The K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter <Emphasis Type="Italic">AhNHX1</Emphasis> improved tobacco tolerance to NaCl stress by enhancing K<Superscript>+</Superscript> retention

High salinity is the one of important factors limiting plant growth and crop production. Many NHX-type antiporters have been reported to catalyze K⁺/H⁺ exchange to mediate salt stress. This study shows that an NHX gene from Arachis hypogaea L. has an important role in K⁺ uptake and transport, which affects K⁺ accumulation and plant salt tolerance. When overexpressing AhNHX1, the growth of tobacco seedlings is improved with longer roots and a higher fresh weight than the wild type (WT) under NaCl treatment. Meanwhile, when exposed to NaCl stress, the transgenic seedlings had higher K⁺/H⁺ antiporter activity and their roots got more K⁺ uptake. NaCl stress could induce higher K⁺ accumulation in the roots, stems, and leaves of transgenic tobacco seedlings but not Na⁺ accumulation, thus, leading to a higher K⁺/Na⁺ ratio in the transgenic seedlings. Additionally, the AKT1, HAK1, SKOR, and KEA genes, which are involved in K⁺ uptake or transport, were induced by NaCl stress and kept higher expression levels in transgenic seedlings than in WT seedlings. The H⁺-ATPase and H⁺-PPase activities were also higher in transgenic seedlings than in the WT seedlings under NaCl stress. Simultaneously, overexpression of AhNHX1 increased the relative distribution of K⁺ in the aerial parts of the seedlings under NaCl stress. These results showed that AhNHX1 catalyzed the K⁺/H⁺ antiporter and enhanced tobacco tolerance to salt stress by increasing K⁺ uptake and transport.     

Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

Identification of a Novel Bacterial K<Superscript>+</Superscript> Channel

In an attempt to explore unknown K⁺ channels in mammalian cells, especially ATP-sensitive K⁺ (K_ATP) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian K_ATP channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K⁺ channel current that was inwardly rectified and had a reversal potential close to K⁺ equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific K_ATP channel openers or blockers did not alter the K⁺ current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of K_ATP channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K⁺ currents were inhibited by protopine (a nonspecific K⁺ channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K⁺ channel in mammalian cells.     

Cloning and functional characterization of the high-affinity K<Superscript>+</Superscript> transporter HAK1 of pepper

High-affinity K⁺ uptake in plants plays a crucial role in K⁺ nutrition and different systems have been postulated to contribute to the high-affinity K⁺ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K⁺ uptake observed in roots. Pepper plants starved of K⁺ for 3 d showed high-affinity K⁺ uptake (K _m of 6 M K⁺) that was very sensitive to NH and their roots expressed a high-affinity K⁺ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast (Saccharomyces cerevisiae), CaHAK1 mediated high-affinity K⁺ and Rb⁺ uptake with K _m values of 3.3 and 1.9 M, respectively. Rb⁺ uptake was competitively inhibited by micromolar concentrations of NH and Cs⁺, and by millimolar concentrations of Na⁺.     

A possible mechanism for low affinity of silkworm Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase for K<Superscript>+</Superscript>

The affinity for K⁺ of silkworm nerve Na⁺/K⁺-ATPase is markedly lower than that of mammalian Na⁺/K⁺-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K⁺ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na⁺/K⁺-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na⁺/K⁺-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase expressed in the cultured cells showed a low affinity for K⁺ and a high affinity for Na⁺, characteristic of the silkworm nerve Na⁺/K⁺-ATPase. These results suggest that the β subunit is responsible for the affinity for K⁺ of Na⁺/K⁺-ATPase.     

K<Superscript>+</Superscript>-induced Ca<Superscript>2+</Superscript> Conductance Responsible for the Prolonged Backward Swimming in K<Superscript>+</Superscript>-agitated Mutant of <Emphasis Type="Italic">Paramecium caudatum</Emphasis>

The K⁺-agitated (Kag) mutant of Paramecium caudatum shows prolonged backward swimming in K⁺-rich solution. To understand the regulation mechanisms of the ciliary motility in P. caudatum, we examined the membrane electrical properties of the Kag mutant. The duration of the backward swimming of the Kag in K⁺-rich solution was about 10 times longer than that of the wild type. In response to an injection of the outward current, the wild type produced an initial action potential and a subsequent membrane depolarization due to I-R potential drop, while the Kag exhibited repetitive action potentials during the depolarization. Under voltage-clamp conditions, the depolarization-activated transient inward current exhibited by the Kag was slightly smaller than that exhibited by the wild type. In response to an application of K⁺-rich solution, both the wild type and the Kag exhibited a depolarizing afterpotential representing the activation of the K⁺-induced Ca²⁺ conductance. The inactivation time course of the K⁺-induced Ca²⁺ conductance of Kag was about 10 times longer than that of the wild type. This difference corresponds well with the difference in behavioral responses between Kag and wild type to K⁺-rich solution. We conclude that the overreaction of the Kag mutant to the K⁺-rich solution is caused by slowing down of the inactivation of the K⁺-induced Ca²⁺ conductance.     

Trk2 transporter is a relevant player in K<Superscript>+</Superscript> supply and plasma-membrane potential control in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In Saccharomyces cerevisiae, TRK1 and TRK2 genes encode partially redundant K⁺ transporters. Direct involvement in K⁺ uptake has been shown for Trk1p since cells growing under limiting environmental K⁺ concentrations demand its presence. The biological role of Trk2p is less understood. In our experiments, TRK2 overexpression improved the ability of trk1 cells to grow in low K⁺ and led to a higher accumulation of K⁺. Using diS-C₃(3) as a potentiometric probe, we revealed a higher hyperpolarization of trk2 cells compared to the wild type. In addition, the deletion of TRK2 in the trk1 genetic background increased the cell sensitivity to hygromycin B, spermine, and TMA. Our studies reinforced the conclusion that Trk1p is the prominent K⁺ uptake transporter and for the first time revealed that though Trk2p is much less effective, its activity contributes significantly to K⁺ supply and the maintenance of plasma-membrane potential.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Ability of multicellular salt glands in <Emphasis Type="Italic">Tamarix</Emphasis> species to secrete Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> selectively

The present study aimed to determine the mechanism of cation-selective secretion by multicellular salt glands. Using a hydroponic culture system, the secretion and accumulation of Na⁺ and K⁺ in Tamarix ramosissima and T. laxa under different salt stresses (NaCl, KCl and NaCl+KCl) were studied. Additionally, the effects of salt gland inhibitors (orthovanadate, Ba²⁺, ouabain, tetraethylammonium (TEA) and verapamil) on Na⁺ and K⁺ secretion and accumulation were examined. Treatment with NaCl (at 0–200 mmol L⁻¹ levels) significantly increased Na⁺ secretion, whereas KCl treatment (at 0–200 mmol L⁻¹ levels) significantly increased K⁺ secretion. The ratio of secretion to accumulation of Na⁺ was higher than that of K⁺. The changes in Na⁺ and K⁺ secretion differed after adding different ions into the single-salt solutions. Addition of NaCl to the KCl solution (at 100 mmol L⁻¹ level, respectively) led to a significant decrease in K⁺ secretion rate, whereas addition of KCl to the NaCl solution (at 100 mmol L⁻¹ level, respectively) had little impact on the Na⁺ secretion rate. These results indicated that Na+ secretion in Tamarix was highly selective. In addition, Na⁺ secretion was significantly inhibited by orthovanadate, ouabain, TEA and verapamil, and K⁺ secretion was significantly inhibited by ouabain, TEA and verapamil. The different impacts of orthovanadate on Na⁺ and K⁺ secretion might be the primary cause for the different Na⁺ and K⁺ secretion abilities of multicellular salt glands in Tamarix.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

High-affinity K<Superscript>+</Superscript> transporter <Emphasis Type="Italic">PhaHAK5</Emphasis> is expressed only in salt-sensitive reed plants and shows Na<Superscript>+</Superscript> permeability under NaCl stress

To understand the mechanism of ion homeostasis in salt tolerant and sensitive plants, we isolated cDNAs for K⁺ transporter PhaHAK1-u and PhaHAK5-u from reed plants. PhaHAK1-u belongs to group I and PhaHAK5-u belongs to group IV by phylogenetic analysis, respectively. PhaHAK5-u is predicted to be a plasma membrane transporter, and shows high-affinity K⁺ transporter. Expression of PhaHAK5 was found in salt-sensitive reed plants, but not in any parts of salt-tolerant reed plants maintained under both control and K⁺ starvation conditions. Under the NaCl stress, the K⁺ uptake ability of the yeast strain expressing PhaHAK5-u was remarkably lower than that of the yeast strain expressing PhaHAK1-u, and PhaHAK5-u showed Na⁺ permeability. These results suggest that PhaHAK5 is one of the routes by which Na⁺ enters cells.     

QTLs for Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> uptake of the shoots and roots controlling rice salt tolerance

An F₂ and an equivalent F₃ population derived from a cross between a high salt-tolerance indica variety, Nona Bokra, and a susceptible elite japonica variety, Koshihikari, were produced. We performed QTL mapping for physiological traits related to rice salt-tolerance. Three QTLs for survival days of seedlings (SDSs) under salt stress were detected on chromosomes 1, 6 and 7, respectively, and explained 13.9% to 18.0% of the total phenotypic variance. Based on the correlations between SDSs and other physiological traits, it was considered that damage of leaves was attributed to accumulation of Na⁺ in the shoot by transport of Na⁺ from the root to the shoot in external high concentration. We found eight QTLs including three for three traits of the shoots, and five for four traits of the roots at five chromosomal regions, controlled complex physiological traits related to rice salt-tolerance under salt stress. Of these QTLs, the two major QTLs with the very large effect, qSNC-7 for shoot Na⁺ concentration and qSKC-1 for shoot K⁺ concentration, explained 48.5% and 40.1% of the total phenotypic variance, respectively. The QTLs detected between the shoots and the roots almost did not share the same map locations, suggesting that the genes controlling the transport of Na⁺ and K⁺ between the shoots and the roots may be different.     

Modulation of Aspartate Release by Ascorbic Acid and Endobain E,an Endogenous Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na⁺, K⁺-ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM d-[³H]aspartate (15 min at 37°C), centrifuged, washed, incubated in the presence of additions (60 s at 37°C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5–5.0 mM ascorbic acid, d-[³H]aspartate release was roughly 135–215% or 110–150%, with or without 40 mM KCl, respectively. The endogenous Na⁺, K⁺-ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5–10.0 mM commercial ouabain enhanced roughly 100% d-[³H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50–60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na⁺, K⁺-ATPase, may well modulate neurotransmitter release at synapses.     

K<Superscript>+</Superscript> transport in the caterpillar intestine epithelium: role of osmolytes for the K<Superscript>+</Superscript>-secretory capacity of the tobacco hornworm midgut

The midgut of the tobacco hornworm, Manduca sexta, actively secretes potassium ions. This can be measured as short-circuit current (I_sc) with the midgut mounted in an Ussing chamber and superfused with a high-K⁺ saline containing as its major osmolyte 166 mM sucrose. Iso-osmotic substitution of sucrose by non-metabolisable compounds (mannitol, urea, NaCl and the polyethylene glycols 200, 400 and 600) led to a dramatic, though reversible, drop in the current. Acarbose, a specific inhibitor of invertase (sucrase) in vertebrates and insects, had no detectable influence on I_sc. Unexpectedly, after replacing sucrose iso-osmotically with the saccharides glucose, fructose, trehalose or raffinose, the K⁺ current could no longer be supported. However, all osmolytes smaller than sucrose (except for NaCl), metabolisable or not, initiated an immediate, quite uniform but transient, increase in I_sc by about 20%, before its eventual decline far below the control value. Hypo-osmotic treatment by omission of sucrose also transiently increased the K⁺ current. Small osmolytes substituted for sucrose caused no transient I_sc stimulation when the epithelium had been challenged before with hypo-osmolarity; however, the eventual decline in I_sc could not be prevented. Our data seem inconsistent with a role of sucrose as energiser or simple osmolyte. Rather, we discuss here its possible role as analogous to that of sucrose in lower eukaryotes or plants, as an extra- and/or intracellular compatible osmolyte that stabilises structure and/or function of the proteins implicated in K⁺ transport.Communicated by G. Heldmaier     

The Administration of Levocabastine,a NTS2 Receptor Antagonist,Modifies Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Properties

Neurotensin behaves as a neuromodulator or as a neurotransmitter interacting with NTS1 and NTS2 receptors. Neurotensin in vitro inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. This effect is prevented by administration of SR 48692 (antagonist for NTS1 receptor). The administration of levocabastine (antagonist for NTS2 receptor) does not prevent Na⁺, K⁺-ATPase inhibition by neurotensin when the enzyme is assayed with ATP as substrate. Herein levocabastine effect on Na⁺, K⁺-ATPase K⁺ site was explored. For this purpose, levocabastine was administered to rats and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in synaptosomal membranes and [³H]-ouabain binding to cerebral cortex membranes were assayed in the absence (basal) and in the presence of neurotensin. Male Wistar rats were administered with levocabastine (50 μg/kg, i.p., 30 min) or the vehicle (saline solution). Synaptosomal membranes were obtained from cerebral cortex by differential and gradient centrifugation. The activity of K⁺-p-NPPase was determined in media laking or containing ATP plus NaCl. In such phosphorylating condition enzyme behaviour resembles that observed when ATP hydrolyses is recorded. In the absence of ATP plus NaCl, K⁺-p-NPPase activity was similar for levocabastine or vehicle injected (roughly 11 μmole hydrolyzed substrate per mg protein per hour). Such value remained unaltered by the presence of 3.5 × 10^?6 M neurotensin. In the phosphorylating medium, neurotensin decreased (32 %) the enzyme activity in membranes obtained from rats injected with the vehicle but failed to alter those obtained from rats injected with levocabastine. Levocabastine administration enhanced (50 %) basal [³H]-ouabain binding to cerebral cortex membranes but failed to modify neurotensin inhibitory effect on this ligand binding. It is concluded that NTS2 receptor blockade modifies the properties of neuronal Na⁺, K⁺-ATPase and that neurotensin effect on Na⁺, K⁺-ATPase involves NTS1 receptor and -at least partially- NTS2 receptor.     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.