Voltage dependence of transient and steady-state Na/K pump currents in myocytes

Experiments are reviewed here in which Na/K pump current was determined as strophanthidin-sensitive current in guinea-pig ventricular myocytes, voltage-clamped and internally-dialyzed via wide-tipped pipettes. In the presence of 150 mM extracellular [Na], both outward and inward pump current, during forward and reverse Na/K exchange respectively, were strongly voltage dependent. But reduction of external [Na] to 1.5 mM severely attenuated the voltage sensitivity of outward Na/K pump current. Voltage jumps elicited large transient pump currents during forward or reverse Na/K exchange, or when pump activity was restricted to Na translocation steps, but not when pumps were presumably engaged in K/K exchange. These findings indicate that Na translocation, but not K translocation, involves net charge movement through the membrane field, and that both forward and reverse Na/K transport cycles are rate-limited not by that voltage-sensitive step but by a subsequent voltage-insensitive step.     

Influence of Lithium Ions on the Transmembrane Potential and Cation Content of Cardiac Cells

The effect of lithium ions on cardiac cells was investigated by recording the changes in transmembrane potential and by following the movement of Li, Na, and K across the cell membrane. Isolated preparations of calf Purkinje fibers and cat ventricular muscles were used. Potentials were measured by intracellular microelectrodes; ion transport was estimated by flame photometric analysis and by using the radioactive isotopes of Na and K. It was shown (a) that Li ions can replace Na ions in the mechanism generating the cardiac action potential but that they also cause a marked depolarization and pronounced changes in action potential configuration; (b) that the resting permeability to Li ions is high and that these ions accumulate in the cell interior as if they were not actively pumped outwards. In Li-Tyrode [K]_i decreases markedly while the K permeability seems to be increased. In a kinetic study of net K and Na fluxes, the outward movement of each ion was found to be proportional to the second power of its intracellular concentration. The effect on the transmembrane potential is explained in terms of changes in ion movement and intracellular ion concentration.     

Conditions for a backward-running Na+/K+ pump in Xenopus oocytes.

Current generated by the electrogenic Na+/K+ pump protein was determined in oocytes of Xenopus laevis as strophantidine-sensitive current measured under voltage clamp. Under conditions of reduced intracellular [Na+] and [ATP], both to values below 1 mM, and in extracellularly K(+)-free medium, the Na+/K+ pump seems to operate in a reversed mode pumping Na+ into the cell and K+ out of the cell. This is demonstrated by strophantidine-induced hyperpolarization of the membrane and inward-directed current mediated by the pump protein. In addition, strophantidine-sensitive uptake of 22Na+ can be demonstrated under these conditions. The pump current decreases with membrane depolarization as expected for a pump cycle that involves inward movement of positive charges during Na+ translocation.     

Electron probe X-ray microanalysis of cellular ions in the eccrine secretory coil cells during methacholine stimulation

Summary Intracellular concentrations of Na, K, Cl ([Na], [K] and [Cl], respectively) and other elements were determined in isolated monkey eccrine sweat secretory coil cells using quantitative electron probe X-ray microanalysis of freeze dried cryosections. The validity of the methodology was partially supported by qualitative agreement of the X-ray microanalysis data with those obtained by micro-titration with a helium glow spectrophotometer. [Na], [K] and [Cl] of the cytoplasm were the same as those in the nucleus in both clear and dark cells. [Na], [K], and [Cl] of the clear cells were also the same as those of the dark cells at rest and after stimulation with methacholine (MCh), suggesting that these two cell types behave like a functional syncytium. MCh stimulation induced a pharmacologically specific, dose-dependent decrease in [K] and [Cl] (as much as 65%), and a 3.7-fold increase in [Na]. In myoepithelial cells, a similar change in [Na] and [K] was noted after MCh stimulation although the decrease in [Cl] was only 20%. The MCh-induced change in [Na], [K] and [Cl] was almost completely inhibited by removal of Ca²⁺ from the medium. 10^–4 m bumetanide inhibited the MCh-induced increase in [Na], reduced the decrease in [K] by about 50%, but slightly augmented the MCh-induced decrease in [Cl]. 10^–4 m ouabain increased [Na] and decreased [K] as did MCh; however, unlike MCh, ouabain increased [Cl] by 56% after 30 min of incubation. Thus the data may be best interpreted to indicate that Ca-dependent K efflux and (perhaps also Ca-dependent) Cl efflux are the predominat initial ionic movement in muscarinic cholinergic stimulation of the eccrine sweat secretory coils and that the ouabain-sensitive Na pump plays an important role in maintenance of intracellular ions and sweat secretion.     

The effects of sodium pump activity on the slow inward current in sheep cardiac Purkinje fibres

The effects of Na pump activity on the slow inward current, Isi, magnitude and twitch tension were investigated in sheep cardiac Purkinje fibres. A two-microelectrode voltage-clamp method was used, tension being measured simultaneously. Na pump activity was lowered either by reducing the extracellular K concentration, [K]O, or by applying the cardiotonic steroid strophanthidin. Reduction of [K]O from 4 to 0 mM leads to time-dependent increases in Isi magnitude and twitch tension. The increases of Isi and tension could be reversed by adding Tl, Rb, Cs or NH4 ions to the K-free superfusate. The actions of these ions are attributed to the known ability of these cations to activate the external site of the Na pump. This conclusion is supported by the observation that such activator cations do not reverse the increases in Isi and tension produced by strophanthidin. We conclude that the effects of low [K]O on Isi are mediated by Na pump inhibition. Similarly the Na pump inhibition produced by strophanthidin increases Isi and tension, although, in this case, other mechanisms may also contribute. Measurements of the activity of the electrogenic Na pump show that elevated intracellular Na ion concentration secondary to Na pump inhibition and not the instantaneous Na pump turnover rate mediates the increase in Isi magnitude.     

[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes

Na/K pump current was determined between -140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] [( Na]pip) in a single cell; the effects of changes in external [Na] [( Na]o) or external [K] [( K]o) were also studied. At 50 mM [Na]pip, 5.4 mM [K]o, and approximately 150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and approximately 150 mM [Na]o, reduction of [Na]pip from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]pip according to the Hill equation with best-fit K0.5 approximately equal to 11 mM and Hill coefficient nH approximately equal to 1.4. At zero [Na]o, reduction of [Na]pip seemed to simply scale down the relatively flat pump I-V relationship: Hill fit parameters for pump activation by [Na]pip at 0 mV were K0.5 approximately equal to 10 mM, nH approximately equal to 1.4. At 50 mM [Na]pip and high [Na]o, reduction of [K]o from 5.4 mM scaled down the sigmoid I-V relationship and shifted it slightly to the right: at 0 mV, K0.5 approximately equal to 1.5 mM and nH approximately equal to 1.0. At zero [Na]o, lowering [K]o simply scaled down the flat pump I-V relationships yielding, at 0 mV, K0.5 approximately equal to 0.2 mM, nH approximately equal to 1.1. The voltage-independent activation of Na/K pump current by both intracellular Na ions and extracellular K ions, at zero [Na]o, suggests that neither ion binds within the membrane field. Extracellular Na ions, however, seem to have both a voltage-dependent and a voltage-independent influence on the Na/K pump: they inhibit outward Na/K pump current in a strongly voltage-dependent fashion, with higher apparent affinity at more negative potentials (K0.5 approximately equal to 90 mM at -120 mV, and approximately 170 mM at -80 mV), and they compete with extracellular K ions in a seemingly voltage-independent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Access channel model for the voltage dependence of the forward-running Na+/K+ pump

The voltage dependence of steady state current produced by the forward mode of operation of the endogenous electrogenic Na+/K+ pump in Na(+)- loaded Xenopus oocytes has been examined using a two-microelectrode voltage clamp technique. Four experimental cases (in a total of 18 different experimental conditions) were explored: variation of external [Na+] ([Na]o) at saturating (10 mM) external [K+] ([K]o), and activation of pump current by various [K]o at 0, 15, and 120 mM [Na]o (tetramethylammonium replacement). Ionic current through K+ channels was blocked by Ba2+ (5 mM) and tetraethylammonium (20 mM), thereby allowing pump-mediated current to be measured by addition or removal of external K+. Control measurements and corrections were made for pump current run-down and holding current drift. Additional controls were done to estimate the magnitude of the inwardly directed pump-mediated current that was present in K(+)-free solution and the residual K(+)- channel current. A pseudo two-state access channel model is described in the Appendix in which only the pseudo first-order rate coefficients for binding of external Na+ and K+ are assumed to be voltage dependent and all transitions between states in the Na+/K+ pump cycle are assumed to be voltage independent. Any three-state or higher order model with only two oppositely directed voltage-dependent rate coefficients can be reduced to an equivalent pseudo two-state model. The steady state current-voltage (I-V) equations derived from the model for each case were simultaneously fit to the I-V data for all four experimental cases and yielded least-squares estimates of the model parameters. The apparent fractional depth of the external access channel for Na+ is 0.486 +/- 0.010; for K+ it is 0.256 +/- 0.009. The Hill coefficient for Na+ is 2.18 +/- 0.06, and the Hill coefficient for K+ (which is dependent on [Na]o) ranges from 0.581 +/- 0.019 to 1.35 +/- 0.034 for 0 and 120 mM [Na]o, respectively. The model provides a reasonable fit to the data and supports the hypothesis that under conditions of saturating internal [Na+], the principal voltage dependence of the Na+/K+ pump cycle is a consequence of the existence of an external high- field access channel in the pump molecule through which Na+ and K+ ions must pass in order to reach their binding sites.     

Na(+) current through KATP channels: consequences for Na(+) and K(+) fluxes during early myocardial ischemia

During early myocardial ischemia, the myocytes are loaded with Na(+), which in turn leads to Ca(2+) overload and cell death. The pathway of the Na(+) influx has not been fully elucidated. The aim of the study was to quantify the Na(+) inward current through sarcolemmal KATP channels (IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential (Vrev) and to estimate the contribution of this current to the Na(+) influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na(+) to K(+) ions, PNa/PK, were close to 0.01. Only in the presence of Na(+) ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/PK, indicative of a Na(+) influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na(+) influx and K(+) efflux is even larger than the respective fluxes by the Na(+) - K(+) pump and all other background fluxes. These results suggest that during early ischemia the Na(+) influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K(+) efflux through KATP channels.     

Acidosis-Induced Enhanced Activity of the Na-K Exchange Pump in the In Vivo Choroid Plexus: An Ontogenetic Analysis of Possible Role in Cerebrospinal Fluid pH Homeostasis

Abstract: Changes in cellular [K] and [Na] in the choroidal epithelium (as a reflection of Na-K pump activity) were analyzed in Sprague-Dawley rats subjected to acute systemic acidosis. In the lateral and 4th ventricle choroid plexus (CP) of adult rats in which metabolic acidosis was induced for 1 h, cell [K] and [Na] increased and decreased by 35 and 15 m m /kg water, respectively, indicating marked stimulation of the Na-K exchange pump in the CSF-facing membrane; in contrast, this striking response of the CP to acidosis could not be elicited in immature animals (1 week old). Since the effects of respiratory acidosis on CP cell [K] and [Na] were similar to those of metabolic acidosis, the reduction in plasma pH (rather than in [HCO₃]) is likely the mechanism underlying the enhanced turnover of Na and K across the CP in adults. The concentration of Na and K in the cerebral cortex, medulla, and CSF was generally not altered during acute acid-base distortions in both mature and immature animals. The striking difference in the response of CNS tissue protected by the blood-CSF barrier (i.e., CP) and the blood-brain barrier (BBB) to systemic acidosis emphasizes a unique role, presumably homeostatic, for the plexus. Since propranolol substantially attenuated the acidosis-induced changes in choroidal cell [K] and [Na], it is possible that there is β-receptor modulation of the Na,K-ATPase (Na-K pump) in the CP. We postulate that the generally observed enhanced electropositivity in the CSF in systemic acidosis is brought about, at least in part, by facilitation of Na-K pumping in the CP, although induced changes in membrane permeability may also be a factor.     

Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.     

Retinoic-acid-induced differentiation of HL-60 cells is associated with biphasic activation of the Na+− K+ pump

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10(-6) M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na]i, which reaches its maximum after 6 h, and an increase in the activity of the Na+-pump, which is reflected by an ouabain-sensitive K+ influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabain-binding sites occurs, this being accompanied by an increase in the number of K+ ions actively transported by each site. The effect of modulation of Na+-pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination, these drugs induced rapid stimulation of the Na+-pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na+]i and in the activity of the Na+-pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.     

Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells

Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chem., 243:1957), the phosphoenzyme formed at 0 degrees C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested that the conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1 approximately P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0 degrees C. Since the ATP:ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1 approximately P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0 degrees C. Simple saturation was observed instead with a K0.5 for Na of approximately 1 mM. The effect of increasing temperature on the ATP:ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0 degrees C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0 degrees C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 degrees C, there was an approximately 300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the E1 approximately P----E2P transition of the Na pump in human red cell membranes is blocked at 0 degrees C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP:ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0 degrees C, where E1 approximately P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme.     

Potassium secretion by the cortical collecting tubule

The isolated perfused rabbit cortical collecting tubule has been shown to actively transport K from bath to lumen. The first step in this process is active uptake of K across the basolateral membrane via and Na:K exchange pump as evidenced by: 1) basolateral localization and Na:K exchange properties of the ouabain-sensitive Na,K-ATPase, 2) ouabain sensitivity of the Na and K fluxes, 3) interdependence of the Na and K fluxes, and 4) ouabain-sensitivity of 42K uptake into the cell across the basolateral membrane. At the luminal border, a significant K permeability of the apical cell membrane has been identified using electrophysiological techniques. This K permeability is insensitive to the diuretic amiloride, and, thus, differs from the pathway for Na entry, which is highly amiloride sensitive. A significant K permeability of the paracellular pathway is not apparent. It is concluded that K secretion by the rabbit cortical collecting tubule occurs via a two-step process: active uptake of K across the basolateral membrane via the Na:K exchange pump, followed by passive efflux of K across the apical membrane via an amiloride-insensitive K conductive pathway.     

Properties of "creep currents" in single frog atrial cells

Changes in membrane current in response to an elevation of [Na]i were studied in enzymatically dispersed frog atrial cells. Na loading by either intracellular dialysis or exposure to the Na ionophore monensin produces changes in membrane current that resemble the "creep currents" originally observed in cardiac Purkinje fibers during exposure to low-K solutions. Na loading induces a transient outward current during depolarizing voltage-clamp pulses, followed by an inward current in response to repolarization back to the holding potential. In contrast to cardiac Purkinje fibers, Na loading of frog atrial cells induces creep currents without accompanying transient inward currents. Creep currents induced by Na loading are insensitive to K channel antagonists like Cs and 4-aminopyridine; they are not influenced by doses of Ca channel antagonists that abolish iCa, but are sensitive to changes in [Ca]o or [Na]o. A comparison of the time course of development of inward creep currents are not tail currents associated with iCa. Inward creep currents can also be induced by experimental interventions that increase the iCa amplitude. Exposure to isoproterenol enhances the iCa amplitude and induces inward creep currents; both can be attenuated by Ca channel antagonists. Both inward and outward creep currents are blocked by low doses of La, independently of La's ability to block iCa. It is concluded that (a) creep currents are not mediated by voltage-gated Na, Ca, or K channels or by an electrogenic Na,K pump; (b) inward creep currents induced either by Na loading or in response to an increase in the amplitude of iCa are triggered by an elevation of [Ca]i; and (c) creep currents may be generated by either an electrogenic Na/Ca exchange mechanism or by a nonselective cation channel activated by [Ca]i.     

Electrophysiology of flounder intestinal mucosa. II. Relation of the electrical potential profile to coupled NaCl absorption

We characterized the hyperpolarization of the electrical potential profile of flounder intestinal cells that accompanies inhibition of NaCl cotransport. Several observations indicate that hyperpolarization of psi a and psi b (delta psi a,b) results from inhibition of NaCl entry across the apical membrane: (a) the response was elicited by replacement of mucosal solution Cl or Na by nontransported ions, and (b) mucosal bumetanide or serosal cGMP, inhibitors of NaCl influx, elicited delta psi a,b and decreased the transepithelial potential (psi t) in parallel. Regardless of initial values, psi a and psi b approached the equilibrium potential for K (EK) so that in the steady state following inhibition of NaCl entry, psi a approximately equal to psi b approximately equal to ECl approximately equal to EK. Bumetanide decreased cell Cl activity (aClc) toward equilibrium levels. Bumetanide and cGMP decreased the fractional apical membrane resistance (fRa), increased the slope of the relation of psi a to [K]m, and decreased cellular conductance (Gc) by approximately 85%, which indicates a marked increase in basolateral membrane conductance (Gb). Since the basolateral membrane normally shows a high conductance to Cl, a direct relation between apical salt entry and GClb is suggested by these findings. As judged by the response to bumetanide or ion replacement in the presence of mucosal Ba, inhibition of Na/K/Cl co-transport alone is not sufficient to elicit delta psi a,b. This suggests the presence of a parallel NaCl co-transport mechanism that may be activated when Na/K/Cl co-transport is compromised. The delta psi a,b response to reduced apical NaCl entry would assist in maintaining the driving force for Na-coupled amino acid uptake across the apical membrane as luminal [NaCl] falls during absorption.     

Evidence for G-Protein Regulation of Inward K+ Channel Current in Guard Cells of Fava Bean

Recent reports have shown that GTP-binding proteins (G-proteins) are present in plants but have given limited indication as to their site of action. G-proteins in animal cells transduce extracellular signals into intracellular or membrane-mediated events, including the regulation of ion channels. Using whole-cell patch clamp, we provide evidence that a G-protein in guard cells of fava bean regulates the magnitude (and not the kinetics) of inward current through K+-selective ion channels in the plasma membrane. GDP[beta]S (100 to 500 [mu]M) increases inward K+ current, whereas GTP[gamma]S (500 [mu]M) has the opposite effect. The control nucleotides ADP[beta]S and ATP[gamma]S (500 [mu]M) do not affect K+ current. Reduction of inward current by GTP[gamma]S is eliminated in the presence of the Ca2+ chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N[prime],N[prime],-tetraacetic acid) (5 mM). When applied intracellularly, the G-protein regulators, cholera toxin and pertussis toxin, both decrease inward K+ current. The entry of K+ (and anions) into guard cells increases their turgor, opening stomatal pores in the leaf epidermis that allow gas exchange with the environment. Our data suggest the involvement of a G-protein in the inhibition of K+ uptake and stomatal opening. Changes in stomatal aperture, vital to both photosynthesis and plant water status, reflect guard-cell responsiveness to a variety of known environmental signals. The results presented here indicate that, in plants as well as animals, ion channel regulation by environmental stimuli may be mediated by G-proteins.     

Identification and characterization of inward K+ -channels in plasma membranes of Arabidopsis root cortex cells

Patch clamping whole-cell reeording techniques were apphed to study the inward K~ channels in Arabidopsis root cortex cells. The inward K~ -channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective tor K~ ions over Na~ ions. The channel activity was significantly inbibited by the external TEA(?) or Ba(?) The changes in cytoplasmic Ca~(2 ) concentrations did not affect the whole-cell inward K~ -currents. The possible asso(?)ation betw(?)en the channel selectivity to K~ and Na(?) ions and plant salt-tolerance was also discussed.     

The Control of Na Uptake into Nitella translucens

Movement of Na into cells of Nitella translucens is a ‘downhill’process; the ions move across the plasmalemma down an electrochemicalpotential gradient. Nevertheless, measurements of Na influxesunder a wide range of experimental conditions have shown thatthere must be links between Na uptake and processes controlledby metabolism. When Ca ions are present in the bathing solution,Na influxes are greatly increased by light under conditionswhere photosynthesis can proceed (i.e. when both photosystemsare active). In the presence of Ca, the influx of Na increasesonly slightly when the external Na concentration is raised above1 mM, and the light-promoted Na influx is considerably inhibitedwhen Cl is removed from the bathing solution. When the Cl concentrationis kept constant, the Na influx in light is determined by theconcentrations of other cations present in solution (K, Ca,or NH₄). In the absence of Ca from the cell wall and solution,the influx is stil enhanced by light, but does not saturatewhen the external Na concentration is raised above 1 mM. Itis suggested that the Na influx in light is partly linked tothe inward Cl pump, but there is also a separate (Cl-independent)effect of light on the permeability of the plasmalemma to Na.Links between Na and Cl uptake could be maintained by effectsof Cl on electrochemical driving forces controlling Na entry;alternatively, chemical coupling between the two processes maybe involved.     

Measurement of Na-K pump current in acinar cells of rat lacrimal glands.

Isolated cells from rat lacrimal glands were voltage clamped using the tight-seal whole-cell recording technique. The intracellular solution contained ATP and an elevated Na concentration (70 mM). Removing external K ions elicited an inward current shift. Ouabain (0.5 mM) induced an inward current shift of identical amplitude, but with slower kinetics. In the presence of ouabain, removal of K ions did not alter the cell current. The potassium- and ouabain-sensitive current was outward between -120 and +20 mV, and its amplitude decreased below -60 mV. This current was highly sensitive to temperature, and was not affected by blockers of the K channels which are present in these cells. It was attributed to an inhibition of the Na-K pump. The Na-K pump current was estimated to be 15 pA for an average acinar cell at physiological temperature, with 70 mM internal Na ions and 20 mM external K ions. Implications of this value in terms of electrolyte secretion are discussed.     

Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase-specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters.