Evidence for a K+-stimulated Na+ efflux at the plasmalemma of barley root cells

Summary The influence of K⁺ on the Na⁺ fluxes of barley root cells was investigated. A increased K⁺ concentration (K⁺ influx) results in a transient increase of the plasmalemma efflux of Na⁺ followed by a decrease, and in a decrease of the cytoplasmic content and the tonoplast influx of Na⁺. These results are consistent with a Na-K-pump at the plasmalemma.     

Na+ and K+ transport in excised soybean roots

Uptake, accumulation and xylem transport of K⁺ and Na⁺ in excised roots of soybean were investigated by use of a perfusion technique. This technique permitted independent quantification of, on the one hand, entry of ions into the roots and their transport through the cortex to the xylem vessels, and on the other hand reabsorption from the xylem vessels to the neighbouring cells and the external medium. Data are consistent with a low degree of selective uptake of K⁺ over Na⁺. However, Na⁺ depletion of the xylem stream by reabsorption limits, although weakly, its translocation to the shoots. Na⁺ reabsorbed is for a great part reexcreted into the external medium. The low efficiency of these processes is discussed in relation to the Na⁺ sensitivity of soybean.     

Compartmental analysis of Rb+ efflux from roots of intact high-salt barley plants

The validity of compartmental analysis of Rb⁺ efflux from roots of intact high-salt barley plants ( Hordeum vulgare L. cv. Salve) was examined. ⁸⁶Rb⁺ was used as a tracer. Rb⁺ (1 m M ) was included together with 3 m M K⁺ in the growth medium, and steady-state conditions were assumed to prevail during the experiment. Three phases of efflux were revealed with half-times of 23 min, 109 min and 12 h, respectively; and the time span of the experiment had to be at least 20 h to make determination of the slow phase possible. We cannot state what compartments in the root the 3 different slopes represent. A comparison of slopes was made between the plots of In efflux vs time and In content vs time. In spite of correction for tracer transport from the roots to the shoot, the slopes for the slow phase did not agree unless up to 85% of the root content of Rb⁺ is assumed not to participate in efflux.     

The regulation of K+ influx in excised barley roots

The electrochemical potential differences for potassium, between excised barley (Hordeum vulgare L.) roots and external media containing 0.05 mM KCl+0.5 mM CaSO₄, were determined over a 4-h period during which initially low-K⁺ roots accumulated K⁺ by pretreatment in 50 mM KCl plus 0.5 mM CaCl₂. This pretreatment resulted in increased internal [K⁺], decreased K⁺ influx (as measured from 0.05 mM KCl+0.5 mM CaSO₄) and decreased values of . These observations indicate that the decline of K⁺ influx associated with increased internal K⁺ concentration cannot be accounted for by passive adjustment to the electrochemical gradient for this ion.     

Toxin-induced K+ efflux through the Na+ channel of neuroblastoma cells

Neurotoxins which modify the gating system of the Na+ channel in neuroblastoma cells and increase the initial rate of 22Na+ influx through this channel also give rise to the efflux of 86Rb+ and 42K+. These effluxes are inhibited by tetrodotoxin and are dependent on the presence in the extracellular medium of cations permeable to the Na+ channel. These stimulated effluxes are not due to membrane depolarization or increases in the intracellular content of Na+ and Ca2+ which occur subsequent to the action of neurotoxins. The relationships of 22Na+ influx and 42K+ (or 86Rb+) effluxes to both the concentration of neurotoxins and the concentration of external permeant cations strongly suggest that the open form of the Na+ channel stabilized by neurotoxins permits an efflux of K+ ions. Our results indicate that for the efflux of each K+ ion there is a corresponding influx of two Na+ ions into the Na+ channel.     

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.     

Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced DA efflux is mediated by DAT and results from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT is voltage-dependent, electrogenic, and dependent on intracellular Na(+) concentration in the recording electrode. Intracellular Na(+) fluorescence, as measured by confocal microscopy using a Na(+)-sensitive dye, was enhanced by AMPH application. Furthermore, the ability of AMPH to induce DA efflux was regulated by intracellular Na(+) concentration and correlated with the size of the DAT-mediated, AMPH-induced ion flux across the plasma membrane. In the absence of intracellular Na(+) but the presence of high intracellular Cl(-), AMPH-induced inward currents elicited DA efflux proportionally to their dimension and duration. Thus, we propose that AMPH-induced DA efflux depends on two correlated transporter processes. First, AMPH binds to the DAT and is transported, thereby causing an inward current. Second, because of this AMPH-induced inward current, Na(+) becomes more available intracellularly to the DAT, thereby enhancing DAT-mediated reverse transport of DA.     

Inhibition of Na+-dependent Ca2+ efflux from heart mitochondria by amiloride analogues

The Na+-induced release of accumulated Ca2+ from heart mitochondria is inhibited by amiloride, benzamil and several other amiloride analogues. These drugs do not affect uptake or release of Ca2+ mediated by the ruthenium red-sensitive uniporter and their effects, like those of diltiazem and other Ca2+-antagonists, appear to be localized principally at the Na+/Ca2+ antiporter of the mitochondrion. Benzamil inhibits Na+/Ca2+ antiport non-competitively with respect to [Na+] with a Ki of 167 microM. In the presence of 1.5 mM Pi the Ki for benzamil inhibition of this reaction is decreased to 87 microM.     

Transport ATPase: thimerosal inhibits the Na+ +K+-dependent ATPase activity without diminishing the Na+-dependent ATPase activity.

A guinea pig kidney membrane preparation was incubated with thimerosal and then thoroughly washed. Comparison of the properties of the native and the modified membranes showed that (a) Na⁺+K⁺-dependent activity is substantially inhibited by thimerosal; (b) thimerosal does not diminish Na⁺-dependent ATPase activity; and (c) the thimerosal treated enzyme, like the native enzyme, is phosphorylated in the presence of Na⁺ and ATP, and dephosphorylated upon the addition of K⁺. It is suggested that thimerosal does not affect the binding of ATP to the high-affinity catalytic site, but that it blocks the binding of ATP to a low affinity modifying site the occupation of which is essential for the dissociation of the stable K⁺-dephosphoenzyme and the recycling of the enzyme.     

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Vanadate selectively inhibits the K0+-activated Na+ efflux in squid axons

The effects of internally applied 1 mM vanadate on the Na⁺ efflux in dialysed squid axons were found to depend on the presence of external K⁺. In K⁺-free artificial sea water, vanadate did not produce any change in the rate of Na⁺ efflux, whereas in the presence of 10 mM K⁺ the Na⁺ efflux was reduced to values even lower than those observed in the absence of K⁺ (inversion of the K⁺-free effect). In vanadate-poisoned axons, K⁺ and NH₄⁺ at low concentrations activated Na⁺ efflux, but at high concentrations both cations were inhibitory. However, NH₄⁺ was always a better activator and a poorer inhibitor than K⁺.     

Na+/K+-dependent adenosinetriphosphatase activity in mammalian kidney.

Microsomal preparations with Na+/K+-dependent ATPase activity from the outer medulla of rabbit and pig kidney were obtained. Purifications were assaied by centrifugation on sucrose discontinuous gradients and gel-filtration on Sepharose 6B, after detergent incubation. Sodium dodecylsulfate, with ATP as protecting agent, can remove a maximal amount of non specific proteins from the membranes and allows the recovery of a fraction with very high specific activity. The approach to purification by affinity chromatography techniques leads to interesting results, which induce us to pursue the present researchs to applicate an affinity method to Na+/K+-ATPase.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Na(+)-dependent Ca2+ efflux inhibits stimulus-induced secretion in bovine chromaffin cells

Stimulations of chromaffin cells with histamine and ionomycin produced rises in cellular free Ca2+ level. The removal of Na+ ions from the medium prolongated the rises without changing the magnitude. The stimulations also facilitated 45Ca2+ efflux from cells by over 3-fold. The facilitation was, however, largely abolished by the Na+ removal, indicating the Na(+)-dependent efflux is a major system to expel Ca2+ from the stimulated cells. The Na+ removal also enhanced secretions evoked by these stimuli. The results suggest the Na(+)-dependent Ca2+ efflux by lowering the elevated cellular Ca2+ plays a role in terminating the stimulus-induced secretion.     

Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

Kinetics of Na+-dependent K+ ion transport in a marine pseudomonad.

The effect of external Na plus concentration on the transport of K plus was studied using K plus-depleted cells of a marine pseudomonad. K plus transport was found to be a saturable process and requires Na plus. The initial rates for K plus transport over a range of external K plus concentrations were measured in suspensions containing various fixed concentrations of Na plus. Reciprocals of the initial rates for K plus transport were plotted against reciprocals of the external concentration of K plus or Na plus to yield two primary Lineweaver-Burk plots. The experimental data were found to fit bisubstrate enzyme kinetics, with a sequential type mechanism. However, the initial rate data did not allow distinction between ordered or random mechanisms. The results suggest that Na plus and K plus form a ternary complex with a specific K plus carrier molecule on the outer surface of the membrane prior to translocation and the release of K plus inside the cell.