Activation of Rb+ and Na+ uptake into yeast by monovalent cations

⁸⁶Rb⁺ uptake by yeast was not only stimulated by Rb⁺ or K⁺ but also by Na⁺. The uptake of ²²Na⁺ was enhanced by both Rb⁺ and K⁺, but not by Na⁺, which was inhibitory at all concentrations applied. Inhibition of ²²Na⁺ uptake by inactive Na⁺ occurred in two phases: one phase refers to inhibition at low Na⁺ concentrations and the other to inhibition at high Na⁺ concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.     

Interaction of phosphate with monovalent cation uptake in yeast.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Interaction of monovalent cations with Rb+ and Na+ uptake in yeast

The concentration dependence of both Rb⁺ uptake and Na⁺ uptake by yeast can be described by a quadratic rate equation. This equation is derived for translocation of cations via a two-site translocation system. In accordance with predictions made for such a two-site translocation system the shape of the uptake isotherm depends both upon the substrate cation species and upon the concentration of other added competing cations. On plotting the rate of Rb⁺ uptake against the quotient of that rate and the Rb⁺ concentration concave, convex and also linear curves are found depending upon the type and the concentration of added monovalent cations. The Na⁺ uptake isotherm plotted in a similar way shows a shift from a concave curve to a straight line on adding increasing amounts of Rb⁺ to the yeast suspension.Decreasing the pH of the medium leads to a more pronounced convex     

Effect of surface potential on Rb+ uptake in yeast: The effect of pH

The apparent K_m of Rb⁺ uptake and the zeta potential of yeast cells are appreciably affected by changes in the pH, variation of the concentration of the buffer cation Tris⁺ and addition of Ca²⁺ to the suspending medium. Irrespective of the way in which the zeta potential is affected, a direct relationship between the apparent K_m of the Rb⁺ uptake and the zeta potential is observed. A reduction of 8 mV in the zeta potential is accompanied by a 20-fold increase in the apparent K_m, which illustrates that electrostatic effects in ion uptake cannot be ignored. Measured zeta potentials are, to a good approximation, linearly related to surface potentials evaluated from a kinetic analysis of the Rb⁺ uptake. This shows the practical use of the zeta potential as a measure of the surface potential in studies of electrostatic effects in ion uptake by yeast. It is concluded that Tris⁺ and the aikaline earth cations inhibit the Rb⁺ uptake in yeast exclusively via a reduction in the surface potential. Protons, in addition, exert a competitive inhibition.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Uptake and effects of several cationic dyes on yeast

Summary Several cationic dyes were found to behave as inhibitors of K⁺ uptake in yeast. When added at high concentrations or in a K⁺-free medium, dyes can also produce and efflux of K⁺. The dyes are taken up by the cells in a process that, in different degrees, for several cations requires glucose and is inhibited to a higher degree by K⁺ than by Na⁺.The inhibition of cation uptake is of the competitive type with EB and close to this type with other dyes. Ca²⁺ inhibits the uptake and effects of dyes and in some cases also seems to change the inhibition kinetics on Rb⁺ uptake closer to a pure competitive type.According to preliminary experiments, the efflux of K⁺ seems to be of the electrogenic type, and not due to the disruption of the cells. The data indicate that, independently of the existence of other types of interaction (which do exist), dyes seem to interact with the system for monovalent cation uptake of yeast in different degrees of specificity and energy requirement. This interaction can be followed by fluorescence or metachromatic changes or reduction of the dyes as observed in the dual wavelength spectrophotometer and can be inhibited specifically by K⁺, but not by Na⁺.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

Uptake by Yeast: Interaction of Rb+, Na+ and Phosphate

It has been shown that addition of phosphate to phosphate deficient yeast gives rise to an immediate increase in the rate of Na⁺ uptake and an immediate decrease in the rate of Rb⁺ uptake. In addition, phosphate uptake is enhanced specifically by Na ions presumably by a process with a very high affinity for phosphate with a K_m of about 2 × 10⁻⁶M at pH 7.2, whereas the K_m for phosphate uptake of the Na⁺ independent process amounts to 1.3 × 10⁻⁴M.     

Effects of monovalent cations on derepression of phosphate transport in yeast

The effect of monovalent cations on derepression of phosphate transport was studied. It was found that ammonium, K⁺ and Rb⁺ accelerate the derepression of phosphate transport produced by glucose in yeast (Saccharomyces cerevisiae). Na⁺ and Li⁺ were ineffective in accelerating derepression; Cs⁺ produced only a minor stimulation. The concentration range of both K⁺ and NH₄⁺ that accelerated derepression was similar to that required for transport to occur. In the case of ammonium, the effects seem to depend exclusively on the so-called low-affinity transport system. The effect was strongly dependent on pH, with an optimum around 6; however, the increase in the pH of the medium did not produce in itself a high increase of the depression. Derepression was dependent on the presence of glucose, and it was very low with ethanol as substrate. The mechanism seems to depend on the ability that both K⁺ and NH₄⁺ have to decrease the membrane potential of the cell while transported, and not on the capacity to produce the alkalinization of the cell interior. In addition, the phenomenon depends on the presence of glucose as substrate, which indicates the involvement of some product of glucose metabolism in the mechanism, and possibly some relation to catabolic repression.     

Mechanisms of gentamicin-induced dysfunction of renal cortical mitochondria: II. Effects on mitochondrial monovalent cation transport

Mitochondrial swelling techniques were used to evaluate the effects of the aminoglycoside antibiotic gentamicin on renal cortical mitochondrial monovalent cation permeability. Gentamicin behaved like EDTA to enhance energy-dependent Na⁺- and K⁺-acetate uptake with a relatively greater effect on Na⁺-acetate uptake. Mg²⁺ prevented and reversed the effects of both EDTA and gentamicin. Neither agent affected energy-independent uptake of Na⁺ and K⁺-acetate. Gentamicin did not enhance energy-independent uptake of K⁺- and Na⁺-nitrate. Gentamicin enhanced energy-dependent swelling in a chloride- and phosphate-containing medium as a function of the medium Na⁺ and K⁺ concentration. This effect occurred simultaneously with gentamicin-induced stimulation of State 4 respiration and was blocked by Mg²⁺. Gentamicin did not affect phosphate transport. The results are taken to indicate a specific action of gentamicin to enhance mitochondrial monovalent cation permeability at an Mg²⁺-sensitive site and it is proposed that this accounts for the effects of gentamicin on mitochondrial respiration.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

Cation-induced asymmetry of choline flux across presynaptic plasma membranes

Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K⁺ and extrude Na⁺ and to synthesise acetylcholien in vitro. Choline uptake is hemicholinium-3 and Na⁺ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na⁺, Li⁺, Rb⁺, Cs⁺ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na⁺ gradient gave a maximal value of about 10². A secondary active transport of choline, where Na⁺ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.     

Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1

The wheat root high-affinity K⁺ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter. At toxic millimolar levels of sodium (Na⁺), HKT1 mediates low-affinity Na⁺ uptake while potassium (K⁺) uptake is blocked. In roots, low-affinity Na⁺ uptake and inhibition of K⁺ uptake contribute to Na⁺ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K⁺ and Na⁺ over Rb⁺, Cs⁺ and Li⁺. In addition, Rb⁺ and Cs⁺, and an excess of extracellular K⁺ over Na⁺, are shown to partially reduce or block HKT1-mediated K⁺-Na⁺ uptake. Furthermore, K⁺, Rb⁺ and Cs⁺ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high-affinity Rb⁺ uptake at approximately 15-fold lower rates than for K⁺. Rb⁺ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35-fold lower HKT1 conductance for Rb⁺. A model for HKT1 activity is presented involving a high-affinity K⁺ binding site and a high-affinity Na⁺ binding site, and competitive interactions of K⁺, Na⁺ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K⁺ and Na⁺ uptake in plants are discussed.     

Cotransport of phosphate and sodium by yeast

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM and is independent of sodium, and a sodium-dependent mechanism with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.6 μM, both $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na⁺, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Rubidium has no effect on the stimulation of phosphate uptake by sodium.Phosphate and arsenate enhance sodium uptake at pH 7.2. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake.The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.     

Closure of the yeast mitochondria unspecific channel (YMUC) unmasks a Mg2+ and quinine sensitive K+ uptake pathway in Saccharomyces cerevisiae

The K⁺ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K⁺-mediated mitochondrial swelling observed in the absence of phosphate (PO₄) and in the presence of a respiratory substrate has led to propose that large K⁺ movements occur in yeast mitochondria. Thus, the uptake of K⁺ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K⁺ transport; these were mitochondrial swelling and the uptake of the radioactive K⁺ analog ⁸⁶Rb⁺. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO₄ concentrations. The high protein concentrations used to measure ⁸⁶Rb⁺ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO₄ but not at 0 or 4 mM PO₄. At 4 mM PO₄ swelling was inhibited while, in contrast, ⁸⁶Rb⁺ uptake was still observed. The results suggest that an energy-dependent K⁺ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K⁺ uptake system, the Mg²⁺ and quinine sensitivity of both swelling and ⁸⁶Rb⁺ uptake were evaluated. Under the conditions where the unspecific pore was closed, K⁺ transport sensitivity to Mg²⁺ and quinine increased. In addition, when Zn²⁺ was added as an antiport inhibitor, uptake of ⁸⁶Rb⁺ increased. It is suggested that in yeast mitochondria, the K⁺ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.     

The acrosome reaction of Strongylocentrotus purpuratus sperm. Ion requirements and movements

The acrosome reaction of sperm of the sea urchin, Strongylocentrotus purpuratus, is accompanied by ion movements. When the reaction is induced by the addition of egg jelly to sperm suspended in sea water, there is an acid release and an uptake (or exchange) of calcium ions. Verapamil and D600, drugs which block Ca²⁺ channels, inhibit induction of the acrosome reaction, acid release, and ⁴⁵Ca²⁺ uptake; this inhibition is reduced at higher concentrations of external Ca²⁺. Although acid release correlates temporally with extension of the acrosome filament, ⁴⁵Ca²⁺ uptake continues after the acrosome reaction has been completed. Neither the acrosome reaction nor acid release is inhibited by cyanide, azide, dinitrophenol (DNP), or carbonyl cyanide m-chlorophenylhydrazone (CCCP), whereas these metabolic inhibitors partially inhibit Ca²⁺ uptake. Tetraethylammonium (TEA) chloride, an inhibitor of delayed axonal potassium currents, inhibits the acrosome reaction. An increase in ⁸⁶Rb⁺ permeability accompanies the acrosome reaction, suggesting that movement of K⁺ is an important effector of the reaction. In support of this, the acrosome reaction may be triggered with nigericin, an ionophore that catalyzes the electrically neutral exchange of K⁺ and H⁺ across membranes. Induction of the acrosome reaction with nigericin can occur with either Na⁺ or K⁺ as the predominant external monovalent cation, while with jelly it requires external Na⁺. With nigericin, there is a delay in acid release, Ca²⁺ uptake, and filament extension, all of which follow a transient proton uptake. Taken together, these data suggest that triggering of the acrosome reaction involves linked permeability changes for monovalent and divalent ions.     

Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na⁺/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na⁺ gradient. The uptake of l-carnitine was of high affinity (K_m=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na⁺/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H⁺ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [¹⁴C]TEA uptake, but unlabeled l-carnitine did not stimulate [¹⁴C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na⁺/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H⁺/organic cation antiporter.     

22Na+ and 86Rb+ fluxes in spermatozoa and oocytes of arbacia punctulata

The fluxes of ²²Na⁺ and ⁸⁶Rb⁺ in Arbacia sperm and oocytes were studied in order to determine how these cells carry out cation exchange with the sea environment. The uptake of these ions by serum followed a pattern of early rapid influx (initial 0.5 min) and subsequent efflux (1–3 min) followed by a gradual uptake (after 3 min). Neither the uptake nor the efflux of these cations by Arbacia sperm were affected by ouabain, suggesting that influx and efflux of ²²Na⁺ and ⁸⁶Rb⁺ in Arbacia sperm occur predominantly by passive transport. The ²²Na⁺ uptake by Arbacia oocytes showed a steady increase after an initial rapid uptake. A slight but significant inhibition of ²²Na⁺ uptake was observed with ouabain. However, ⁸⁶Rb⁺ uptake by the oocytes reached an early equilibrium and was not affected by ouabain. The uptake of Rb⁺ by Arbacia oocyte is by passive transport while that of Na⁺ is both by passive and active transport.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.     

The influence of monovalent cations upon influx and efflux of Ca2+ in barley roots

The effects of monovalent cations, inhibitors of metabolism dinitrophenol (DNP), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and KCN and temperature variations upon Ca²⁺ fluxes in intact roots of barley (Hordeum vulgare L. cv. Fergus and Herta) seedlings were investigated. ⁴⁵Ca²⁺ influx was depressed in CaSO₄-grown (low-salt) plants by the presence of NH₄⁺, K⁺, or Na⁺ in the uptake medium. In contrast Ca²⁺ influx was slightly increased by Li⁺. In low-salt roots pretreated with KCN and in roots preloaded with K⁺ (high-K⁺ plants), the presence of K⁺ in the medium had no significant effect on Ca²⁺ influx, while in roots preloaded with Na⁺, the presence of K⁺ in the medium depressed Ca²⁺ influx. In absolute terms, Ca²⁺ influx was significantly greater in high-salt (both K⁺ or Na⁺ preloaded) than in low-salt roots.Patterns of ⁴⁵Ca²⁺ efflux in the absence and in the presence of K⁺, NH₄⁺, or Li⁺ in the external medium showed that these monovalent cations caused stimulation of ⁴⁵Ca²⁺ efflux both from the cytoplasmic and vacuolar phases.It was noted that these modifications of Ca²⁺ fluxes by monovalent cations are transient and characteristic of a transitional stage of cation uptake by low-salt roots. We conclude that, together with stimulated active H⁺ efflux (another characteristic of this transitional stage), modifications of Ca²⁺ fluxes during monovalent cation uptake by low-salt roots is a response directed towards the maintenance of electrical neutrality.Determination of net fluxes revealed that the plants were close to Ca²⁺ flux equilibrium in the growth medium (0.5 mM CaSO₄). Transfer of these plants to 0.5 mM CaSO₄ + 0.25 mM K₂SO₄ caused a net release of CA²⁺ into the external medium.