K+-permeability of the outer border of the frog skin (R. temporaria)

Summary Skins fromRana temporaria, investigated with microelectrode techniques in the absence of Na uptake across the outer border (Na-free epithelial solution or amiloride), were found to be permeable to K⁺ at the apical membrane in 10–20% of the experiments. Full development of the K⁺-permeable state requires the absence of Na⁺ uptake for certain periods of time, which suggests that the K⁺-permeability of the apical membrane is higher at lower intracellular [Na]. The addition of Ba⁺⁺ reduces the K⁺-permeability of the apical membrane. These skins may provide a model for the study of transcellular K⁺ movements.     

A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode disks

Internode disks of tomato (Lycopersicon esculentum cv. Moneymaker) were shaken in glutamine and sucrose solutions. At low external pH (<±5.5), the uptake of these substances was accompanied with K⁺ efflux, at high pH (>±5.5) with K⁺ influx. Low concentrations of external K⁺ (2 mmol l^-1) stimulated the uptake of glutamine, which was strongly inhibited by the supply of high K⁺ concentrations (20 mmol l^-1). The effect of K⁺ was particularly pronounced at high pH-values. Addition of CCCP in light reduced the uptake of glutamine to the same level as in the dark, and stopped the K⁺ fluxes which coincided with the uptake. A model is presented wherein the movements of K⁺ across the membrane are related to co-transport, depending on the membrane potential and the Nernst potential of K⁺.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone     

Effect of sodium on potassium fluxes at the cell membrane and vacuole membrane of red beet

Slices of red beet (Beta vulgaris) washed for 5 to 6 days are known to accumulate Na⁺ in preference to K⁺ from solutions containing both ions. The present work, using ion concentrations of 1.0 mm or less, with Ca²⁺ added in some cases, shows that Na⁺ strongly inhibits K⁺ influx at the cell membrane (plasmalemma) while K⁺ efflux is increased to a lesser extent. This result from compartmental analysis is confirmed by short (15-minute) influx experiments, which indicate an immediate inhibitory effect of Na⁺ on K⁺ influx at the cell membrane. It is concluded that cation selectivity, even when Na⁺ is favored for uptake, is primarily determined at the cell membrane. Nevertheless, a high level of K⁺ in the cytoplasm is maintained during Na⁺ influx, by an inhibition of K⁺ transfer to the vacuole.     

Potassium-activated phosphatase from human red blood cells

Summary The cell membrane K⁺-activated phosphatase activity was measured in reconstituted ghosts of human red cells having different ionic contents and incubated in solutions of varying ionic composition. When K⁺-free ghosts are suspended in K⁺-rich media, full activation of the phosphatase is obtained. Conversely, very little ouabainsensitive activity is detected in K⁺-rich ghosts suspended in K⁺-free media. These results, together with the fact that Na⁺ competitively inhibits the effects of K⁺ only when present externally, show that the K⁺ site of the membrane phosphatase is located at the outer surface of the cell membrane. The Mg⁺⁺ requirements for K⁺ activation of the membrane phosphatase are fulfilled by internal Mg⁺⁺. Addition of intracellular Na⁺ to ATP-containing ghosts raises the apparent affinity of the enzyme for K⁺, suggesting that the sites where ATP and Na⁺ produce this effect are located at the inner surface of the cell membrane. The asymmetrical features of the membrane phosphatase are those expected from the proposed role of this enzyme in the Na⁺–K⁺-ATPase system.The authors are established investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Transmembrane potential-mediated coupling between H+ pump operation and K+ fluxes in Elodea densa leaves hyperpolarized by fusicoccin, light or acid load

In isolated Elodea densa leaves, the relationships between H⁺ extrusion (-ΔH⁺), K⁺ fluxes and membrane potential (E_m) were investigated for two different conditions of activation of the ATP-dependent H⁺ pump. The ‘basal condition’ (darkness, no pump activator present) was characterized by low values of-ΔH⁺ and K⁺ uptake (ΔK⁺), wide variability of the ?ΔH⁺/ΔK⁺ ratio, relatively low membrane polarization and E_m values more positive than EK for external K⁺ concentrations (|K⁺]_o of up to 2mol m^?3. A net K⁺ uptake was seen already at [K⁺]_o below 1 mol m^?3, suggesting that K⁺ influx in this condition was a thermodynamically uphill process involving an active mechanism. When the H⁺ pump was stimulated by fusicoccin (FC), by cytosol acidification, or by light (the ‘high polarization condition’), K⁺ influx largely dominated K⁺ and C^? efflux, and the ?ΔH⁺/ΔK⁺ ratio approached unity. In the range 50 mmol m^?3?5 mol m^?3 [K⁺]₀, E_m was consistently more negative than E_K. The curve of K⁺ influx at [K⁺]₀ ranging from 50 to 5000mmol m^?3 fitted a monophasic, hyperbolic curve, with an apparent half saturation value = 0–2 mol m^?3. Increasing |K⁺]₀ progressively depolarized E_m, counteracting the strong hyperpolarizing effect of FC. The effects of K⁺ in depolarizing E_m were well correlated with the effects on both K⁺ influx and ?ΔH⁺, suggesting a cause-effect chain: K⁺₀ influx → depolarization → activation of H⁺ extrusion. Cs⁺ competitively inhibited K⁺ influx much more strongly in the ‘high polarization’ than in the ‘basal’ condition (50% inhibition at [Cs⁺]/[K⁺]₀ ratios of 1:14 and 1:2, respectively) thus confirming the involvement of different K⁺ uptake systems in the two conditions. These results suggest that in E. densa leaves two distinct modes of interactions rule the relationships between H⁺ pump, membrane polarization and K⁺ transport. At low membrane polarization, corresponding to a low state of activation of the PM H⁺-ATP^ase and to E_m values more positive than E_K, K⁺ influx would mainly     

Resting potential of the mouse neuroblastoma cells: I. The presence of K+ channels activated at high K+ concentration but closed at low K+ concentration including the physiological concentration

The effects of changes in extracellular K⁺ concentration ([K⁺]_o) on the resting membrane potential, the input resistance and ⁸⁶Rb efflux (as a marker of K⁺ efflux) were examined with use of the cultured mouse neuroblastoma cells (N-18 clone). The results obtained are as follows. (1) The membrane potential was depolarized, with an increase in [K⁺]_o at concentrations above 10–20 mM at a rate of 55–58 mV per 10-fold change in [K⁺]_o, but practically unchanged with varying [K⁺]_o below this concentration. (2) Above the critical [K⁺]_o of 10–20 mM, the input membrane resistance decreased sharply by a factor of $\text{1}\text{4}\text{?}\text{1}\text{5}$ with an increase in [K⁺]_o. A similar decrease in the resistance occurred even under the conditions that the membrane potential was held at control level (about ?55 mV) by a steady-state current passage. (3) Elimination of Na⁺ and Cl^? from the external solution brought about practically no change in the membrane potential. (4) A fractional escape rate of ⁸⁶Rb from N-18 cells remained constant at relatively low level (0.125%/min on average) in the low [K⁺]_o range, but increased sharply with increasing [K⁺]_o above 15 mM (e.g., approx. 3.4- and 4.5-fold at 30 and 100 mM [K⁺]_o, respectively). (5) The high K⁺-induced ⁸⁶Rb efflux was not practically inhibited by 1 mM tetraethylammonium or 0.1 mM 4-aminopyridine, indicating that the K⁺ channels activated by an elevation of [K⁺]_o are not the delayed (voltage-dependent) K⁺ channels. The present results favoured the conclusion that N-18 cells carry K⁺ channels which open at high [K⁺]_o but are closed at low [K⁺]_o including the physiological range for the mouse neuroblastoma cells (around 5.4 mM). This conclusion leads to the notion that in the mouse neuroblastoma N-18 cells the K⁺ permeability does not mainly contribute to determining the resting membrane potential under physiological conditions.     

Ba2+-sensitive potassium permeability of the apical membrane in newt kidney proximal tubule

Summary The apical membrane K⁺ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V _a change) during alteration of luminal K⁺ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V _a change/decade for K⁺ was 50 mV at K⁺ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K⁺ concentration was increased from 2.5 to 25mm. Ba²⁺ (1mm in the lumen) reducedV _a change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba²⁺-sensitive K⁺ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K⁺ activity measured by K⁺-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K⁺ across both cell membranes. Thus, it is concluded that cell K⁺ passively diffuses, at least in part, through the K⁺ conductive pathway of the apical membrane.     

Mechanisms by which Li+ stimulates the (Na++K+)-dependent ATPase

The addition of LiCl stimulated the (Na⁺+K⁺)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na⁺/low K⁺ media and at low Mg. ATP concentrations. Apparent affinities for Li⁺ were estimated at the α-sites (moderate-affinity sites for K⁺ demonstrable in terms of activation of the associated K⁺-dependent phosphatase reaction), at the β-sites (high-affinity sites for K⁺ demonstrable in terms of activation of the overall ATPase reaction), and at the Na⁺ sites for activation. The relative efficacy of Li⁺ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li⁺ was substituted for K⁺, and also in terms of the relative effect of Li⁺ on the apparent K_M for Mg· ATP. With these data, and previously determined values for the apparent affinities of K⁺ and Na⁺ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li⁺ stimulates by relieving inhibition due to K⁺ and Na⁺ (i) by competing with K⁺ for the α-sites on the enzyme through which K⁺ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na⁺ at low-affinity inhibitory sites, which may represent the external sites at which Na⁺ is discharged by the membrane NA⁺/K⁺ pump that this enzyme represents. Both these sites of action for Li⁺ would thus lie, in vivo, on the cell exterior.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

The action of androgens on Na+,K+-ATPase activity of erythrocyte membranes

The effect of androgens (testosterone, androsterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) on erythrocyte membrane during their nonspecific binding was investigated. The change in erythrocyte membrane Na⁺,K⁺-ATPase activity was measured at different hormone concentration in a suspension. It is shown that the dependence has dome-shaped character: at the elevated hormone concentration Na⁺,K⁺-ATPase activity starts to increase, reaches its maximum, and then decreases. The hypothesis is put forward that an increase in microscopists of erythrocyte membrane first intensifies Na⁺,K⁺-ATPase activity due to the growth of the maximum energy of membrane phonons, and then decreases it due to hindering conformational transitions in the enzyme molecule.     

Identification of Electric-Field-Dependent Steps in the Na,K-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Kinetics of Na+-, K+-ATPase Inhibition by an Endogenous Modulator (II-A)

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na⁺, K⁺-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na⁺, K⁺-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na⁺ (3.1–100 mM), or K⁺ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases V_max in all cases; K_M value decreased for ATP but remained unaltered for Na⁺ and K⁺, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K⁺ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K⁺ concentration.     

Intracellular potential and K+ activity in rat kidney proximal tubular cells in acidosis and K+ depletion

Summary Techniques were developed for the measurement of intracellular potentials and potassium activities in rat proximal tubule cells using double barreled K⁺ liquid-ion-exchanger microelectrodes. After obtaining measurements of stable and reliable control values, the effects of K⁺ depletion and metabolic and respiratory acidosis on the intracellular potential and K⁺ activity in rat kidney proximal tubular cells were determined. At a peritubular membrane potential of –66.3±1.3 mV (mean±se), intracellular K⁺ activity was 65.9±2.0 mEq/liter in the control rats. In metabolic acidosis [70 mg NH₄ Cl/100 g body wt) the peritubular membrane potential was significantly reduced to –47.5±1.9 mV, and cellular K⁺ activity to 53.5±2.0 mEq/liter. In contrast, in respiratory acidosis (15% CO₂) the peritubular membrane potential was significantly lowered to –46.1±1.39 mV, but the cellular K⁺ activity was maintained at an almost unchanged level of 63.7±1.9 mEq/liter. In K⁺ depleted animals (6 weeks on low K⁺ diet), the peritubular membrane potential was significantly higher than in control animals, –74.8±2.1 mV, and cellular K⁺ activity was moderately but significantly reduced to 58.1±2.7 mEq/liter. Under all conditions studied, cellular K⁺ was above electrochemical equilibrium. Consequently, an active mechanism for cellular K⁺ accumulation must exist at one or both cell membranes. Furthermore, peritubular HCO₃ ^– appears to be an important factor in maintaining normal K⁺ distribution across the basolateral cell membrane.     

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K⁺]_o. When the membrane voltage was more positive than −50 millivolts, the membrane conductance in the whole-cell mode was low, and K⁺ channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than −50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K⁺ compared with Cs⁺, Na⁺, choline or Cl⁻. Low concentrations of [Cs⁺]_o or [Na⁺]_o blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K⁺ channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K⁺]_o about 10 millimolar, the influx through these channels is sufficient to increase the internal [K⁺] by 2 millimolar per minute. These K⁺ channels are activated by membrane voltages in the normal physiological range and could contribute to K⁺ uptake whenever the membrane is more negative than the K⁺ equilibrium potential.     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium

Summary We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in anin vitro preparation of bullfrog RPE-choroid. Changes in RPE [K⁺] _i alter the current produced by this pump. Increasing [K⁺] _o in the solution perfusing thebasal membrane increases RPE [K⁺] _i (measured with a K⁺-specific microelectrode), and also depolarizes theapical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0mm [K⁺] _o outside the apical membrane. Removal of Cl^– from the solution perfusing the basal membrane abolishes the K⁺-evoked apical depolarization by preventing the entry of K⁺ (as KCl) into the cell. We conclude that the increase in [K⁺] _i causes the decrease in pump current. This result is consistent with the finding that [K⁺] _i is a competitive inhibitor of the Na⁺–K⁺ pump in red blood cells.It is possible that the light-evoked changes in [K⁺] _o in the distal retina could alter RPE [K⁺] _i , and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.     

Evidence for a highly specific k/h antiporter in membrane vesicles from oil-seed rape hypocotyls

We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substrate-specific, K⁺/H⁺ antiport in both tonoplast and plasma membrane-enriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K⁺ (with Cl⁻ or SCN⁻) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H⁺-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K⁺/H⁺ exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCl was supplied, even in the absence of 100 millimolar Cl⁻ and, second, efflux of K⁺ from K⁺-loaded vesicles drives intravesicular accumulation of H⁺ against the electrochemical potential gradient. Neither was the exchange due to competition between K⁺ and quinacrine for membrane sites, nor to inhibition of the H⁺-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K⁺ (at micromolar concentrations); Na⁺/H⁺ antiport was detected only at concentrations two orders of magnitude higher. Rb⁺, Li⁺, or Cs⁺ were ineffective. Dependence of tonoplast K⁺/H⁺ antiport on K⁺ concentration was complex, showing saturation at 10 millimolar K⁺ and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar NO₃⁻ and showed no sensitivity to vanadate or oligomycin) and with plasma membrane-enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol-sensitive but showed no sensitivity to NO₃⁻ or oligomycin). The possible physiological role of such a K⁺/H⁺ exchange mechanism is discussed.     

Effect of potential-dependent potassium uptake on calcium accumulation in rat brain mitochondria

The effect of potential-dependent potassium uptake at 0–120 mM K⁺ on matrix Ca²⁺ accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K⁺ content in the medium was observed due to K⁺ uptake into the mitochondria. The accumulation of Ca²⁺ was shown to depend on K⁺ concentration in the medium. At K⁺ concentration ?30 mM, Ca²⁺ uptake is decreased due to K⁺-induced membrane depolarization, whereas at higher K⁺ concentrations, up to 120 mM K⁺, Ca²⁺ uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K⁺ uptake. Mitochondrial K _ATP ⁺ -channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K⁺ uptake as well as K⁺-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca²⁺ uptake, i.e. increase in Ca²⁺ uptake at low K⁺ content in the medium due to the smaller membrane depolarization and decrease in Ca²⁺ uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K _ATP ⁺ channel, in the regulation of calcium accumulation in rat brain mitochondria.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.