xylem par enchyma cells of young barley roots also contain a number of in ward rectifying K⁺ channels that are controlled by G-proteins. The involvement of G-proteins emphasises once more that po tassium transport at the symplast/xylem boundary is under hor monal control. The role of the electrical potential difference across the symplastxylem boundary in controlling potassium release is discussed.