The Na+,K+,2Cl- -cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway

We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl- -cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+,K+,2Cl- -cotransport system. LK1 cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide-sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1 cells (increase in Vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)-bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)-bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1 cells present, 1.44 pmol/mg protein, specifically bound (3H)-bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)-ouabain binding sites as well as an increase in the activity of the Na+,K+-ATPase, expressed as a function of ouabain-sensitive 86Rb+ uptake. Furthermore, LK1 cells were different in the concentrations of intracellular Na+ (increases) and K+ (decreases) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.     

Low-affinity potassium uptake system in the archaeon Methanobacterium thermoautotrophicum: overproduction of a 31-kilodalton membrane protein during growth on low-potassium medium.

During growth on low-K+ medium (1 mM K+), Methanobacterium thermoautotrophicum accumulated K+ up to concentration gradients ([K+]intracellular/[K+]extracellular) of 25,000- to 50,000-fold. At these gradients ([K+]extracellular of < 20 microM), growth ceased but could be reinitiated by the addition of K+ or Rb+. During K+ starvation, the levels of a protein with an apparent molecular weight of 31,000 increased about sixfold. The protein was associated with the membrane and could be extracted by detergents. Cell suspensions of M. thermoautotrophicum obtained after K+-limited growth catalyzed the transport of both K+ and Rb+ with apparent Km and Vmax values of 0.13 mM and 140 nmol/min/mg, respectively, for K+ and 3.4 mM and 140 nmol/min/mg, respectively, for Rb+. Rb+ competitively inhibited K+ uptake with an inhibitor constant of about 10 mM. Membranes of K+-starved cells did not exhibit K+-stimulated ATPase activity. Immunoblotting with antisera against Escherichia coli Kdp-ATPase did not reveal any specific cross-reactivity against membrane proteins of K+-starved cells. Cells of M. thermoautotrophicum grown at a high potassium concentration (50 mM) catalyzed K+ and Rb+ transport at similar apparent Km values (0.13 mM for K+ and 3.3 mM for Rb+) but at significantly lower apparent Vmax values (about 60 nmol/min/mg for both K+ and Rb+) compared with K+-starved cells. From these data, it is concluded that the archaeon M. thermoautotrophicum contains a low-affinity K+ uptake system which is overproduced during growth on low-K+ medium.     

Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis

The complex interrelationships between the transport of inorganic cations and C4 dicarboxylate were examined using mutants defective in potassium transport and retention, divalent cation transport, or phosphate transport. The potassium transport system, studied using 86Rb+ as a K+ analogue, kinetically appeared as a single system (Km 200 microM for Rb+, Ki 50 microM for K+), the activity of which was only slightly reduced in K+ retention mutants. Divalent cation transport, studied using 54Mn2+, 60Co2+, and 45Ca2+, was more complex being represented by at least two systems, one with a high affinity for Mn2+ (Km 2.5 microM) and a more general one of low affinity (Km 1.3-10 mM) for Mg2+, Mn2+, Ca/2+, and Co2+. Divalent cation transport was repressed by Mg2+, derepressed in K+ retention mutants, and defective in Co2+-resistant mutants. Phosphate was required for both divalent cation and succinate transport, and phosphate transport mutants (arsenate resistant) were found to be defective in both divalent cation and succinate transport. Divalent cations, especially Mg2+ and Co2+, decreased Km for succinate transport approximately 20-fold over that achieved with K+; neither cation was required stoichiometrically for succinate transport. The loss of divalent cation transport in cobalt-resistant mutants has been correlated with the loss of a 55,000 molecular weight membrane protein. Similarly, the loss of phosphate transport in arsenate-resistant mutants has been correlated with the loss of a 35,000 molecular weight membrane component.     

Membrane ionic currents in Rhodobacter capsulatus. Evidence for electrophoretic transport of K+, Rb+ and NH4+

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

Divalent cation transport systems of Rhodopseudomonas capsulata.

Separate divalent cation transport systems for energy-dependent uptake of Mg2+ and Mn2+ were found both with aerobically and heterotrophically grown and with photosynthetically grown cells of Rhodopseudomonas capsulata. The maximum rate of Mg2+ uptake differed between photosynthetic and aerobic cells, while the Km for the Mg2+ transport system was constant. Photosynthetic midlog-phase cells exhibited Km's for uptake of about 55 micrometer Mg2+ and 0.5 micrometer Mn2+. The Vmax's also differed between the two systems: 0.6 to 1.8 mumol/min per g (dry weight) of cells for Mg2+, but only 0.020 mumol/min per g for Mn2+, making the distinction between a "macro-requirement" system and a system functioning at trace nutrient levels. Calcium was not normally taken up by intact cells of R. capsulata. However, chromatophore membranes isolated from photosynthetic cells took up Ca2+ by an energy-dependent process.     

Catalytic properties of phenol carboxylase. In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Potassium Fluxes in Chlamydomonas reinhardtii (I.Kinetics and Electrical Potentials)

Potassium influx and cellular [K+] were measured in the unicellular green alga Chlamydomonas reinhardtii after pretreatment in either 10 or 0 mM external K+ ([K]0). K+ (42K+ or 86Rb+) influx was mediated by a saturable, high-affinity transport system (HATS) at low [K+]0 and a linear, low-affinity transport system at high [K+]o. The HATS was typically more sensitive to metabolic inhibition (and darkness) than the low-affinity transport system. Membrane electrical potentials were determined by measuring the equilibrium distribution of tetraphenylphosphonium. These values, together with estimates of cytoplasmic [K+] (B. Malhotra and A.D.M. Glass [1995] Plant Physiol 108: 1537-1545), demonstrated that at 0.1 mM [K+]0 K+ uptake must be active. At higher [K+]0 (>0.3 mM) K+ influx appeared to be passive and possibly channel mediated. When cells were deprived of K+ for 24 h, the Vmax for the HATS increased from 50 x 10-6 to 85 x 10-6 nmol h-1 cell-1 and the Km value decreased from 0.25 to 0.162 mM. Meanwhile, cellular [K+] declined from 24 x 10-6 to 9 x 10-6 nmol cell-1. During this period influx increased exponentially, reaching its peak value after 18 h of K+ deprivation. This increase of K+ influx was not expressed when cells were exposed to inhibitors of protein synthesis. The use of 42K+ and 86Rb+ in parallel experiments demonstrated that Chlamydomonas discriminated in favor of K+ over Rb+, and this effect increased with the duration of K+ deprivation.     

Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. Evidence for the cotransport of Na+, K+, and Cl-

Confluent monolayer cultures of the Madin-Darby canine kidney (MDCK) cell line have been shown to possess a furosemide and bumetanide-sensitive (Na+,K+)-cotransport system. We have studied the effect of anion substitutions on (Na+,K+)-cotransport. In Na+-depleted cells, bumetanide-sensitive uptake of 22Na+ or 86Rb+ exhibited an absolute requirement for extracellular Cl-. Chloride could be replaced in the buffers by Br-, but not by F-, I-, acetate, nitrate, thiocyanate, sulfate, or gluconate. The effect of Cl- was saturating, and Na+-stimulated 86RB+ uptake as well as K+-stimulated 22Na+ uptake was shown to be dependent on the square of the Cl- concentration. The concentration of Cl- which gave half-maximal stimulation of cation cotransport varied between 58 and 70 mM. There was a small degree of cooperativity between the binding affinities for Cl- and K+ at constant Na+ concentrations. Bumetanide-sensitive 36Cl- uptake could be demonstrated when extracellular Na+ and K+ were present simultaneously. Uptake through this system was unaffected by changes in the membrane potential or by the imposition of pH gradients. Together these data strongly suggest that the bumetanide-sensitive transport system in Madin-Darby canine kidney cells co-transports Na+, K+, and Cl- in a ratio of 1:1:2.     

Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP

We have measured the time course of release of 42K and 86Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42K is released with a rate constant of approximately 45 s-1 and 86Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42K or 86Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42K and 86Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle.     

Dual system for potassium transport in Saccharomyces cerevisiae.

In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.     

Physiological basis for the high salt tolerance of Debaryomyces hansenii.

The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K+, Na+ was able to substitute for K+, restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na+ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na+ and Li+ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb+ uptake in D. hansenii (Vmax, 4.2 nmol mg [dry weight]-1 min-1; K(m), 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb+ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb+ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na+ than is that of S. cerevisiae, that Na+ substitutes for K+ when K+ is scarce, and that the transport of K+ is favored by the presence of Na+. In low K+ environments, D. hansenii behaved as a halophilic yeast.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.     

Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.