Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

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.     

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.     

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium,the midgut of tobacco hornoworm (Manduca sexta)

Summary Transbasal electrical potential (V _b) and intraepithelial potassium chemical activity ((K⁺) _i ) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofV _b ranging from +8 to –48 mV (relative to blood side) were recorded. For all sites, (K⁺) _i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than –20 mV show relatively high sensitivity ofV _b to changes in blood side K⁺ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, dye-coupling of several adjacent cells is seen. Low potential sites — those withV _b less negative than –20 mV —typically do not show high sensitivity ofVb to changes of external K⁺, but rather (K⁺) _i rapidly approaches the K⁺ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K⁺) _i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.     

The voltage-dependent potassium-uptake channel of corn coleoptiles has permeation properties different from other K+ channels

The initial response of coleoptile cells to growth hormones and light is a rapid change in plasma-membrane polarization. We have isolated protoplasts from the cortex of maize (Zea mays L.) coleoptiles to study the electrical properties of their plasma membrane by the patch-clamp techniqueUsing the whole-cell configuration and cell-free membrane patches we could identify an H⁺-ATPase, hyperpolarizing the membrane potential often more negative than -150 mV, and a voltage-dependent, inward-rectifying K⁺ channel (unit conductance 5–7 pS) as the major membrane conductan-ces Potassium currents through this channel named CKC1_in (for Coleoptile K ⁺ Channel inward rectifier) were elicited upon voltage steps negative to -80 mV, characterized by a half-activation potential of -112 mV. The kinetics of activation, well described by a double-exponential process, were strongly dependent on the degree of hyperpolarization and the cytoplasmic Ca²⁺ level. Whereas at nanomolar Ca²⁺ concentrations K⁺ currents increased with a t_1/2=16 ms (at -180 mV), higher calcium levels slowed the activation process about fourto fivefoldUpon changes in the extracellular K⁺ concentration the reversal potential of the K⁺ channel followed the Nernst potential for potassium with a 56-mV shift for a tenfold increaseThe absence of a measurable conductance for Na⁺, Rb⁺, Cs⁺ and a permeability ratio P_{NH 4} ⁺ /P_K+ around 0.25 underlines the high selectivity of CKC1_in for K⁺In contrast to Cs⁺, which at submillimolar concentration blocks the channel in a voltage-dependent manner, Rb⁺, often used as a tracer for K+, does not permeate this type of K⁺ channelThe lack of Rb⁺ permeability is unique with respect to other K⁺ transporters. Therefore, future molecular analysis of CKC1_in, considered as a unique variation of plant inward rectifiers, might help to understand the permeation properties of K⁺ channels in general.Abbreviations CKC1_in Coleoptile K ⁺ Channel inward rectifier - U membrane voltage - I_ss steady-state currents - I_tail tail currents Experiments were conducted in the laboratory of F.G. during the stay of RHas a guest professor sponsored by Special Project RAISA, subproject N2.1, paper N2155.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

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 ⁺ .     

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.     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Selective block by α-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

The efficacy and mechanism of -dendrotoxin (DTX) block of K⁺ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K⁺ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K⁺ (K⁺) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K⁺ current (I_K,in), but was ineffective in blocking current through the outward-rectifying K⁺ channels (I_K,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for I_K,in, neither altering its activation at voltages negative of –120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of I_K,in followed a simple titration function with an apparent K_1/2 for block of 2.2 nM in 3 mm K _o ⁺ . However, DTX block was dependent on the external K⁺ concentration. Raising K⁺ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K _i = 10 mm in 10 nm DTX). The effect of K⁺ in protecting I _K,in was competitive with DTX and specific for permeant cations. A joint analysis of I_K,in block with DTX and K⁺ concentration was consistent with a single class of binding sites with a K _d for DTX of 240 pm. A K _d of 410 m for extracellular K⁺ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K⁺ (K_1/2 1 mm) for I_K,in activation; they parallel features of K⁺ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K⁺ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K⁺ inward rectifier.We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

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.     

Na+ and K+ fluxes stimulated by Na+-coupled glucose transport: Evidence for a Ba2+-insensitive K+ efflux pathway in rabbit proximal tubules

Summary Addition of glucose or the nonmetabolizable analogue -methyl-d-glucoside to rabbit proximal tubules suspended in a glucoseand alanine-free buffer caused a sustained increase in intracellular Na⁺ content (+43±7 nmol · (mg protein)^–1) and a concomitant but larger decrease in K⁺ content (–72±11 nmol· (mg protein)^–1). A component of the net K⁺ efflux was Ba²⁺ insensitive, and was inhibited by high (1mm) but not low (10 m) concentrations of the diuretics, furosemide and bumetanide. The increase in intracellular Na⁺ content is consistent with the view that the increased rates of Na⁺ and water transport seen in the proximal tubule in the presence of glucose can be attributed (at least in part) to a stimulation of basolateral pump activity by an increased [Na⁺] _i .     

Potassium-dependent,bipolar gating of K+ channels in guard cells

Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K⁺, fluxes thought to pass through K⁺ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K⁺ channels, its pharmacology and dependence on external K⁺ (K _v ⁺ ) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary electrogenic transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V _m ) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K⁺ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K⁺ current rose with voltage positive toV _m but it decayed over two voltage ranges, one negative toV _m and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K _v ⁺ and followed the K⁺ equilibrium potential. Against a (presumably) low background of primary ion transport, the K⁺ current contributed appreciably to charge balance atV _m in 0.1mm as well as in 1 to 10mm K _v ⁺ . Thus, gating of these K⁺ channels compensates for the prevailing K⁺ conditions to ensure net K⁺ movement out of the cell.     

Ba2+-sensitive potassium permeability of the apical membrane in newt kidney proximal tubule

Summary The apical membrane K⁺ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V _a change) during alteration of luminal K⁺ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V _a change/decade for K⁺ was 50 mV at K⁺ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K⁺ concentration was increased from 2.5 to 25mm. Ba²⁺ (1mm in the lumen) reducedV _a change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba²⁺-sensitive K⁺ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K⁺ activity measured by K⁺-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K⁺ across both cell membranes. Thus, it is concluded that cell K⁺ passively diffuses, at least in part, through the K⁺ conductive pathway of the apical membrane.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

Summary The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na⁺-enriched cells. Subsequent incubation in K⁺-containing NaCl medium results in the reaccumulation of K⁺, Cl^–, water and the extrusion of Na⁺. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (K _M for K ₀ ⁺ =3.5mm;J _max=30.1 mEq/kg dry min), which in these experiments was coupled 1K⁺/1 Na⁺. The second is the Cl^–-dependent (Na⁺+K⁺) cotransport system (K _M for K ₀ ⁺ =6.8mm;J _max=20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K⁺1Na⁺2Cl^–, the exchange of K _i ⁺ for K ₀ ⁺ . The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.