External KCl Concentration Research Articles

Transporter networks can serve plant cells as nutrient sensors and mimic transceptor-like behavior

SummarySensing of external mineral nutrient concentrations is essential for plants to colonize environments with a large spectrum of nutrient availability. Here, we analyzed transporter networks in computational cell biology simulations to understand better the initial steps of this sensing process. The networks analyzed were capable of translating the information of changing external nutrient concentrations into cytosolic H+ and Ca2+ signals, two of the most ubiquitous cellular second messengers. The concept emerging from the computational simulations was confirmed in wet-lab experiments. We document in guard cells that alterations in the external KCl concentration were translated into cytosolic H+ and Ca2+ transients as predicted. We show that transporter networks do not only serve their primary task of transport, but can also take on the role of a receptor without requiring conformational changes of a transporter protein. Such transceptor-like phenomena may be quite common in plants.

Directional Potassium Transport through a Unimolecular Peptide Channel

AbstractThree unimolecular peptide channels have been designed and prepared by using the β‐helical conformation of gramicidin A (gA). The new peptides bear one to three NH3+ groups at the N‐end and one to three CO2− groups at the C‐end. These zwitterionic peptides were inserted into lipid bilayers in an orientation‐selective manner. Conductance experiments on planar lipid bilayers showed that this orientation bias could lead to observable directional K+ transport under multi‐channel conditions. This directional transport behavior can further cause the generation of a current across a planar bilayer without applying a voltage. More importantly, in vesicles with identical external and internal KCl concentrations, the channels can pump K+ across the lipid bilayer and cause a membrane potential.

Directional Potassium Transport through a Unimolecular Peptide Channel.

Three unimolecular peptide channels have been designed and prepared by using the β-helical conformation of gramicidin A (gA). The new peptides bear one to three NH3+ groups at the N-end and one to three CO2- groups at the C-end. These zwitterionic peptides were inserted into lipid bilayers in an orientation-selective manner. Conductance experiments on planar lipid bilayers showed that this orientation bias could lead to observable directional K+ transport under multi-channel conditions. This directional transport behavior can further cause the generation of a current across a planar bilayer without applying a voltage. More importantly, in vesicles with identical external and internal KCl concentrations, the channels can pump K+ across the lipid bilayer and cause a membrane potential.

Suppression of sensitivity to drugs and antibiotics by high external cation concentrations in fission yeast.

BackgroundPotassium ion homeostasis plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics and chemotherapeutic agents in Schizosaccharomyces pombe and other yeasts. However, the precise relationship between drug resistance in S. pombe and external potassium concentrations (particularly in its natural habitats) remains unclear. S. pombe can tolerate a wide range of external potassium concentrations which in turn affect plasma membrane polarization. We thus hypothesized that high external potassium concentrations suppress the sensitivity of this yeast to various drugs.MethodsWe have investigated the effect of external KCl concentrations on the sensitivity of S. pombe cells to a wide range of antibiotics, antimicrobial agents and chemotherapeutic drugs. We employed survival assays, immunoblotting and microscopy for these studies.ResultsWe demonstrate that KCl, and to a lesser extent NaCl and RbCl can suppress the sensitivity of S. pombe to a wide range of antibiotics. Ammonium chloride and potassium hydrogen sulphate also suppressed drug sensitivity. This effect appears to depend in part on changes to membrane polarization and membrane transport proteins. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated.ConclusionsHigh concentrations of external potassium and other cations suppress sensitivity to a wide range of drugs in S. pombe. Potassium-rich environments may thus provide S. pombe a competitive advantage in nature. Modulating potassium ion homeostasis may sensitize pathogenic fungi to antifungal agents.

The influence of gap junction network complexity on pulmonary artery smooth muscle reactivity in normoxic and chronically hypoxic conditions

Experiments on intrapulmonary arteries (IPAs) isolated from rats maintained in normoxia and chronic hypobaric hypoxia showed that in normoxia, the IPA contractile sensitivity to KCl was not modified by gap junction inhibition. In contrast, chronic hypoxia induced an endothelium-independent hypersensitivity, which was suppressed by gap junction inhibition. For the theoretical analysis of these results, we developed a model of interconnected myocytes. Given that smooth muscle cells in IPAs are known to communicate via gap junctions, we regard the cytoarchitecture of the IPA as a spatial network, in which nodes represent individual smooth muscle cells and the links signify intercellular communication. A single-cell model that drives the dynamics of individual nodes includes the major elements of voltage-dependent Ca(2+) signalling. In addition, interindividual variability of SMCs is introduced by distributing the reversal potentials for K(+). Cell-to-cell connection consists of passive Ca(2+) diffusion and electrical coupling, and connection between cells is determined by the topology of the intercellular network. Model predictions indicate that the experimental results can be explained by topological modifications and not by changes in the number of gap junctions. According to the model, in normoxia the myocytes are connected in a complex network, whereas chronic hypoxia is related to loss of complexity, leading to hypersensitivity. Our results thus indicate that chronic hypoxia entails gap junction network rearrangements, leading to disturbances in the intercellular communication pathways.

Pharmacological characterization of N-methyl-d-aspartic acid (NMDA)-like receptors in the single-celled organismParamecium primaurelia

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.

Biophysical effects of the natural product euplotin C on the Paramecium membrane

The effect of euplotin C--a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus--on the voltage-dependent Ca(2+) channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca(2+) channel blockers, we demonstrate that euplotin C modulates Ca(2+) channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca(2+) channel functions through the reduction of cell membrane fluidity.

Potassium/Proton Antiport System of Escherichia coli

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.

Effect of euplotin C on swimming behavior in Paramecium

The effect of euplotin C—a lipophilic toxic metabolite produced by the protist ciliate Euplotes crassus—on the swimming behavior was studied in a single‐celled system represented by the ciliate Paramecium primaurelia. When in P. primaurelia internal Ca2+ concentration coupled to membrane depolarization increases, a reversal in the direction of ciliary beating and consequently in swimming direction occurs. The ciliary reversal is correlated to Ca2+ influx amount. In this study, evidence was given that continuous ciliary reversal (CCR) duration, induced by high external KCl concentrations, is longer in euplotin C‐treated cells than in control cells. To test the hypothesis that euplotin C increases CCR duration by modulating a specific subtype of Ca2+ channel, selective Ca2+ channel blockers were used. Blocking L‐type channels by nimodipine and verapamil, N‐ and Q‐type channels by Ω‐conotoxins, fractions GVIA and MVIIC, significantly reduced the CCR duration evoked by membrane depolarization, suggesting an involvement of these channels in ciliary reversal in Paramecium. The effect of euplotin C on CCR duration persisted when Ω‐conotoxin GVIA or Ω‐conotoxin MVIIC were applied, conversely, it disappeared when L‐type channel blockers were used. The magnitude of the block by nimodipine and verapamil in the presence of euplotin C was comparable with that observed in the absence of euplotin C, suggesting that the Ca2+ channels modulated by euplotin C were dihydropyridine‐sensible calcium channels similar to L‐type channels found in mammalian cells.

A membrane potential-sensitive dye for vascular smooth muscle cells assays

Changes in membrane potential of rat aorta smooth muscle cells were investigated using the bis-oxonol sensitive probe DIBAC 2(3). We compared the changes in membrane potential induced by a high external KCl concentration in aorta smooth muscle cells from normotensive 2 kidney (2K) and from renal hypertensive 2 kidney-1 clip (2K-1C) rats. The spectral properties of the membrane potential were first characterized in aqueous buffers and in cultured smooth muscle cells from 2K and 2K-1C rat aortas. Fluorescence emission and the images were recorded using a laser scanning confocal microscope. The relationship between fluorescence intensity (FI) and membrane potential ( ψ m) as a function of the increasing extracellular KCl concentration was linear in the 5–40 mmol/L KCl range in both 2K and 2K-1C rat aorta cells. Cell membranes from 2K-1C rat aorta cells were more depolarized (−55 mV) than 2K rat aorta cells (−65 mV). The results show that in 2K-1C aorta cells only 10 mmol/L KCl was needed to induce complete membrane depolarization while in 2K cells 40 mmol/L KCl was needed to induce a similar effect. This study clearly shows that the method is suitable to measure the membrane potential in cultured smooth muscle cells.

Verapamil-induced kinetics of ion flux in oat seedlings

Verapamil application in ion transport studies on plant cells is widespread; however, the mechanism of its action is still poorly understood. Net flux of Ca 2+ , K + , Na + , H + and Cl – were measured in solution around oat seedlings using the non-invasive ion-selective microelectrode MIFE technique. The verapamil effect on intact plant tissues was a distinct immediate influx of monovalent cations, H + , K + or Na + . Pre-treatment with tetraethyl-ammonium, Cs + , Ba 2+ or Ca 2+ did not affect K + flux changes, whereas DMSO, Cd 2+ and vanadate increased verapamil-induced K + influx. Verapamil-induced K + flux increased with increased external KCl concentration or pH. Verapamil concentration up to 1 mM failed to shift Ca 2+ flux to efflux. Pre-treatment with adrenaline and dopamine agonists and antagonists led to changes in verapamil-induced ion flux, especially for Ca 2+ . We suggest that a transporter that allows entry of K + and Na + was the main system providing monovalent ion influx after verapamil application.

Microfabricated Liquid Junction Ag/AgCl Reference Electrode and Its Application to a One-Chip Potentiometric Sensor

A liquid-junction Ag/AgCl reference electrode was microfabricated. A silver thin-film pattern was covered with a polyimide protecting layer with a 50-μm-wide slit at the center of the pattern, and the AgCl layer was grown from there into the silver layer. A liquid junction was formed with a photocurable hydrophilic polymer. The electrolyte layer was formed by screen-printing a paste containing fine KCl powder. A silicone rubber passivation covered the entire area except for the pad and the end of the junction. The novel thin-film Ag/AgCl element could maintain its expected potential for longer than 30 h in a saturated KCl solution. The completed miniature liquid-junction reference electrode could maintain a stable potential level within ±1 mV for longer than 100 h with the aid of a poly(vinylpyrrolidone) (PVP) matrix in the electrolyte layer. Fluctuation of the potential was less than 0.1 mV at each moment. Dependency on external KCl concentration and pH was shown to be insignificant. The miniature refere...

Resting potential of rat cerebellar granule cells during early maturation in vitro.

The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca2+ channels and a membrane potential that can respond to changes in external K+. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca2+ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from -40 mV at the first day in vitro to -60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca2+ influx through L-type and N-type voltage-activated Ca2+ channels, functional at day 0. Gross estimations of the permeabilities of Na+ and Cl- were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K+ ions seems to underlie the evolution of the resting potential with time.

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid‐insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi‐1 and abi‐2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis. The largest light‐induced opening of A. thaliana stomates was found at an external KCl concentration of 50 mM. Stomatal apertures were increased by lowering external Ca2+ to 0.05 mM. The apertures of stomates incubated with 10 μM ABA were not altered by changes in Ca2+ from 0.05 to 1.0 mM.Stomates of all abi mutants showed a light‐stimulated stomatal opening. The opening of wild type and abi‐3 stomates was inhibited by ABA, while stomates of abi‐1 and abi‐2 did not respond to ABA. The insensitivity of abi‐1 and abi‐2 stomates to ABA may thus explain the observed disturbed water relations.

Regulation of Sucrose Efflux from Soybean Leaf Discs

Net sucrose efflux from discs of fully expanded leaves of soybean (Glycine max [L.] Merr.) plants was studied to characterize sucrose efflux into the apoplast. Net sucrose efflux had a Q(10) of 2.3, was linear for at least 3.5 hours, and was selective for sucrose over glucose. Sulfhydryl group inhibitors reduced sucrose efflux by up to 80%. There was a biphasic promotion of sucrose efflux by KCl with an apparent saturable component up to about 20 millimolar, above which the effect was linear. Sucrose efflux was promoted by NaCl as a linear function of concentration. Monovalent cation ionophores did not affect sucrose efflux, regardless of external KCl concentration. Light in the absence of added HCO(3)-increased sucrose efflux by about 20%. Sucrose efflux was promoted by increasing pH from 4 to about 8, above which no additional effect was observed. When leaf discs were bathed at pH 6.0, the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased sucrose efflux by about 25%. CCCP in the presence of valinomycin had the same effect as CCCP alone. Inhibition of plasmalemma ATPase activity with N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, or orthovanadate increased sucrose efflux. These data indicate that sucrose efflux from soybean leaf discs is not a result of simple leakage but is a regulated process.

The stoichiometry of the cardiac sodium-calcium exchange system.

A thermodynamic approach was adopted for determining the stoichiometry of the cardiac Na-Ca exchange system. Vesicles were equilibrated with 0.1 mM 45CaCl2 in a medium containing 30 mM NaCl, 20 mM KCl, and 110 mM LiCl. The vesicles were then treated with valinomycin and diluted into media containing the same 45CaCl2 and NaCl concentrations as in the equilibration medium but with the external KCl concentration adjusted so as to impose either positive or negative membrane potentials (delta psi). 45Ca2+ uptake ensued upon establishing a positive (inside) potential whereas 45Ca2+ efflux was observed for a negative delta psi. These delta psi-dependent Ca2+ movements did not occur if NaCl was omitted from the media, indicating that they were mediated by the Na-Ca exchanger. High concentrations of either NaCl or CaCl2 inhibited the delta psi-dependent Ca2+ movements. To determine the exchange stoichiometry, a series of Na+ gradients were established across the vesicle membrane so as to oppose the effects of a constant delta psi (either positive or negative) on Ca2+ movements. The stoichiometry n could be determined from the magnitude of the Na+ gradient that exactly compensated for delta psi such that no net Ca2+ movement occurred. This point is defined by the relation (n - 2)delta psi = nENa where ENa is the equilibrium potential for Na+ under these conditions. The value of n (+/- S.E.) determined in this way was 2.97 +/- 0.03 (n = 9).

Properties of the Isolated Intact Chloroplast at Cytoplasmic K+ Concentrations

Photosynthesis, stroma-pH, and internal K(+) and Cl(-) concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K(+), H(+), Cl(-)) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).Isolated intact chloroplasts are a Donnan system which accumulates cations (K(+) or added Tetraphenylphosphonium(+)) and excludes anions (Cl(-)) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium(+) distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K(+) or Na(+) and the release of protons all become very small. Light-induced K(+) uptake is not abolished by valinomycin suggesting that the K(+) uptake is not primarily active. Intact chloroplasts contain higher K(+) concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO(2) fixation at high K(+) concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO(2) fixation in intact chloroplasts.

The effects of cholesterol inclusion on the molecular organisation of bimolecular lipid membranes

Dielectric measurements on planar egg phosphatidylcholine bilayers formed from n- hexadecane solutions indicate that these bilayers contain very low equilibrium concentrations of alkane. In 100 mM KCl the capacitance of the hydrophobic region was found to be 7.0 ±0.2 mF/m 2 . The addition of cholesterol (at 2:1 mole ratio) was found to affect only marginally the capacitance of the hydrophobic region of such bilayers. Precise measurements of the frequency dependence of the bilayer impedance at very low frequencies now allow the resolution of several electrically distinct substructural regions within the bilayer. Examination of the effects of cholesterol inclusion upon the electrical parameters of these substructural regions indicate that cholesterol spans the acetyl region (i.e. the region containing the glycerol bridge of the phosphatidylcholine molecules in the bilayer) with the hydroxyl group of the cholesterol molecules located inbetween the phosphate group and the glycerol oxygens of the phosphatidylcholine molecules. The capacitance of the hydrophobic region of both phosphatidylcholine and phosphatidylcholine/cholesterol bilayers formed from n- hexadecane solutions was found to decrease slightly as the external KCl concentration was decreased.

Study of Ion Transport across Amphoteric Ion Exchange Membrane. I. Electroconductive Membrane Permeability

Abstract Basic ion transport characteristics of a homogeneous amphoteric ion exchange membrane (Kanegafuchi Chem. Ind. 1.0-PA-29) were studied by measuring the membrane potentials and conductances. Concentration cell systems of NaCl, KCl, CaCl2, and SrCl2 with a concentration range of 10−1–10−3 mol dm−3 were studied and the electroconductive membrane permeabilities were estimated from the electrochemical data. Generally, Cl− is slightly more permeable than cations under the present experimental conditions, except the result that K+ is more permeable at low concentrations. Unlike the usual uniform cation exchange membrane of which transport characteristics to ions strongly depend upon the number of ionic charges, a “1.0-PA-29” amphoteric membrane exhibits different transport properties for the NaCl and KCl systems. In the NaCl system, the membrane was constantly anionic selective, while in the KCl system, biphasic ion selectivities were observed depending upon the external KCl concentration. Fundamental electrochemical properties of the membrane systems were discussed by examining the dependencies of membrane permeability on external electrolyte concentrations and on differences in ionic species. The present result suggests that the ionic size is one of the critical factors determining the transport characteristics across amphoteric ion exchange membrane.

A light-dependent current associated with chloroplast aggregation in the alga Vaucheria sessilis

Local irradiation of the alga Vaucheria sessilis (Vauch.) D.C. with blue light, which stimulates cortical fiber reticulation and chloroplast aggregation (M.R. Blatt and W.R. Briggs, 1980, Planta 147, 355-362), also induces an outward-directed current from the irradiated region of the cell. This current appears in conjunction with cortical fiber reticulation and precedes chloroplast aggregation. The current is not photosynthetic in origin, as indicated by experiments with 3(3,4-dichlorophenyl)-1,1-dimethyl urea and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP). It shows a wavelength-dependence similar to that of chloroplast aggregation and reaches a maximum of 500 nA cm(-2) with saturating light intensities. The current is not dependent upon the presence of Na(+), K(+), or Cl(-) in a test medium containing only Na(+), K(+), Ca(2+), and Cl(-), but is inhibited, apparently nonspecifically, in the absence of external calcium. Both the light-induced current and chloroplast aggregation are stimulated by increases in the external KCl concentration and are inhibited by sub-micromolar concentrations of CCCP or by external pHs below approximately 5.5. We suggest that blue light stimulates the local extrusion of cations, possibly of protons, at the plasma membrane, an event which may act to destabilize the cortical fibers in Vaucheria, disrupt cytoplasmic streaming, and eventually lead to organelle aggregation in the light.