Carrier-mediated Potassium Efflux Across the Cell Membrane of Red Beet

The flux ratio (influx/efflux) of K⁺ across the plasmalemma of beet cells at an external potassium concentration of 0.6 mm does not respond to changes of membrane potential in the manner expected for the free diffusion of ions. The K⁺ efflux is affected by the presence of adsorbed Ca²⁺, but is apparently unrelated to the electrical potential or to the net uptake of potassium. The K⁺ efflux is greater than the efflux of the sulfate and organic anions which are accumulated with potassium, and is partially dependent on the presence of external potassium. Thus the loss of ⁴²K from the cell does not appear to be a leakage of freely diffusing K⁺ ions, nor a leakage of ion pairs, but a carrier-mediated transport or exchange of potassium across the cell membrane.     

Ion transport by heart mitochondria. XXII. Spontaneous, energy-linked accumulation of acetate and phosphate salts of monovalent cations

The accumulation of monovalent cations by isolated beef heart mitochondria has been studied by evaluating the efficiency of energy-dependent osmotic swelling. Extensive osmotic swelling occurs spontaneously when isolated heart mitochondria are suspended in 0.1 m acetate or phosphate salts. The swelling and ion uptake depend on either respiration or the presence of exogenous ATP, and the initial rate of swelling is proportional to the initial rate of respiration or ATP hydrolysis, respectively. The efficiency of the reaction varies somewhat from preparation to preparation but approaches a limit of about 2 cations accumulated per pair of electrons traversing a phosphorylation site. All monovalent cations tested support the reaction, but the most efficient energy-dependent swelling occurs with K⁺. Weak acid anions are required for the ion accumulation and swelling and the reaction appears to depend on the amount of free acid available in the suspension. Permeant strong acid anions, such as NO₃⁻, fail to support the swelling reaction in the presence of energy. Valinomycin increases both the amount and the efficiency of ion uptake under these conditions. Mg²⁺ decreases both of these values whereas p-chloromercuriphenyl sulfonate increases both. These responses are discussed in terms of current models of mitochondrial ion transport.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

The kinetics of sodium extrusion in striated muscle as functions of the external sodium and potassium ion concentrations

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na⁺ efflux that depends upon the external sodium ion concentration can be abolished by 10^-5 M glycoside. The glycoside-insensitive but external sodium-dependent Na⁺ efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10^-5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K]_o to the left and upwards.     

Relation between sodium-coupled amino acid and sugar transport and sodium/potassium pump activity in isolated intestinal epithelial cells

Cells isolated from the epithelium of the small intestine are used to study the relationship between amino acid or sugar-coupled sodium transport and potassium uptake through the sodium/potassium pump. Potassium influx is a saturable function of the external potassium concentration. Uptake in the presence of ouabain, a specific pump inhibitor, is greatly reduced. This remaining influx is linearly related to the concentration up to 6 mM potassium. Sugars and amino acids are actively accumulated by the intestinal cells. Their transport is accompanied by an initial extra influx of sodium. Although cells seem to regulate their internal sodium concentrations, this is not accompanied with a concomitant increase in potassium uptake through the pump. Thus L-alanine, 3-0-methyl-D-glucoside, and alpha-methyl-D-glucoside all fail to increase the rate of ouabain-sensitive potassium uptake. A very high coupling ratio of sodium efflux to potassium influx through the pump would be a likely explanation of the present results though they cannot be regarded as conclusive.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes.

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.     

The effect of ions, ion channel blockers, and ionophores on uptake of vitellogenin into cockroach follicles

Since calcium plays an important role in vitellogenin binding and uptake in Nauphoeta cinerea and because calcium channels have been described in follicles of this species, we investigated the effect of various ions, ionophores, and ion channel blockers on vitellogenin uptake in vitro. Calcium significantly stimulated vitellogenin uptake; this effect could be substituted best by barium and less well by strontium and magnesium. The stimulatory effect of calcium, and to a certain extent also that of barium, was dependent on the vitellogenin concentration, whereas the effect of strontium and magnesium was not. In the presence of calcium, vitellogenin uptake was inhibited by barium, strontium, and magnesium as well as by the transition elements nickel, cobalt, and zinc, but not by manganese which had a stimulatory effect. Valinomycin, verapamil, tetraethylammonium, and atropine reduced vitellogenin uptake, while amiloride and ouabain were ineffective. Our results indicate that calcium inward (and possibly potassium outward) fluxes play an important role in vitellogenin uptake.     

Structure-activity relationships of several cardiotonic steroids with respect to inhibition of ion transport in frog muscle

Cesium uptake by sodium-loaded frog sartorius muscles was inhibited 100% by 10^-6 M ouabain and 10^-6 M cymarin. The doses for 50% inhibition of cesium uptake by five cardiotonic aglycones were 1.5 x 10^-6 M for strophanthidin, 2 x 10^-7 M for telocinobufagin, 1.6 x 10^-6 for digitoxigenin, 2.4 x 10^-6 M for periplogenin, and 6.3 x 10^-6 M for uzarigenin. Because of the limited solubility of sarmentogenin the maximum concentration studied was 2 x 10^-6 M which inhibited cesium uptake about 36%. Inhibition of cesium uptake by cymarin was not reversed during a 3.5 hr incubation in fresh solution while the muscles treated with ouabain and strophanthidin recovered partly during this time. Cymarin was a more potent inhibitor of sodium efflux than strophanthidin and periplogenin was less potent. Increased cesium ion concentration in the external solution decreased the strophanthidin inhibition of cesium uptake but 25 mM cesium did not overcome the inhibition by 10^-8-10^-6 M strophanthidin. Increased potassium ion concentration in the external solution decreased but did not completely overcome inhibition of sodium efflux by strophanthidin. It is concluded that potassium or cesium ions do not compete with these drugs for a particular site on the ion transport complex. The same structural features of the drugs are necessary for inhibition of ion transport in frog muscle as are required for inhibition of ion transport in other tissues, inhibition of sodium-potassium-stimulated ATPases, and toxicity to animals.     

Effects of pH Changes on Ion and 2,4-D Uptake of Wheat Roots

The quantitative relationships between pH-dependent ion and 2,4-D uptake in winter wheat seedlings (Triticum aestivum L. cv. Yubileynaya 50) have been investigated. The movement of various ions (potassium, phosphate, nitrate and ammonium) and 2,4-D across the root membranes was monitored with radioactive and stable isotope tracer methods. It was found that the H₊ ion concentration of the absorption solution strongly influences the 2,4-D uptake of the roots. Simultaneously, the 2,4-D uptake stimulates secretion of H⁺ into the absorption solution, that is, a H⁺ efflux can accompany the uptake of 2,4-D. This finding is consistent with the acid secretion theory of auxin and fusicoccin action. At pH 4 the 2,4-D uptake was much higher than at pH 6, thereby inhibiting the ion uptake and increasing the phytotoxicity in the plant. The results indicate that 2,4-D enters the root cells rapidly at the lower pH, mostly as undissociated molecules. With reference to the 2,4-D concentration in the roots at pH 4, a possible transport mechanism of the auxin herbicide is briefly discussed.     

Control of the induction of ion transport through mitochondrial membranes by the enzymes of the oxidative phosphorylation system

It has been shown that the induction of earlier described system of potassium-dependent transport of hydrogen ions in mitochondria at low pH values of the incubation medium is inhibited by the inhibitors of mitochondria respiratory chain and ATPase. It has been found that antimycin and oligomycin suppress the efflux of potassium ions from mitochondria in the presence of succinic acid. The uncoupler (FCCP) turns the effect of ATPase inhibitors to the efflux of potassium ions and acceleration of mitochondria respiration under experimental conditions. At the same time TMPD removes the effect of antimycin on potassium ion efflux from uncoupled FCCP of mitochondria. The data obtained are explained in terms of the postulate that under experimental conditions along with the system of potassium-dependent ion transport there appears leakage of protons through the ATPase channel. A conclusion is made concerning the control of ion transport induction in mitochondria by the enzymes of oxidative phosphorylation system.     

Mechanisms of citrate transport and exchange in corn mitochondria

Previous work (Birnberg, Jayroe, Hanson 1982 Plant Physiol 70: 511-516) demonstrated that corn mitochondria (Zea mays L.) can accumulate citrate by a malate- and phosphate-independent proton symporter. This uptake and symport of other ions were investigated. Passive swelling experiments indicated that corn mitochondria can accumulate several other anions by proton symport, but only isocitrate is taken up nearly as effectively as citrate. At the optimal pH (4.5), active uptake of carrier-free [¹⁴C]citrate in 50 micromolar mersalyl is inhibited by fourteen anions, but only the I₅₀ (the concentration of inhibitor required to reduce uptake of carrier-free [¹⁴C]citrate by 50%) values of citrate (0.08 millimolar) and d-and l-isocitrate (0.5 millimolar) are less than 4 millimolar. Isocitrate is a competitive inhibitor of citrate uptake and [¹⁴C]isocitrate is accumulated with a K_m similar similar to its I₅₀. Valinomycin reduces net active citrate accumulation at pH 7.5, consistent with the relatively low V_max for citrate uptake. At pH 4.5, mersalyl reduces the rate of citrate uptake without changing the affinity of the carrier for citrate. Thus, the corn mitochondria have a high-affinity, mersalyl-insensitive carrier selective for citrate that also transports isocitrate.     

The use of a continuous assay system to show the stimulation of phosphoenolpyruvate production in intact mitochondria by valinomycin and by Mn2+

1. A method for the direct recording of the PEP efflux from isolated mitochondria is described.
2. This method has been used to show the stimulation of PEP efflux by externally added Mn⁺⁺ ions.
3. Valinomycin, uncoupler and oleate were also shown to stimulate PEP efflux.
4. Valinomycin caused an increase in the internal concentration of both PEP and citrate.
5. The results indicate that the major pathway of PEP synthesis in isolated mitochondria is via PEP carboxykinase and the results do not call for an unknown pathway of metabolism.
6. Two interactions between PEP and citrate are described; competition for the mitochondrial interior and the stimulation of PEP production by citrate.

     

The Stoichiometry of Respiration-driven Potassium Transport in Corn Mitochondria

Determinations were made for corn (Zea mays L., WF9-Tms × M14) mitochondria of the stoichiometric relationship between K⁺ transport and bond energy produced in respiration (K⁺/~ ratio). With inward pumping of potassium acetate activated by NADH oxidation, the initial rate of K⁺ transported into the sucrose inaccessible space varied between 0.58 and 0.97 K⁺/~, assuming 2 high energy bond equivalents per NADH oxidized. Only small amounts of H⁺ were ejected. Valinomycin did not alter the ratio.     

Effects of prostaglandins on the interaction of Ca2+ with mitochondria

Ca²⁺ stimulates the binding of a variety of prostaglandins (PG) to liver mitochondria. Optimal binding is observed at slightly acidic pH and at concentrations of Ca²⁺ between 200 and 500 μm. The stimulation of the binding requires the active transport of Ca²⁺ in mitochondria and is only observed in the absence of permeant anions. The maximal amount of PG bound is about 3 nmol/mg of mitochondrial protein. All PG tested induce efflux of the Ca²⁺ taken up by mitochondria without impairing the ability of mitochondria to actively accumulate it. Optimal stimulation of the efflux of Ca²⁺ requires concentrations of PG higher than those used in the PG-binding experiments and is associated with an evident uncoupling of the respiration that follows a Ca²⁺-induced O₂ uptake jump. The “uncoupling” by PG is explained by postulating the entrance of protonated PG into mitochondria, followed by their exit from the organelle as 2:1 complexes with Ca²⁺.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Sodium and potassium transport to the xylem are inherited independently in rice, and the mechanism of sodium: potassium selectivity differs between rice and wheat

The heritability of sodium and potassium transport to the xylem was measured by the regression of F_n+1, on F_n means in two segregating breeding populations of rice (Oryza sativa L.). The narrow-sense heritabilities of shoot sodium concentration were 0.42 and 0.43 in the two populations, respectively, and the corresponding values for the heritability of shoot potassium concentration were 0.46 and 0.52. The sodium: potassium ratio was apparently heritable (0.36 and 0.40) because it was regressed positively on sodium concentration and negatively on potassium concentration. There was no significant relationship between the shoot sodium and potassium concentrations themselves. It is concluded that sodium and potassium uptake in rice are controlled by different genes which segregate independently. The magnitude of the transpirational bypass flow was estimated to be some 10 times greater in rice than in wheat (Triticum aestivum L.) and was found to be highly correlated with sodium uptake in rice but not in wheat. It is concluded that the bypass flow provides an additional pathway for sodium uptake in rice and that this accounts for the functional and genetic independence of sodium and potassium uptake in rice and consequently for the lesser prominence of potassium:sodium discrimination in rice than in wheat.     

Magnesium transport by mitochondria

The pathways for the uptake and extrusion of Mg²⁺ by mitochondria are not well defined. the present evidence suggests that uptake occurs by nonspecific diffusive pathways in response to elevated membrane potential. There is disagreement as to some of the properties of Mg²⁺ efflux from mitochondria, but the reaction resembles K⁺ efflux in many ways and may occur in exchange for H⁺. Matrix free magnesium ion concentration, [Mg²⁺], can be measured using fluorescent probes and is set very close to cytosol [Mg²⁺] by a balance between influx and efflux and by the availability of ligands, such as P_i. There are indications that matrix [Mg²⁺] may be under hormonal control and that it contributes to the regulation of mitochondrial metabolism and transport reactions.     

Quantitative trait loci for component physiological traits determining salt tolerance in rice

Rice (Oryza sativa) is sensitive to salinity, which affects one-fifth of irrigated land worldwide. Reducing sodium and chloride uptake into rice while maintaining potassium uptake are characteristics that would aid growth under saline conditions. We describe genetic determinants of the net quantity of ions transported to the shoot, clearly distinguishing between quantitative trait loci (QTL) for the quantity of ions in a shoot and for those that affect the concentration of an ion in the shoot. The latter coincide with QTL for vegetative growth (vigor) and their interpretation is therefore ambiguous. We distinguished those QTL that are independent of vigor and thus directly indicate quantitative variation in the underlying mechanisms of ion uptake. These QTL independently govern sodium uptake, potassium uptake, and sodium:potassium selectivity. The QTL for sodium and potassium uptake are on different linkage groups (chromosomes). This is consistent with the independent inheritance of sodium and potassium uptake in the mapping population and with the mechanistically different uptake pathways for sodium and potassium in rice under saline conditions (apoplastic leakage and membrane transport, respectively). We report the chromosomal location of ion transport and selectivity traits that are compatible with agronomic needs and we indicate markers to assist selection in a breeding program. Based upon knowledge of the underlying mechanisms of ion uptake in rice, we argue that QTL for sodium transport are likely to act through the control of root development, whereas QTL for potassium uptake are likely to act through the structure or regulation of membrane-sited transport components.