Effect of ATPase inhibitors on cell potential and k influx in corn roots

Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Ψ) and K⁺ (⁸⁶Rb) influx of corn root tissue over a wide range of K⁺ activity. N,N′Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K⁺ influx and depolarized Ψ at low, but not at high, K⁺ activity (K°). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K⁺ influx over a wide range of K°, with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Ψ_p) above 0.2 millimolar K°. Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K⁺ influx and Ψ_p at low K° can be attributed to inhibition of the plasmalemma ATPase.     

H and k electrogenic exchanges in corn roots

The membrane potential difference, the net H⁺ exchange rate, the K⁺ net flux, and the K⁺ (⁸⁶Rb⁺) influx were measured in excised corn roots as functions of the K⁺ concentration in the medium at various pH values, in the presence of poorly permeant anions. The roots behaved as a K⁺/H⁺ exchange system. By comparing the results in normal or hypoxic conditions, or in the presence of vanadate, it was possible to distinguish the active components of membrane potential and transports from the passive ones. The magnitude of the electrogenic potential was not related to the active H⁺ extrusion rate. At pH 6, the variations of the electrogenic potential resulted from variations of the stoichiometry of the active H⁺/K⁺ exchange. The same relationship between this stoichiometry and the K⁺ concentration was observed in conditions ensuring different membrane polarizations (pH 6, pH 4, or pH 6 with fusicoccin). Both metabolic and Mg-ATPase specific inhibitors stopped the active H⁺ transport and the net K⁺ influx. Nevertheless, the tracer influx in the presence of vanadate remained higher than the passive influx calculated from the permeability coefficient determined in hypoxia. It is proposed that vanadate uncouples the K⁺ moiety of the H⁺/K⁺ antiport and allows it to mediate isotopic exchanges.     

The Influence of the Intracellular Potential on Potassium Uptake by Beetroot Tissue

Intracellular potentials were measured in beetroot tissue during the steady-state uptake of K⁺ from various solutions. In solutions containing bicarbonate, the membrane potential becomes up to 70 mv more negative than the estimated equilibrium potential for K⁺. The uptake of K⁺ from such solutions is correlated with variations in the potential, both when the bicarbonate concentration is changed and also when the metabolic activity of the tissue is changed by washing in water for various periods. However, the estimated permeability to K⁺ varies from 0.4 x 10^-7 to 1.5 x 10^-7 cm·sec^-1. It is postulated that the change of potential arises from the metabolic transport of HCO₃^- into the cell or H⁺ outwards, and that the associated uptake of K⁺ is partly or entirely by passive diffusion across the cell membrane. In contrast, K⁺ uptake from KCl solutions is not accompanied by any significant change in the membrane potential, which remains relatively close to the K⁺ equilibrium potential. In solutions containing both KHCO₃ and KCl, it appears that an amount of K⁺ equal to the influx of Cl^- is taken up independently of the potential, while the component of K⁺ uptake which is not balanced by Cl^- uptake is related to the potential in the manner described. These results suggest that K⁺ uptake is linked to Cl^- uptake in an electrically neutral active transport process.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Complexity of potassium acquisition: How much flows through channels?

The involvement of potassium (K⁺)-selective, Shaker-type channels, particularly AKT1, in primary K⁺ acquisition in roots of higher plants has long been of interest, particularly in the context of low-affinity K⁺ uptake, at high K⁺ concentrations, as well as uptake from low-K⁺ media under ammonium (NH₄⁺) stress. We recently demonstrated that K⁺ channels cannot mediate K⁺ acquisition in roots of intact barley (Hordeum vulgare L.) seedlings at low (22.5 µM) external K⁺ concentrations ([K⁺]_ext) and in the presence of high (10 mM) external NH₄⁺, while the model species Arabidopsis thaliana L. utilizes channels under comparable conditions. However, when external NH₄⁺ was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K⁺ influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K⁺ influx, illustrating a hitherto unexplored magnitude and rapidity of K⁺-uptake capacity and plasticity. Here, we expand on our previous work by offering further characterization of channel-mediated K⁺ fluxes in intact barley, with particular focus on anion effects, root respiration and pharmacological sensitivity and highlight key additions to the current model of K⁺ acquisition.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Ion fluxes and phytochrome protons in mung bean hypocotyl segments: I. Fluxes of potassium

K⁺ [⁸⁶Rb⁺] uptake by Phaseolus aureus Roxb. hypocotyl segments cut immediately below the hook is inhibited by the active form of phytochrome (Pfr). Short load-short wash experiments indicate that the inhibition of uptake occurs across the plasmalemma. A maximal inhibition of short term uptake occurs in 10 to 50 millimolar KCI. Low temperature had only a small effect on influx and the inhibition of influx from 50 millimolar KCI. A consideration of the electrochemical gradient for K⁺ suggests that passive K⁺ fluxes may predominate under these conditions. Red light induces small depolarizations of membrane potential in subhook cells. Far red light antagonizes this effect. Pfr inhibits efflux of K⁺[⁸⁶Rb⁺] from subhook segments. This effect is also relatively insensitive to low temperature. This inhibition of efflux may reflect inhibition of a K⁺ -K⁺ exchange process, or reduced passive permeability of the plasmalemma to K⁺. In contrast, Pfr enhances short term uptake of K⁺[⁸⁶Rb⁺] in apical hypocotyl hook segments of Phaseolus aureus Roxb. Short load-short wash experiments indicate that fluxes across the plasmalemma are modified by Pfr. A maximal enhancement of short term influx occurs in 50 millimolar KCI. Influx and the red light enhancement of influx from 50 millimolar KCI are relatively insensitive to low temperature. Pfr also enhances efflux of K⁺[⁸⁶Rb⁺] from preloaded apical hook segments. This increased influx may reflect enhancement of a K⁺ -K⁺ exchange process or increased passive permeability of the plasmalemma to K⁺.     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots

The influx of K⁺ into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K⁺ was measured in K⁺ solutions ranging in concentration from 0.01 to 50 mM. In both species the K⁺ influx was lower in the roots with high K⁺ content. The extent of reduction by high internal [K⁺] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.     

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.     

A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

Astrocytes fulfill a central role in regulating K⁺ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na⁺ ions and one proton and the efflux of one K⁺ ion. Thus, intracellular K⁺ concentration ([K⁺]_i) is potentially influenced both by extracellular K⁺ concentration ([K⁺]_o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K⁺ ion movements on [K⁺]_i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K⁺]_i using the recently developed K⁺ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K⁺]_i at 133±1 mM. We first investigated the dependency of [K⁺]_i levels on [K⁺]_o. We found that [K⁺]_i followed [K⁺]_o changes nearly proportionally in the range 3–10 mM, which is consistent with previously reported microelectrode measurements of intracellular K⁺ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K⁺]_i that depended on the glutamate concentration with an apparent EC₅₀ of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K⁺]_i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K⁺ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K⁺]_i regulation with excellent spatial resolution.     

Plasma Membrane-associated Adenosine Triphosphatase Activity of Isolated Cortex and Stele from Corn Roots

The plasma membrane fractions from separated cortex and stele of primary roots of corn (Zea mays L. WF9 × M14) contained cation ATPase activity at similar levels but with somewhat different properties. ATPase activity from cortex was optimum at pH 6.5, showed a simple Michaelis-Menten saturation with increasing ATP·Mg, and showed complex kinetic data for K⁺ stimulation similar in character to the kinetic data for K⁺-ATPase and K⁺ influx in primary roots. The results for cortex indicate that homogenates of primary roots are dominated by membranes from cortical cells.

ATPase activity from stele was optimum at pH 6.5 and showed another maximum at pH 9. At pH 6.5, activity from stele had properties similar to that from cortex except that the kinetics of K⁺ stimulation closely approached that expected for a Michaelis-Menten enzyme. At pH 9, the enzyme activity from stele was inhibited by 5 μg/ml oligomycin, suggesting that a significant portion of the activity was of mitochondrial origin. Sucrose density gradient analysis indicated some contamination of mitochondrial membranes in the plasma membrane fraction from stele. The results for stele are consistent with the view that stelar parenchyma cells are not deficient in ion pumps.

     

The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K _u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.     

Simultaneous Measurements of Cytoplasmic K Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina

The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K⁺-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (K_o⁺), the resting conductance (membrane conductance G_m, 0.85 ± 0.25 Siemens per square meter (±standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K⁺ activities (a_K+), judged most reliable, gave values of 117 ± 67 millimolar; stable a_K+ values were 77 ± 31 millimolar. Equilibrium potentials for K⁺ (Nernst equilibrium potential) (E_K) calculated, using either of these data sets, were near the mean membrane potential (V_m). On a cell-to-cell basis, however, E_K was generally negative of the V_m, despite an electrogenic contribution from the Chara proton pump. When K_o⁺ was increased to 1.0 millimolar or above, G_m rose (by 8- to 10-fold in 10 millimolar K_o⁺), the steady state I-V characteristics showed a region of negative slope conductance, and V_m followed E_K. These results confirm previous studies which implicated a K_o⁺-induced and voltage-dependent permeability to K⁺ at the Chara plasma membrane. They provide an explanation for transitions between apparent K_o⁺-insensitive and K_o⁺-sensitive (`K⁺ electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells.     

Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth

Root temperature strongly affects shoot growth, possibly via “nonhydraulic messengers” from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25° and 35°C, respectively. Hydraulic conductance (L_p) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25°C, remaining high with further warming. In sorghum, the L_p of intact plants and of detached roots peaked at 35°C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L_p. Extreme temperatures greatly decreased ion flux, particularly K⁺ and NO₃⁻, to the xylem of detached roots of both species. Removing external K⁺ did not alter short-term K⁺ flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently “uncoupled” from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions

1. The adenosine-triphosphatase activity of rat-brain microsomes was measured between 0° and 37°. The stimulatory effect of Na⁺ plus K⁺ on the Mg²⁺-dependent adenosine-triphosphatase activity decreased sharply with decreasing temperature and became negligible at 0°. An Arrhenius plot drawn from the experimental data showed two discontinuities: one at about 6° and the other at about 20°. 2. The increment in activity induced by Na⁺ plus K⁺ was more sensitive to oligomycin at lower than at higher temperatures, but the opposite was observed for ouabain. The action of oligomycin showed a biphasic character, since below a certain concentration it caused slight activation of Na⁺-plus-K⁺-activated adenosine triphosphatase. 3. Where oligomycin increased the activity of the enzyme, it also enhanced the accumulation of an acid-precipitable phosphorylated compound formed through the transfer of the γ-phosphate group of [³²P]ATP to the enzyme system. Stimulatory concentrations of oligomycin did not interfere with K⁺-mediated dephosphorylation of the intermediate, though high concentrations of oligomycin counteracted the effect of K⁺. 4. The temperature profile of K⁺-stimulated microsomal phosphatase qualitatively resembled that of microsomal adenosine triphosphatase.     

Electrogenic pump activity in red beet: Its relation to ATP levels and to cation influx

Summary In storage tissue ofBeta vulgaris L., carbonyl cyanidem-chlorophenylhydrazone or cyanide+salicylhydroxamic acid reduce cell electropotentials from about –200 to below –100 mV. The relationship between potential and cellular ATP level is examined during treatment with different concentrations of inhibitiors. At low ATP levels the potential rises sharply with increases in ATP, but above an ATP level of approximately 50% of the uninhibited level the potential changes very little with ATP concentration. A plot of membrane potentialvs.⁸⁶Pb⁺ influx or of potentialvs. net K⁺ uptake indicates that as the level of inhibition is decreased, the potential tends to reach a limit while cation influx and net uptake continue to increase. Resistance measurements, although subject to difficulties of interpretation, indicate no change in conductance with potential, ion flux, or ATP level. Thus the membrane potential should directly reflect electrogenic pump activity, attributed to active uncoupled H⁺ efflux. K⁺ uptake can occur against its electrochemical gradient and is attributed to a coupled K⁺ influx/H⁺ efflux pump. The results show that the electrogenic pump activity is independent of the K⁺/H⁺ exchange rate. Thus electrogenic H⁺ efflux and K⁺/H⁺ exchange may represent different transport systems, or different modes of operation of a single pump with variable stoichiometry.     

Regulation of Rubidium Uptake in Sunflower Roots

Uptake of Rb⁺ was investigated in 12-day-old intact plants of sunflower (Helianthus annum L. var. californicus) which had been cultivated or pretreated in nutrient solutions with various K⁺ concentrations. The relationship between Rb⁺ influx and K⁺ concentration of the roots indicated regulation of Rb⁺ uptake by allosteric inhibition of the uptake mechanism. A constant passive influx occurred contemporaneously with the active uptake as shown by experiments at 0°C or with 2,4-dinitrophenol. The allosteric regulation of ion carrier activity occurred after a time lag of up to 1 h after the change of external solution. In experiments involving Rb⁺ treatments of K⁺-deficient plants, the synthesis of carriers for transport of Rb⁺ could be demonstrated. A model including allosteric regulation of Rb⁺ uptake in roots is discussed.