Ion transport by heart mitochondria: XXVII. The relation of mercurial-dependent adenosine triphosphatase activity to ion movements

The properties of a mercurial-dependent adenosine triphosphatase activity have been examined in isolated beef heart mitochondria. The reaction differs from that induced by uncouplers in that it is associated with extensive ion uptake and osmotic swelling, is highly specific for K⁺ over Na⁺, and is enhanced by respiration. Evidence is presented which suggests that the following events can account for the observations: (1) The mercurial blocks the phosphate transporter so that phosphate hydrolyzed from ATP is trapped in the matrix. (2) This interior negative potential causes cations to move inward and swelling results. (3) Permeability to K⁺ but not to Na⁺ is enhanced greatly by the reaction of the mercurial with the membrane. The inward movement of K⁺ closely resembles that produced by valinomycin, in that it is accompanied by proton ejection into the medium and it rapidly establishes a condition in which ion gradients cannot be maintained. This marked increase in permeability may be related to the pH gradient and is manifest as additional passive swelling in the absence of sucrose and passive contraction when sucrose is present. A comparison of the kinetics of swelling and of ATP hydrolysis shows that the elevated rates of ATPase are correlated with this condition of high permeability. When a corresponding condition of high permeability to Na⁺ is established by treatment with gramicidin or EDTA, the mercurial-dependent ATPase is nearly as rapid in Na⁺ as in the K⁺ medium. It appears, therefore, that the K⁺ specificity resides at the level of membrane permeability and is not a feature of the ATPase reaction per se. (4) Respiration appears to affect the ATPase reaction by virtue of its ability to extrude ions from the matrix in the presence of the mercurial. p-Chloromercuriphenyl sulfonate causes a switch from respiration-dependent ion accumulation to respiration-dependent ion extrusion to occur. A model to explain these reactions is presented.     

Effects of haemolymph free cations on blowfly taste receptor responses

Ionic composition of the haemolymph and electrophysiological responses of tarsal taste hairs were determined for individual flies of the species Calliphora vicina, in order to test the hypothesis that electrophysiological response values of individual flies are correlated with the ionic composition. Instead of flame photometry we used isotachophoresis to determine the ionic composition; only non-bound ions contribute to the result. Because of this we found lower concentration values than those reported so far. There was a strong correlation between the concentrations of the cations Na⁺, K⁺ and Mg²⁺, while Ca²⁺ was not related with any other cation. A significant correlation was shown to exist between the response of taste cells and the Na⁺, K⁺ and Ca²⁺ content in the haemolymph. These correlations explain, at least partly, the systematic differences in taste cell responses between flies, as reflected in interindividual variability.     

Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro: general procedures for the determination of water,sodium, potassium and chloride in protoscoleces

Reisin I.L. and Rotunno C.A. 1981. Water and electrolyte balance in protoscoleces of Echimcoccus granulosus incubated in vitro: General procedures for the determination of water, sodium, potassium and chloride in protoscoleces. International Journal for Parasitology11: 399–404. Protoscoleces of E. granulosus (sheep strain) were incubated in vitro at 37°C in Ringer Krebs solution (RKS) for up to 3 h. When they were briefly washed in sucrose 0.3 M at 4°C, the water and electrolyte contents were: 1.768 ± 0.034 mlg^?1 d.w. for water content and 123 ± 2, 209 ± 2 and 78 ± 2 μmolg^?1 d.w. for Na⁺, K⁺ and Cl^? respectively. When protoscoleces were not washed in sucrose solution but were spun down from RKS, the K⁺ content suffered a very small change but larger values for Na⁺ and Cl⁺ contents were obtained. These higher Na⁺ and Cl^? contents are attributed to the RKS ions retained in the trapping space. The steady state distribution of Na⁺ and K⁺ in the protoscoleces incubated at 37°C indicates the activity of an active transport mechanism.     

Acetylcholine receptor-mediated sodium ion efflux after rapid hypoosmotic loading of radiotracer

Torpedo membrane vesicles (microsacs) enriched in acetylcholine receptors can be loaded with radiotracer permeants such as ²²Na⁺ and [³H]sucrose in only a few minutes. The fast loading technique employs exposure of vesicles to hypoosmotic solutions containing the radiotracers. Subsequent to loading of permeants, their efflux from the vesicles can be determined by a Millipore filtration technique. Efflux rates following hypoosmotic loading are comparable to those following the conventional loading technique which employs isotonic solutions. Hence, efflux measurements can be begun immediately after vesicle preparation. Furthermore, simultaneous loading of ²²Na⁺ and [³H]sucrose allows a normalization of ²²Na⁺ efflux with respect to vesicle volume, since ²²Na⁺, but not [³H]sucrose, is released specifically by cholinergic effectors. Such double-isotope measurements increase the reliability of the data derived.     

Studies on stimulus-response coupling in human neutrophils: I. Role of monovalent cations in the respiratory and secretory response to N-formylmethionylleucylphenylalanine

Human blood neutrophils suspended in Na⁺-free, high-K⁺, phosphate-buffered solution exhibit respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) much higher than those suspended in phosphate-buffered solution containing physiological concentration of K⁺ and Na⁺. The differences between the responses are very marked at low doses of fMet-Leu-Phe (10^?9, 10^?8 M), progressively decrease at higher doses, and disappear at the maximal stimulatory concentration of the peptide (10^?6 M). The higher responses of human neutrophils to fMet-Leu-Phe are not dependent on the membrane depolarization, that occurs when the cells are suspended in high-K⁺ buffered solution, but on the absence, or on the low concentration, of Na⁺ in the suspending medium. In fact: (i) the higher respiratory and secretory responses progressively decrease by substituting K⁺ with Na⁺ in the suspending solution, without change of the state of depolarization; (ii) the replacement of extracellular Na⁺ with choline ions does not affect the transmembrane potential of neutrophils but induces higher respiratory and secretory responses to fMet-Leu-Phe; (iii) the membrane depolarization induced by gramicidin and by ouabain does not result in a higher respiratory response to chemotactic peptide. These results indicate that in human neutrophils Na⁺ plays a regulative role in the stimulation of the respiratory burst and in the secretion induced by the chemotactic peptide. This regulation does not influence the maximal responses, but the threshold of the responses. K⁺ is also involved at least in the respiratory response, since the effect of the absence of Na⁺ is potentiated when the concentration of K⁺ of the suspending solution is high. Furthermore, the finding that a very high respiratory burst and the secretion of β-glucuronidase and vitamin B-12-binding protein can be induced by fMet-Leu-Phe in human neutrophils in the absence of external Na⁺ indicates that the entry of this cation and the consequent decrease in transmembrane potential are not necessary events for the activation of respiration and secretion by the peptide. The mechanism underlying the effect of the modification of ionic composition of the external medium is discussed in terms of the molecular events triggered by the stimulus at the level of the plasma membrane and of the recognition phenomena at the cell surface, that are common steps for the induction of the respiratory and secretory responses in neutrophils.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Na+ (Li+)-proline cotransport inEscherichia coli

Summary Na⁺ and Li⁺ were found to stimulate the transport ofl-proline by cells ofEscherichia coli induced for proline utilization. The gene product of the put P gene is involved in the expression of this transport activity since the put P⁺ strains CSH 4 and WG 148 show activity and the put P^– strain RM 2 fails to show this cation coupled transport. The addition of proline was found to stimulate the uptake of Li⁺ and of Na⁺. Attempts to demonstrate proline stimulated H⁺ uptake were unsuccessful. It is concluded that the proline carrier (coded by the put P gene) is responsible for Na⁺ (or Li⁺)-proline cotransport.     

Salt tolerance in suspension cultures of sugar beet : induction of na/h antiport activity at the tonoplast by growth in salt

Cell suspension cultures of sugar beet were grown at various salinities (0-200 millimolar NaCl). Their tolerance to Na⁺ was comparable to that of the intact plant. Tonoplast vesicles were prepared by sucrose density gradient centrifugation of microsomal membranes and shown to be highly purified. The vesicles were subjected to a pH jump in the presence of acridine orange and the rate of recovery of fluorescence after addition of Na⁺ was used as a measure of Na⁺-dependent H⁺ efflux. In the presence of K⁺ and valinomycin, the Na⁺/H⁺ antiport showed saturation kinetics. Increasing Na⁺ in the growth medium did not change the apparent K_m for Na⁺, but increased V_max to about twice the control value, suggesting a specific induction of antiport synthesis by salt.     

Involvement of na in active uptake of pyruvate in mesophyll chloroplasts of some c(4) plants : na/pyruvate cotransport

An artificial Na⁺ gradient across the envelope (Na⁺ jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C₄ plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219:347). In the present study, ²²Na⁺ and pyruvate uptake were examined in mesophyll chloroplasts of several species of C₄ plants. Enhancement of pyruvate uptake by a Na⁺ jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in turn enhanced Na⁺ uptake in the dark when added together with Na⁺. When flux of endogenous Na⁺ was measured in these mesophyll chloroplasts preincubated with ²²Na⁺, pyruvate addition induced Na⁺ influx, and the extent of the pyruvate-induced Na⁺ influx positively correlated with that of pyruvate uptake. A Na⁺/H⁺ exchange ionophore, monensin, nullified all the above mutual effects of Na⁺ and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na⁺ uptake and increased equilibrium level of chloroplast ²²Na⁺. Measurements of initial uptake rates of pyruvate and Na⁺ gave a stoichiometry close to 1:1. These results point to Na⁺/pyruvate cotransport into mesophyll chloroplasts of some C₄ plants.     

Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes

Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na⁺ flux measurements was limited due to poor selectivity for Na⁺ ions of commercially available Na⁺ cocktails. Often, these cocktails cannot discriminate between Na⁺ and other interfering ions such as K⁺ and Ca²⁺, leading to inaccurate measurements of Na⁺ concentration and, consequently, inaccurate Na⁺ flux calculations. To overcome this problem, three Na⁺-selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na⁺ cocktail for selectivity for Na⁺ ions over other ions (particularly K⁺ and Ca²⁺). Among the three calixarene-based Na⁺ cocktails tested, cocktail 2 [in % w/w: Na⁺ ionophore (4-tert-butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na⁺ ions over K⁺ and Ca²⁺ ions and was highly stable over time (up to 10 h). Na⁺ flux measurements under a wide range of NaCl concentrations (25-150 mM) using Na⁺ cocktail 2 established a clear dose-response relationship between severity of salt stress and magnitude of Na⁺ influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na⁺ cocktail 2 was compared with commercially available ETH 227-based Na⁺ cocktail by measuring Na⁺ fluxes at the two Arabidopsis root zones in response to 100 mM NaCl treatment. With calixarene-based Na⁺ cocktail 2, a large decreasing Na⁺ influx (0-15 min) followed by small Na⁺ influx (15-45 min) was measured, whereas with ETH-based Na⁺ cocktail Na⁺ influx was short-lived (1-3 min) and was followed by Na⁺ efflux (3-45 min) that might have been due to K⁺ and Ca²⁺ efflux measured together with Na⁺ influx. In conclusion, Na⁺-selective calixarene-based microelectrodes have excellent potential to be used in real-time Na⁺ flux measurements in plants.     

Acid-sensitive TWIK and TASK Two-pore Domain Potassium Channels Change Ion Selectivity and Become Permeable to Sodium in Extracellular Acidification

Two-pore domain K⁺ channels (K2P) mediate background K⁺ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K⁺ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH_o) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH_o affects these acid-sensitive K2P channels is not well understood. Here we show that in Na⁺-based bath solutions with physiological K⁺ gradients, lowered pH_o largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K⁺ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na⁺ to K⁺ relative permeability. Low pH_o-induced inhibitions in these acid-sensitive K2P channels are more profound in Na⁺-based bath solutions than in channel-impermeable N-methyl-d-glucamine-based bath solutions, consistent with increases in the Na⁺ to K⁺ relative permeability and decreases in electrochemical driving forces of outward K⁺ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K⁺ channels change ion selectivity in response to lowered pH_o, provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.     

Regulation of sodium transport in erythrocytes

The kinetic response of swine erythrocyte (Na + K)-ATPase to Na⁺ concentration was hyperbolic in low KCl (5–25 mm) but became increasingly sigmoidal (n = 2.2) as KCl was increased to 150 mm. The addition of 150 mm LiCl did not cause an increase in sigmoidicity although it decreased the apparent affinity for Na⁺. The dependence of ouabain-inhibited efflux of Na⁺ on internal Na⁺ concentration was measured in intact cells with intracellular cation concentrations altered by incubation in p-ehloromercuriphenyl sulfonate. The response to Na⁺ was sigmoidal (n = 2.2) in cells containing high K⁺ but hyperbolic in preparations in which most of the intracellular K⁺ was replaced by Li⁺, even in the presence of 150 mm external KCl. The data are consistent with a model in which internal K⁺ is an allosteric (feedback) inhibitor of Na⁺ efflux and there are three Na⁺ sites which interact cooperatively.     

Effect of Salt Stress on Growth,Na+ Accumulation and Proline Metabolism in Potato (Solanum tuberosum) Cultivars

Potato (Solanum tuberosum) is a major crop world-wide and the productivity of currently used cultivars is strongly reduced at high soil salt levels. We compared the response of six potato cultivars to increased root NaCl concentrations. Cuttings were grown hydroponically and treated with 0 mM, 60 mM and 180 mM NaCl for one week. Growth reduction on salt was strongest for the cultivars Mozart and Mona Lisa with a severe senescence response at 180 mM NaCl and Mozart barely survived the treatment. The cultivars Desiree and Russett Burbank were more tolerant showing no senescence after salt treatment. A clear difference in Na⁺ homeostasis was observed between sensitive and tolerant cultivars. The salt sensitive cultivar Mozart combined low Na⁺ levels in root and stem with the highest leaf Na⁺ concentration of all cultivars, resulting in a high Na⁺ shoot distribution index (SDI) for Mozart as compared to Desiree. Overall, a positive correlation between salt tolerance and stem Na⁺ accumulation was found and the SDI for Na⁺ points to a role of stem Na⁺ accumulation in tolerance. In stem tissue, Mozart accumulated more H₂O₂ and less proline compared to the tolerant cultivars. Analysis of the expression of proline biosynthesis genes in Mozart and Desiree showed a clear reduction in proline dehydrogenase (PDH) expression in both cultivars and an increase in pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Desiree, but not in Mozart. Taken together, current day commercial cultivars show promising differences in salt tolerance and the results suggest that mechanisms of tolerance reside in the capacity of Na⁺ accumulation in stem tissue, resulting in reduced Na⁺ transport to the leaves.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

Cloning and Characterization of a Novel Vacuolar Na+/H+ Antiporter Gene (Dgnhx1) from Chrysanthemum

Plant vacuolar Na⁺/H⁺ antiporter genes play significant roles in salt tolerance. However, the roles of the chrysanthemum vacuolar Na⁺/H⁺ antiporter genes in salt stress response remain obscure. In this study, we isolated and characterized a novel vacuolar Na⁺/H⁺ antiporter gene DgNHX1 from chrysanthemum. The DgNHX1 sequence contained 1920 bp with a complete open reading frame of 1533 bp encoding a putative protein of 510 amino acids with a predicted protein molecular weight of 56.3 kDa. DgNHX1 was predicted containing nine transmembrane domains. Its expression in the chrysanthemum was up-regulated by salt stress, but not by abscisic acid (ABA). To assess roles of DgNHX1 in plant salt stress responses, we performed gain-of-function experiment. The DgNHX1-overexpression tobacco plants showed significant salt tolerance than the wild type (WT). The transgenic lines exhibited more accumulation of Na⁺ and K⁺ under salt stress. These findings suggest that DgNHX1 plays a positive regulatory role in salt stress response.     

Chronic exposure to soil salinity in terrestrial species: Does plasticity and underlying physiology differ among specialized ground-dwelling spiders?

In salt marshes, the alternation of low and high tides entails rapid shifts of submersion and aerial exposure for terrestrial communities. In these intertidal environments, terrestrial species have to deal with an osmotic loss in body water content and an increase in sodium chloride concentration when salt load increases. In salt marshes, spiders represent an abundant arthropod group, whose physiological ecology in response to variations of soil salinity must be further investigated. In this study, we compared the effect of salinity on the survival and physiology of three species of Lycosidae; two salt marsh species (Arctosa fulvolineata and Pardosa purbeckensis) and one forest species (P. saltans). Spiders were individually exposed at three salinity conditions (0‰, 35‰ and 70‰) and survival, changes in body water content, hemolymph ions (Na⁺, Ca²⁺, Mg²⁺, K⁺; ICP-MS technique) and metabolites (mainly amino acids, polyols, sugars; LC and GC techniques) were assessed. The survival of the forest species P. saltans was very quickly hampered at moderate and high salinities. In this spider, variations of hemolymph ions and metabolites revealed a quick loss of physiological homeostasis and a rapid salt-induced dehydration of the specimens. Conversely, high survival durations were measured in the two salt-marsh spiders, and more particularly in A. fulvolineata. In both P. purbeckensis and A. fulvolineata, the proportion of Na⁺, Ca²⁺, Mg²⁺, K⁺ remained constant at the three experimental conditions. Accumulation of hemolymph Na⁺ and amino acids (mainly glutamine and proline) demonstrated stronger osmoregulatory capacities in these salt-marsh resident spiders. To conclude, even if phylogenetically close (belonging to the same, monophyletic, family), we found different physiological capacities to cope with salt load among the three tested spider species. Nevertheless, physiological responses to salinity were highly consistent with the realized ecological niches of the spiders.     

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na⁺ channel (ENaC) is modulated by Na⁺ self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na⁺. A His residue within the extracellular domain of γENaC (γHis²³⁹) was found to have a critical role in Na⁺ self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na⁺ self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na⁺ self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na⁺ self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the γH239F mutant showed that, in the absence of Na⁺ self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that γHis²³⁹ functions within an extracellular allosteric regulatory subdomain of the γ subunit that has an important role in conferring the response of the channel to external Na⁺.