An osmotically induced cytosolic Ca2+ transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance of Saccharomyces cerevisiae

Hyperosmotic stress caused by NaCl, LiCl, or sorbitol induces an immediate and short duration ( approximately 1 min) transient cytosolic Ca(2+) ([Ca(2+)](cyt)) increase (Ca(2+)-dependent aequorin luminescence) in Saccharomyces cerevisiae cells. The amplitude of the osmotically induced [Ca(2+)](cyt) transient was attenuated by the addition of chelating agents EGTA or BAPTA, cation channel pore blockers, competitive inhibitors of Ca(2+) transport, or mutations (cch1Delta or mid1Delta) that reduce Ca(2+) influx, indicating that Ca(ext)(2+) is a source for the transient. An osmotic pretreatment (30 min) administered by inoculating cells into media supplemented with either NaCl (0.4 or 0.5 m) or sorbitol (0.8 or 1.0 m) enhanced the subsequent growth of these cells in media containing 1 m NaCl or 2 m sorbitol. Inclusion of EGTA in the osmotic pretreatment media or the cch1Delta mutation reduced cellular capacity for NaCl but not hyperosmotic adaptation. The stress-adaptive effect of hyperosmotic pretreatment was mimicked by exposing cells briefly to 20 mm CaCl(2). Thus, NaCl- or sorbitol-induced hyperosmotic shock causes a [Ca(2+)](cyt) transient that is facilitated by Ca(2+) influx, which enhances ionic but not osmotic stress adaptation. NaCl-induced ENA1 expression was inhibited by EGTA, cch1Delta mutation, and FK506, indicating that the [Ca(2+)](cyt) transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance.     

Molecular cloning of cDNA coding for kidney aldose reductase. Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress

Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.     

Changes in phosphatidylinositol metabolism in response to hyperosmotic stress in Daucus carota L. cells grown in suspension culture.

Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeled PIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (PI) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.     

Expression Analysis of Proline Metabolism-related Genes From Halophyte Arabis stelleri under Osmotic Stress Conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.     

Expression analysis of proline metabolism-related genes from halophyte Arabis stelleri under osmotic stress conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.     

Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12

Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift.     

Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.     

The externally derived portion of the hyperosmotic shock-activated cytosolic calcium pulse mediates adaptation to ionic stress in suspension-cultured tobacco cells

The influx of Ca(2+) into the cytosol has long been suggested to serve as a signaling intermediate in the acquisition of tolerance to hyperosmotic and/or salinity stresses. Here we use aequorin-transformed suspension-cultured tobacco cells to directly assess the role of cytosolic calcium (Ca(2+)(cyt)) signaling in salinity tolerance acquisition. Aequorin luminescence recordings and (45)Ca influx measurements using inhibitors of Ca(2+) influx (Gd(3+) and the Ca(2+)-selective chelator EGTA), and modulators of organellar Ca(2+) release (phospholipase C inhibitors U73122 or neomycin) demonstrate that hyperosmolarity, whether imposed by NaCl or by a non-ionic molecule sorbitol, induces a rapid (returning to baseline levels of Ca(2+) within 10 min) and complex Ca(2+)(cyt) pulse in tobacco cells, deriving both from Gd(3+)-sensitive externally derived Ca(2+) influx and from U73122- and neomycin-sensitive Ca(2+) release from an organelle. To determine whether each of the two components of this brief Ca(2+) signal regulate adaptation to hyperosmotic shock, the Ca(2+) pulse was modified by the addition of Gd(3+), U73122, neomycin, or excess Ca(2+), and then cells were treated with salt or sorbitol. After 10 min the cell culture medias were diluted with additional hyperosmotic media to reduce the toxic affects of the modulators, and the growth of cells was measured after 1 week. Gd(3+) treatment reduced growth in salt relative to control cells but not in sorbitol, and exposure to excess Ca(2+) increased growth in salt but not in sorbitol. In contrast, exposure to inhibitors of IP(3) formation had no effect on growth in salt or sorbitol. Therefore, although hyperosmotic treatment stimulates both Ca(2+) influx and Ca(2+) release from an internal Ca(2+) depot, only Ca(2+) influx has a measurable impact on ionic stress tolerance acquisition in tobacco cell suspensions. In contrast, osmoadaptation in these cells appears to occur independent of Ca(2+) signaling.     

Hyperosmotic stress signaling to the nucleus disrupts the Ran gradient and the production of RanGTP

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10-20 min) and late (30-60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response.     

Protein patterns,osmolytes, and aldose reductase of L-929 cells exposed to hyperosmotic media

L-929 cells acclimated to media made hyperosmotic (600 mosmol/kgH₂O) by addition of NaCl, sorbitol, or mannitol show, on SDS-polyacrylamide gels, a markedly enhanced protein band at 40 kDa, most likely corresponding to the enzyme aldose reductase. The effect was not observed in cells acclimated to a medium rendered hyperosmotic by addition of proline. The major organic osmolyte accumulated is sorbitol in cells acclimated to high-sorbitol or high-NaCl medium, proline in cells acclimated to high-proline medium. Cells acclimated to any of these hyperosmotic media display unaltered Na⁺ levels and similarly increased K⁺ levels and decreased Cl⁻ levels. These results are interpreted in terms of the mechanisms involved in aldose reductase induction and in regulation of the enzyme activity in long-term acclimation to hyperosmotic media. © 1996 Wiley-Liss, Inc.     

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 μM) or higher (150 μM) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 μM choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K_m's with different affinities.The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.     

Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover

In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus.     

Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks, and the relation to toxicity

Substantial increases in chromosome aberrations were induced in Chinese hamster ovary cells by medium made hyperosmotic with NaCl, KCl, sucrose, sorbitol or dimethyl methylphosphonate. The increases were associated with cytotoxicity but occurred in the range (e.g., 70% survival) commonly included in in vitro tests for 'genotoxicity'. The relation between increased osmotic pressure and chromosome aberrations is compound-dependent, e.g., some compounds may have a direct effect in addition to an effect mediated by osmotic pressure/ionic strength. Also, glycerol at high osmolality was not toxic and did not induce aberrations, probably because rapid equilibration across the cell membrane precluded severe osmotic stress to the cells. Weak increases in DNA single-strand breaks (NaCl and KCl) and double-strand breaks (NaCl) were also detectable, at higher concentrations and more toxic levels than those required to produce aberrations. Slight elevations in sister-chromatid exchange frequencies caused by hyperosmotic medium were found in the presence of toxicity and severe cell cycle delay. Our data on cell growth inhibition suggest that this is the result of increased incorporation of bromodeoxyuridine per cell due to decreased numbers of growing cells, although other mechanisms cannot be ruled out. The observations on chromosome aberrations demonstrate the need for keeping in vitro test conditions in the physiological range, and provide a means for investigation of indirect DNA damage.     

The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation

The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 microM choline, washed, resuspended, and then incubated for 10 min in choline-free buffer containing 4.74 (Krebs-Ringer bicarbonate, KRB) or 25 mM KCl. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 microM choline did not lead to a further increase, but preincubation with 25 or 50 microM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 microM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 mM KCl, ACh release from slices preincubated with 50 microM choline was greater than from slices preincubated with 0, 10, or 25 microM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KCl, the ACh content of slices preincubated with 0 or 10 microM choline decreased significantly, whereas that of slices preincubated with 25 or 50 microM choline did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of short-term temperature changes on the adhesivity of nerve cells. Participation of DNA molecules on non-specific cellular adhesion

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and sieving. Immediately after dissociation the cells were preincubated in a PBS solution at -6 to +54 degrees C for 3 and 20 min. After this preincubation cells were rotated for 60 min at 37 degrees C in the PBS solution. Cellular adhesivity was estimated during this time period and EM pictures of organized in vitro aggregates after 24-28 h were taken. In a separate series of experiments, freshly dissociated were treated with DNAase before the rotation procedure. Preincubation in a cold or a warm medium did not alter the inhibition of cellular adhesivity significantly. Distinct inhibition of cellular adhesion was observed in cells preincubated above 53 degrees C. Adhesion was also inhibited below -5 degrees C, however, this effect was mainly dependent on the rate of freezing and thawing. Digestion of dissociated cells with DNAase (20 micrograms/ml) decreased cell adhesion. At 37 degrees C the adhesivity decreased by about 20%. Aggregates of cells preincubated at 0 degrees C for 20 min did not exhibit marked EM changes after 24-28 h in vitro. The present results have shown the rather high resistance of molecules responsible for cellular adhesion and its reversibility to temperature changes. Furthermore, non-specific cellular adhesion was shown on physically active DNA molecules.     

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.     

Development of high heat resistance of Arizona neotype by preincubation at 35 degrees C in media containing NaCl.

When cells of Arizona neotype were preincubated at 35 degrees C in a medium containing NaCl (1 to 10%), they were found to be much more heat resistant upon being tested at 57 degrees C in a Trypticase (BBL Microbiology Systems) soy-yeast extract broth medium containing 10% NaCl than cells that had not been preincubated. Although no growth takes place during preincubation in the presence of 10% NaCl, some metabolic activity is necessary, since the presence of 2,4-dinitrophenol during incubation reduced the amount of gain in heat resistance. Incubation in the presence of N2, instead of air, abolished the effect. Chloramphenicol and rifampin, however, had no effect. Preincubated cells were just as heat resistant when separated by centrifugation and suspended in a fresh medium as when suspended in the incubated supernatant liquid. Conversely, the incubated supernatant liquid did not confer increased heat resistance upon unincubated cells. The increase in heat resistance, therefore, is probably a result of the salt osmotically plasmolyzing the cells by removing intracellular water, thus rendering the cellular contents more stable to heat. It is not known, however, why metabolic activity is necessary.     

Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp. PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock

Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na(+)/K(+) transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na(+) sensitivity, and inhibits HCO(3)(-) uptake. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K(+) transport activity. They encode an NAD(+)-binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K(+) transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABE-mediated K(+) uptake occurred with a moderately high affinity (K(m) of about 60 microm), and depended on both Na(+) and a high membrane potential, but not on ATP. KtrABE neither mediated Na(+) uptake nor Na(+) efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K(+) uptake required Na(+) and was inhibited by protonophore. A Delta ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol. Hyperosmotic shock led initially to loss of net K(+) from the cells. The Delta ktrB cells shocked with sorbitol failed to reaccumulate K(+) up to its original level. These data indicate that in strain PCC 6803 K(+) uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock.