Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Closure of the yeast mitochondria unspecific channel (YMUC) unmasks a Mg2+ and quinine sensitive K+ uptake pathway in Saccharomyces cerevisiae

The K⁺ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K⁺-mediated mitochondrial swelling observed in the absence of phosphate (PO₄) and in the presence of a respiratory substrate has led to propose that large K⁺ movements occur in yeast mitochondria. Thus, the uptake of K⁺ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K⁺ transport; these were mitochondrial swelling and the uptake of the radioactive K⁺ analog ⁸⁶Rb⁺. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO₄ concentrations. The high protein concentrations used to measure ⁸⁶Rb⁺ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO₄ but not at 0 or 4 mM PO₄. At 4 mM PO₄ swelling was inhibited while, in contrast, ⁸⁶Rb⁺ uptake was still observed. The results suggest that an energy-dependent K⁺ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K⁺ uptake system, the Mg²⁺ and quinine sensitivity of both swelling and ⁸⁶Rb⁺ uptake were evaluated. Under the conditions where the unspecific pore was closed, K⁺ transport sensitivity to Mg²⁺ and quinine increased. In addition, when Zn²⁺ was added as an antiport inhibitor, uptake of ⁸⁶Rb⁺ increased. It is suggested that in yeast mitochondria, the K⁺ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Kinetic study on the effects of intracellular K++ and Na++ on Na++, K++, Cl+− cotransport of HeLa cells by Rb++ influx determination

Summary The effects of intracellular K⁺ and Na⁺ (K⁺ c, Na⁺ c) on the Na⁺,K⁺,Cl^+– cotransport pathway of HeLa cells were studied by measuring ouabain-insensitive, furosemide-sensitive Rb⁺ influx (JRb) at various intracellular concentrations of K⁺ and Na⁺ ([K⁺]c, [Na⁺]c). When [K⁺]c was increased and [Na⁺]c was decreased, keeping the sums of their concentrations almost constant, JRb as a function of the extracellular Rb⁺ or Na⁺ concentration ([Rb⁺]e, [Na⁺]e) was stimulated. However, the apparent K _0.5 for Rb⁺ e or Na⁺ e remained unchanged and the ratio of the apparent K ^+0.5 for K⁺ c and the apparent K _i for Na⁺ c was larger than 1. When JRb was increased by hypertonicity by addition of 200 mM mannitol, the apparent maximum JRb increased without change in the apparent K _0.5 for Rb⁺ e. These results show that K⁺ c stimulates and Na⁺ c inhibits JRb, without change in the affinities of the pathway for Rb⁺ e and Na⁺ e. The affinity for K⁺ c is slightly lower than that for Na⁺ c. Hypertonicity enhances JRb without any change in the affinity for Rb⁺ e. We derived a kinetic equation for JRb with respect to K⁺ c and Na⁺ c and proposed a general and a special model of the pathway. The special model suggests that, in HeLa cells, JRb takes place when Rb⁺ e binds to the external K⁺ binding site of the pathway after the binding of K⁺ c to the internal regulatory site.We thank Mr. T. Masuya for technical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 03202136) from the Japanese Ministry of Education, Science and Culture.     

Role of the sodium pump and the background K+ channel in passive K+(Rb+) uptake by isolated cardiac sarcolemmal vesicles

Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of⁸⁶Rb⁺ by the preparation was measured in the presence of an opposing K⁺ gradient using a rapid ion exchange technique. At low extravesicular Rb⁺ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb⁺ uptake under these conditions, and produced a complete block of Rb⁺ influx in the presence of 1mm ouabain. When⁸⁶Rb⁺ was used as a tracer of extravesicular K⁺ (Rb ₀ ⁺ 40 m K ₀ ⁺ =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba²⁺ (K _0.5=20 m). At a constant internal K⁺ concentration (K _in ⁺ =50mm) the magnitude of the Ba²⁺-sensitive K⁺ uptake was found to depend on K ₀ ⁺ in a manner that closely resembles the K⁺ concentration dependence of the background K⁺ conductance (I _Kl) observed electrophysiologically in intact cardiac cells. We conclude that K⁺ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Potassium-dependent changes in the expression of membrane-associated proteins in barley roots : I. Correlations with k(rb) influx and root k concentration

Barley (Hordeum vulgare L. cv Halcyon) seedlings which had been grown in full strength complete inorganic nutrient media (containing 6 millimolar K⁺) had high internal K⁺ concentrations and low values of K⁺ (⁸⁶Rb⁺) influx when influx was measured from solutions containing 100 micromolar K⁺. Transfer of these plants to solutions lacking K⁺ resulted in significant reductions of root and shoot K⁺ concentrations and values of K⁺ (⁸⁶Rb⁺) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K⁺, the changes induced by K⁺ deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K⁺ (⁸⁶Rb⁺) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K⁺ status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washed in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar.     

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Summary Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K⁺ (and Cl^–) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K⁺-free media a net influx of Na⁺ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K⁺ and triphenylmethyl phosphonium⁺, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time⁸⁶Rb⁺ fluxes were measured:⁸⁶Rb⁺ influx was not affected by hypotonicity, implying an unchanged operation of the Na⁺–K⁺-ATPase. On the other hand,⁸⁶Rb⁺ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.Abbreviations and symbols DW dry weight - E extracellular (polyethylene glycol) space - E Nernst potential - H₂O_e H₂O_i tissue water, extra- and intracellular - TPMP ⁺ triphenyl methyl phosphonium salt - WW wet weight     

Ba2+-inhibitable86Rb+ fluxes across membranes of vesicles from toad urinary bladder

Summary ⁸⁶Rb⁺ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba²⁺ required for half-maximal inhibition was 0.6mm. The effects of externally added cations on⁸⁶Rb⁺ influx and efflux have established that this pathway is conductive, with a selectivity for K⁺, Rb⁺ and Cs⁺ over Na⁺ and Li⁺. the Rb⁺ uptake is inversely dependent on external pH, but not significantly affected by internal Ca²⁺ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb⁺ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K⁺ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na⁺ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca²⁺ activity directly blocks apical Na⁺ entry.     

Ca2+-Activated K+ channel from human erythrocyte membranes: Single channel recification and selectivity

Summary The Ca²⁺-activated K⁺ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K⁺, Rb⁺, and NH ₄ ⁺ , respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K⁺ (23.5 pS±3.2)>NH ₄ ⁺ (14.2 pS±1.2)>Rb⁺ (11.4 pS±1.8). With low extracellular K⁺ concentrations (substitution with Na⁺) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K⁺ and Na⁺ enhanced rectification were observed due to a Na⁺ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (P _K/P _X) was calculated: K⁺ (1.0)>Rb⁺ (1.4±0.1)>NH ₄ ⁺ (8.5±1.3)>Li⁺(>50); Na⁺ (>110); Cs⁺ (5). Li⁺, Na⁺, and Cs⁺ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl⁺ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process.     

Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Errata

1. (1) The significance of the specific (ouabain-sensitive) ⁸⁶Rb⁺ or ⁴²K⁺ uptake by cardiac muscle preparations which are not ‘sodium-loaded’ was studied.

2. (2) In left atrial preparations of guinea-pig heart, resting ⁸⁶Rb⁺ uptake was relatively low. It was markedly increased by electrical stimulation. This stimulated uptake was further enhanced by isoproterenol and inhibited by verapamil.

3. (3) In rat atria, the resting ⁸⁶Rb⁺ uptake was somewhat higher than in guinea-pig atria, and the increase in uptake caused by electrical stimulation was smaller. In guinea-pig right ventricular papillary muscle, the resting uptake was highest among those tissues studied, and the response to electrical stimulation was smallest. In the latter tissue, verapamil produced only a minimal inhibition of the specific ⁸⁶Rb⁺ uptake.

4. (4) The effect of the frequency of electrical stimulation on ⁸⁶Rb⁺ uptake paralleled its influence on the force of contraction, suggesting the involvement of intracellular sodium in both events.

5. (5) In both left atrial and right papillary muscle preparations of guinea-pig heart, specific ⁴²K⁺ uptake observed with 5.8 mM K⁺ was relatively high, and was increased only slightly by electrical stimulation. This electrical stimulation, however, increased ouabain-induced inhibition of ⁴²K⁺ uptake, suggesting that the stimulation increases the amount of Na⁺ available to the sodium pump.

6. (6) When the K⁺ concentration was 1 mM, the resting ⁴²K⁺ uptake was low, and could be enhanced by electrical stimulation.

Delta endotoxin inhibits Rb+ uptake,lowers cytoplasmic pH and inhibits a K+-ATPase inManduca sexta CHE cells

Summary Delta endotoxin, a 68 kilodalton protein isolated fromBacillus thuringiensis spp.Kurstaki, is a potent entomocidal agent that alters a K⁺ current across midgut tissue of many phytophagous insects. This toxin completely inhibited the vanadate-sensitive⁸⁶Rb⁺ uptake and mimicked the vanadate-induced decrease in cytosolic pH in a cell line (CHE) originating fromManduca sexta embryonic tissue. The toxin also inhibited a K⁺-sensitive-ATPase in the plasma membranes isolated from these cells. Using the K⁺-sensitive-ATPase substratp-nitrophenyl phosphate, delta endotoxin was found to have aK _i of 0.4 m. These data suggest that the toxin inhibits a K⁺-ATPase responsible for⁸⁶Pb⁺ uptake in the CHE cells. The relationship between the toxin inhibition of K⁺-ATPase and toxin-altered K⁺ current is discussed.     

Rapid auxin- and fusicoccin-enhanced Rb+ uptake and malate synthesis in Avena coleoptile sections

The short-term effects of auxin (indole-3-acetic acid) and fusicoccin (FC) on Rb⁺ uptake and malate accumulation in Avena sativa L. coleoptile sections have been investigated. FC stimulates ⁸⁶Rb⁺ uptake within 1 min while auxin-enhanced uptake begins after a 15–20-min lag period. Auxin has little or no effect on ⁸⁶Rb⁺ uptake at external pHs of 6.0 or less, but substantial auxin effects can be observed in the range of pH 6.5 to 7.5. Competition studies indicate that the uptake mechanism is specific for Rb⁺ and K⁺. After 3 h of auxin treatment the total amount of malate in the coleoptile sections is doubled compared to control sections. FC causes a doubling of malate levels within 60 min of treatment. Auxin-induced malate accumulation exhibits a sensitivity to inhibitors and pH which is similar to that observed for the H⁺-extrusion and Rb⁺-uptake responses. Both auxin- and FC-enhanced malate accumulation are stimulated by monovalent cations but this effect is not specific for K⁺.Abbreviations FC fusicoccin - IAA indole-3-acetic acid     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

Thiol-dependent passive K+Cl− transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes

Summary Ouabain-resistant (OR), volume-or N-ethylmaleimide (NEM)-stimulated K⁺(Rb⁺)Cl^– fluxes were measured in low-K⁺ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K⁺ effluxes and Rb⁺ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K⁺Cl^– flux, metabolic depletion did not reduce K⁺Cl^– flux of normal or swollen cells. The anion preference of OR K⁺ efflux in NEM-treated cells was Br^–>Cl^–>HCO ₃ ^– =F^–I^–=NO ₃ ^– =CNS^–, and hence consistent with a reported Br^–>Cl^–>NO ₃ ^– sequence of the volume-dependent K⁺Cl^– transport. Alloimmune anti-L_l antibodies known to decrease passive K⁺ fluxes in low K⁺ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive Rb⁺Cl^– fluxes suggesting their immunologic identity, a conclusion also supported by anti-L₁ absorption studies. Since pretreatment with anti-L₁ prevented the activation of Rb⁺ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb⁺Cl^– influx, the NEM reactive SH groups must be located apart from the L₁ antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K⁺Cl^– transport path(s) regulated by a protein with two functional subunits or domains; a chemically (C _s) and a volume (V _s)-stimulated domain, both interfacing with the L₁ surface antigen. Attachment of alloanti-L₁ from the outside reduces K⁺Cl^– transport stimulated throughC _s by NEM orV _s by cell swelling.     

Acclimation of potassium influx in rye (Secale cereale) to low root temperatures

The influx of K⁺(⁸⁶Rb⁺) into intact roots of rye (Secale cereale L. cv. Rheidal) exposed to a differential temperature (DT) between the root (8° C) and shoot (20° C) is initially reduced compared with warm-grown (WG) controls with both shoot and root maintained at 20° C. Over a period of 3 d, however, K⁺-influx rates into DT plants are restored to levels similar to or greater than those of the WG controls, the absolute rates of K⁺ influx being strongly dependent upon the shoot/root ratio. Acclimation in DT plants results in a reduction of K⁺ influx into the apical (0–2 cm) region of the seminal root which is associated with a compensatory increase in K⁺ influx into the more mature, basal regions of the root. Values of V _max and apparent K _m for K⁺ influx into DT plants were similar to those for WG plants at assay temperatures of 8° C and 20° C except for an increase in the apparent K _m at 8° C. The influx of K⁺ from solutions containing 0.6 mol·m^-3 K⁺ into both WG and DT plants was found to be linearly related to assay temperature over the range 2–27° C, and the temperature sensitivity of K⁺ influx to be dependent upon shoot/root ratio. At high shoot/root ratios, the ratio of K⁺ influx at 20° C:K⁺ influx at 8° C for WG plants approached a minimum value of 1.9 whereas that for DT plants approached unity indicating that K⁺ influx into DT plants has a large temperature-insensitive component. Additionally, when plants were grown in solutions of low potassium concentration, K⁺ influx into DT plants was consistently greater than that into WG plants, in spite of having a greater root potassium concentration ([K⁺]int). This result indicates some change in the regulation of K⁺ influx by [K⁺]int in plants exposed to low root temperatures. We suggest that K⁺ influx into rye seedlings exposed to low root temperatures is regulated by the increased demand placed on the root system by a proportionally larger shoot and that the acclimation of K⁺ influx to low temperatures may be the result of an increased hydraulic conductivity of the root system.Abbreviations DT differential temperature pretreatment - [K⁺]int root potassium concentration - [K⁺]ext potassium concentration of nutrient medium - WG warm-grown pretreatment