Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria

The blockage of the apical K⁺ channels in frog species Rana temporaria by Ba²⁺ and Cs⁺ is strongly voltage-dependent. The interaction of both blockers with the K⁺ channels was studied by recording relations between the K⁺ currents (I _K) and the transepithelial and intracellular potential. Mucosal Ba²⁺ and Cs⁺ depress I _K, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba²⁺ and Cs⁺ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fR _a, and the voltage divider ratio, fV _a, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fV _a in the presence of Ba²⁺ deviate markedly from the expected fR _a values. The inhibitory effect of Ba²⁺ and Cs⁺ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (), referred to the extracellular side of the channel. We obtained for Ba²⁺, =0.34±0.05 and for Cs⁺, =0.81±0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of .     

Effect of Cs+, Li+ and Na+ on the potassium conductance and gating kinetics in the frog node of Ranvier

The effects of monovalent internal cations Cs⁺, Li⁺ and Na⁺ on potassium channel conductance in the frog node of Ranvier were studied by means of the voltage clamp. As previously reported, when 10–80% of the internal K⁺ was replaced by one of the above cations, the steady-state current-voltage relationship was significantly modified. The main effect was a voltage-dependent attenuation of the currents. We demonstrate that the current attenuation is associated with a change in the channel gating kinetics. For small depolarizations the kinetics can be described by the usual potassium conductance activation time constant, τ _n . However, under certain experimental conditions (e.g. substitution of the intracellular K⁺ with 10% Cs⁺), during larger depolarizations, stepping the membrane potential to values above 40–60 mV, the conductance develops with two time constants: τ _n and a new, slower time constant that, in contrast to τ _n , grows with membrane potential. These results can be explained by assuming that the catins may occupy two different sites in the channel; when the first site is occupied the channel is blocked, while occupation of the second site results in slowing of the gating kinetics in the affected channels.     

Chloride transport in isolated frog (Rana temporaria) skin

Summary In a large number of isolated frog skins, with potential differences of from 20–92mV (mean, 55.3±3.6 S.E.M.), the chloride influx was found to be slightly greater than chloride efflux under shortcircuit conditions, but the difference was not statistically significant. However, if skins of low potential (less than 50 mV) were selected, chloride influx was significantly higher than chloride efflux (P<0.05). In this low potential group furosemide, (10^–3 M, applied to the solution bathing the mucosal surface) was found to produce a) a small increase in short-circuit current, which was generally apparent within 1 min, maximal in 5 min; and thereafter declined towards the control value; b) a marked increase in potential difference, apparent within 1 min and sustained for at least 30 min; c) a large and sustained increase in the calculated d.c. resistance of the skin and d) a decrease in the influx of chloride, such that influx and efflux were equalized.     

Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH

With the single-channel patch-clamp technique we have identified Ca²⁺-sensitive, high-conductance (maxi) K⁺ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K⁺ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K⁺. In excised inside-out patches and with symmetrical 140 mmol/l K⁺, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K⁺ over Na⁺. Depolarization of the BLM increased maxi K⁺ channel activity. Increasing cytosolic free Ca²⁺ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca²⁺ directly activated channel activity in a voltage-dependent manner. Maxi K⁺ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K⁺ channel outward current was reversibly blocked by micromolar concentrations of Ba²⁺ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.     

Procaine has opposite effects on passive Na and K permeabilities in frog skin

Procaine has opposite effects on the active transport of Na⁺ when applied on the mucosal side of the frog skin [where it produces a stimulation of the short-circuit current (I _sc)] or when added on the serosal side (where it produces an inhibition ofI _sc). In an attempt to reveal and localize the primary effect of procaine on either the apical or latero-basal membranes of the epithelial cells, we have tried to chemically dissect both membrane functions with inhibitors and ionophores. When applied on the apical side of the latero-basally depolarized epithelium, 25 mmol/l procaine increasesI _sc andV _oc (transepithelial open-circuit potential), while decreasing the transepithelial resistance. TheE ₁–E ₂ linearity domain of the I–V curves is narrowed. On the serosal side of the depolarized epithelium, the same concentration of procaine does not affectI _sc andV _oc (which are already inhibited) but it produces an increase in the transepithelial resistance (R _t). Procaine influence on the passive K⁺ permeability was studied by using the ionophore nystatin, which is assumed to form channels permeable to K⁺, when applied on the amiloride blocked apical membrane. In nystatin-treated epithelia, 25 mmol/l procaine on the apical side decreaseI _sc,V _oc andR _t. In parallel experiments during Cl^– substitution by SO ₄ ^2– , the procaine effects onI _sc andV _oc are no longer maintained, but transient. The results suggest that procaine positively influences the Na⁺ transient through the apical Na⁺-channels, and inhibits the epithelial permeability for K⁺, possibly by reducing K⁺-ions accessibility to the K⁺-channels.     

Effects of standard diuretics and RPH 2823 on transepithelial Na+ transport in isolated frog skin

Summary Short-circuit current (SCC) techniques were used to monitor the effects of various diuretic agents on Na⁺ transport in isolated frog skin, a model for the late distal tubule and the collecting duct of the mammalian kidney. Acctazolamide, hydrochlorothiazide, torasemide, and ethacrynic acid did not affect sodium transport (as indicated by the SCC) or transepithelial electrical resistance when added either to the apical (outer) or to the inner (basolateral, corial) bathing solution of the tissue. However, Na⁺ transport was sensitive to amiloride, the triamterene derivate dimethylamino-hydroxypropoxytriamterene (RPH 2823), and to furosemide. Whereas apical amiloride, and RPH 2823 induced a dose-dependent decrease in SCC and increase in transepithelial electrical resistance, apical furosemide resulted in a dose-dependent increase in SCC and a decrease in electrical resistance. None of the three diuretic agents caused a significant change in SCC when applied to the inner bathing Ringer's solution. The small furosemide-induced decrease in resistance compared with the huge increase in SCC suggests that furosemide affects Cl^– permeability as well as Na⁺ permeability. Evidence for this notion was achieved by the following findings: (a) The decrease in resistance after furosemide was more pronounced in tissues bathed in Cl^–-free solutions compared with Cl^–-containing solutions. (b) In contrast, SCC stimulation by apical furosemide is Cl^–-ion independent, but strongly Na⁺-ion dependent. (c) SCC stimulation by furosemide is amiloride-sensitive. With respect to the onset, locus, and reversibility of action, it seems reasonable to assume that amiloride, RPH 2823, and furosemide all influence transepithelial Na⁺ transport by interacting with the Na⁺ channel or a regulator site of it within the apical membrane. The stoichiometry of the amiloride (RPH 2823)-receptor site interaction revealed Hill-coefficient(s) of less than 1, indicating a negative cooperativity among the receptor sites. The interaction between Na⁺ ions and amiloride or RPH 2823 displayed mixed competitive-noncompetitive inhibition. Taken together, these results support the hypothesis that amiloride and Na⁺ as well as RPH 2823 and Na⁺ may act at different loci on the apical entry mechanism inRana esculenta skin.Abbreviations R Transepithelial electrical resistance - R _x /R₀ Transepithelial electrical resistance at timex (the time after manipulation) divided by the value at time 0 (the time before manipulation) - SCC Short-circuit current - SCC _x /SCC₀ Short-circuit current at timex (the time after manipulation) divided by the value at time 0 (the time before manipulation) Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthday     

Trans- and paracellular K+ transport in diluting segment of frog kidney

In frog diluting segment transepithelial K⁺ net flux (J _te ^K ) occurs via trans- and paracellular transport routes. Inhibition of transcellular K⁺ transport disclosesJ _te ^K across the shunt-pathway. By means of K⁺-sensitive microelectrodes we have measured secretoryJ _te ^K induced by an acute K⁺ load, in the diluting segment of the isolated and doublyperfused frog kidney. Transcellular K⁺ transport was inhibited by blocking the luminal K⁺ permeability either directly by barium or indirectly by the diuretic drug amiloride (via intracellular acidification induced by inhibition of Na⁺/H⁺ exchange), by the the Na⁺/K⁺ pump inhibitor ouabain or by inducing an acute acid load. All experimental maneouvers led to a reduction of secretoryJ _te ^K to about 50% of the controlJ _te ^K . The apparent permeability coefficient for K⁺ of this nephron portion after inhibition of transcellular secretoryJ _te ^K was reduced to a similar extent. We conclude: In frog diluting segment the ratio of trans- over paracellularJ _te ^K is close to unity. This ratio represents a minimum estimate because inhibition of the transcellular K⁺ pathway by barium, amiloride or an acute acid load may have been incomplete. Acidosis and/or amiloride exert large antikaliuretic effects due to the inhibition of the luminal K⁺ permeability.     

Oxytocin stimulates the apical K+ conductance in frog skin

We measured the effects of oxytocin (0.1 U/ml) on the current (I _sc) recorded through skins ofRana temporaria incubated with an isotonic K⁺ solution on the api al side while the transepithelial potential was clamped to zero. Under these conditions,I _sc is carried by inward K⁺ movements. Oxytocin markedly stimulated this inward K⁺ current. When the spontaneous fluctuations were analyzed we found that oxytocin increased the plateau (S _o ⁽¹⁾) of the spontaneous Lorentzian component without modifying the corner frequency (f _c ⁽¹⁾). Addition of Ba²⁺ to the mucosal solution blockedI _sc both in the presence and absence of oxytocin. Moreover, with mucosal Ba²⁺ a characteristic blocker-induced Lorentzian component appeared in the power spectrum. Analysis of this blocker-induced noise showed that oxytocin increased the number of active K⁺ channels in the apical membrane, while the changes in single channel current were in agreement with expected alterations of the electrochemical driving force.     

Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues

We have investigated the possible existence of a Na⁺/H⁺ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues: 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na⁺ influxes (J ₁₃) with aK _I of 4·10^–7 mol/l and the Na⁺ pool (control: 77±4 neq·h^–1·cm^–2; phenamil: 21±1 neq·h^–1·cm^–2). On the contrary EIPA (10^–5 mol/l) had no effect onJ ₁₃ nor on the apical Na⁺ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO ₃ ^– , 5% CO₂, pH 7.34) to a HCO ₃ ^– -free Ringer (5% CO₂, pH 6.20) while blocking the Na⁺ conductance with phenamil, produced a large stimulation of Na⁺ influxes exclusively across the basolateral membranes (J ₃₂), after return to a normal Ringer (J ₃₂=706±76 and 1635±199 neq·h^–1·cm^–2 in control and acid-loaded epithelia respectively). The stimulation ofJ ₃₂ was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (K _I=7·10^–6 mol/l) and EIPA (K _I=5·10^–7 mol/l) whereas phenamil had no effect. In na⁺-loaded epithelia (ouabain treated) the Na⁺ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10^–5 mol/l) or amiloride (10^–4 mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na⁺ permeability mediated by a Na⁺/H⁺ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.     

Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin

The voltage-dependence of the inhibitory effect of mucosal Cs⁺ on the inward K⁺ current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaCl and KCl Ringer solutions on the serosal and mucosal side respectively (contron skins), as well as with tissues incubated with K₂SO₄ Ringer solutions on both sides (depolarized skins). Studies of the dose-depedence of the Cs⁺ block showed that under both experimental conditions the apparent affinity of Cs⁺ increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs⁺ required to block 50% of the K⁺ current (K_Cs) recorded while the transepithelial voltage was clamped at zero mV was 16 mmol/1. K_Cs decreased exponentially with muscosal positive voltages. The dependence of K_Cs on the membrane potential was analyzed with Eyring rate theory in which Cs⁺ was assumed to block the K⁺ transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance =0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis wasn=3.1. Experiments with K⁺-depolarized tissues showed that only inward K⁺ currents recorded with positive transepithelial voltages were depressed by external Cs⁺. Also under these conditions K_Cs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed =0.09 andn=1.7. The difference in found in control and depolarized tissues can be explained by the influence of the basolateral membrane resistance on theI–V relations.     

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na⁺ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3–4 days or 7–10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na₂SO₄ (25 mmol/l) solutions modified the Na⁺ transport rate and the morphology of the epithelium. The highest Na⁺ transport rates were found for the animals adapted to the Na⁺ free solutions and were correlated with an increase in the total MR cell pit-surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO₂ increase and HCO ₃ ^– decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10^–6 M) or during acidification of the epithelium by serosally applied CO₂. Na⁺ transport was enhanced by AVT (10^–6 M) or oxytocin (100mU/ml) when the skin was bathed on its apical side with a high Na⁺ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na⁺ transport when the apical solution was low in Na⁺ (0.5mmol/l). It is concluded that MR cells play a key role in Na⁺ and H⁺ transport through the frog skin epithelium when bathed on its apical side with a low Na⁺ containing solution. Distinct pathways for sodium transport through two cell types (MR cells and granular cells) are proposed depending on the Na⁺ concentration of the solution bathing the apical side of the epithelium.     

Kinetic studies on the effects of ouabain on Na+ fluxes in frog skin

Among 48 pieces of paired frog skins ofRana pipiens in Ringer's solution, 10 pieces showed a strictly monotone decrease in the short circuit current (SCC) following ouabain treatment (10^–4 M). In 9 cases a transient attenuation, and in 27 cases a distinct wave in the ebb of the SCC, was seen. In 2 instances, two waves were seen. Associated with the not-monotone events was a transient rise in electrical skin conductance. The reasons for these mixed skin responses are unknown. One possible reason is considered here: Early during the ouabain action, some of the Na⁺ entering from the mucosal side is trapped in the skin by electroneutral processes, in keeping with the already known fact that ultimately cellular KCl is partly replaced by NaCl. Computer assisted model studies show how monotone, and not-monotone transepithelial net Na⁺ flux curves can be generated. Essential conditions for the generation of notmonotone Na⁺ flux curves are: 1. Presence of two distinct cellular, active Na⁺ pools in the model. 2. Presence of a loop pathway in which a principal transepithelial Na⁺ transport compartment, and a constitutent Na⁺/K⁺ maintenance compartment, are connected to each other and to the extracellular compartment. The model, then, predicts under which kinetic conditions monotone and not-monotone transepithelial Na⁺ flux curves will be seen.     

Intracellular calcium modulates basolateral K+-permeability in frog skin epithelium

Cytosolic calcium ([Ca²⁺]_i) has been suggested as a key modulator in the regulation of active sodium transport across electrically tight (high resistance) epithelia. In this study we investigated the effects of calcium on cellular electrophysiological parameters in a classical model tissue, the frog skin. [Ca²⁺]_i was measured with fura-2 in an epifluorescence microscope setup. An inhibition of basolateral potassium permeability was observed when cytosolic calcium was increased. This inhibition was reversible upon removal of calcium from the serosal solution.     

Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na⁺ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was –140 mV and test potentials ranged from –100 to + 40 mV (pulse duration 100 or 1000 ms). AP-A (50–100 nM) markedly slowed the rate of decay of Na⁺ current (I _Na) and increased peak I _Na conductance (g _Na) by 38±5.5% (mean±SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the g _Na/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state I _Na availability (h ) demonstrated an increase in slope factor from 5.9±0.8 mV in control to 8.0±0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified I _Na showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (–15±2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (–3±1%). This use-dependent effect was probably the result of a decrease in the rate of recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1±0.3 ms in control to 6.0±1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2±6.5 ms in control to 188.9±36.4 ms (P< 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca²⁺ or Mg²⁺) to 10 mM abolished the effects of AP-A on the rate of I _Na decay. These results demonstrate that modification of cardiac Na⁺ channels by AP-A markedly slowed I _Na inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in g _Na presumably by increasing the number of channels open at peak I _Na. AP-A slows the rate of recovery of I _Na from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations. Thus, increasing [Mg²⁺]_o or [Ca²⁺]_o displaces AP-A from binding, suggesting that they share related binding sites on the external surface of the Na⁺ channel.     

K+-dependent stability and ion conduction of Shab K+ channels: a comparison with Shaker channels

K⁺ depletion exerts dramatically variable effects on different potassium channels. Here we report that Shab channels are rather stable in the absence of either internal or external K⁺ alone; however, its stability is greater with K⁺ outside the cell. In contrast, with 0 K⁺ (non-added) solutions on both sides of the membrane, the conductance (G_K) is rapidly and irreversibly lost. G_K is lost with the channels closed and regardless of the composition of the 0 K⁺ solutions. In comparison, it is known that the Shaker B G_K collapses only if the channels are gated in 0 K⁺, Na⁺-containing solutions. In order to compare the behavior of Shab to that of Shaker, we show that after extensively gating the channels in 0 K⁺ N-methyl-D-glucamine solutions, most Shaker channels remain stable, and in a conformation where G_K collapses as soon as there is Na⁺ in the solutions. Regarding ion conduction, in contrast to Kv2.1 and Shaker A463C that have a sizable G_Na in 0 K⁺, Shab, which shares a 463-cysteine and an identical signature sequence with these channels, does not appreciably conduct Na⁺, although it presents a significant Cs⁺ conductance. The observations suggest that there are at least two sites where K⁺ binds and thus maintains Shab G_K stable, one internal and the other(s) most likely located outside the selectivity filter.     

Membrane potentials in Rana temporaria muscle fibres in strongly hypertonic solutions

Conventional microelectrode methods were used to measure variations in resting membrane potentials, E _m, of intact amphibian skeletal muscle fibres over a wide range of increased extracellular tonicities produced by inclusion of varying extracellular concentrations of sucrose. Moderate increases in extracellular tonicity to up to 2.6× normal (2.6τ) under Cl⁻ free conditions produced negative shifts in E _m that followed expectations for the K⁺ Nernst equation (E _K) applied to a perfect osmometer containing a conserved intracellular K⁺ content despite any accompanying cell volume change. In contrast, E _m remained stable in fibres studied in otherwise similar Cl⁻ containing solutions, consistent with E _m stabilization despite negative shifts in E _K through inward cation-Cl⁻ co-transport activity. Short exposures to higher tonicities (>3τ) similarly produced negative shifts in E _m in Cl⁻ free but not Cl⁻ containing solutions. However, prolonged exposures to solutions of >3τ caused gradual net positive changes in E _m in both Cl⁻ containing and Cl⁻ free solutions suggesting that these changes were independent of cation-Cl⁻ transport. Indeed, there was no evidence of cation-Cl⁻ co-transport activity in strongly hypertonic solutions despite its predicted energetic favourability, suggesting its possible regulation by E _m in muscle. Additional findings implicated a failure to maintain greatly increased transmembrane [K⁺] gradients in these E _m changes. Thus: (1) halving or doubling [K⁺]_e produced negative or positive shifts␣in E _m, respectively in isotonic or moderately hypertonic (<2.7τ), but not strongly hypertonic (>3τ) solutions; (2) subsequent restoration of isotonic extracellular conditions produced further positive changes in E _m consistent with a dilution of the depleted [K⁺]_i by fibres regaining their original resting volumes; (3) quantitative modelling similarly predicted a gradual net efflux of K⁺ as the balance between active and passive [K⁺] fluxes altered due to increased transmembrane [K⁺] gradients in hypertonic and low [K⁺]_e solutions. However, the observed positive changes in E _m in the most strongly hypertonic solutions eventually exceeded these predictions suggesting additional limitations on␣Na⁺/K⁺-ATPase activity in strongly hypertonic solutions.James A. Fraser and Kai Yuen Wong have equally contributed to this paper.     

ATP maintains ATP-inhibited K+ channels in an operational state

In patch-clamp records of K⁺ _ATP channels in an insulin-secreting cell line (RINm5F) inhibition evoked by exposing the internal surface of the membrane to ATP is followed not just by the recovery of K⁺ _ATP channel activity when the ATP is removed but by a marked activation of K⁺ _ATP channels. This phenomenon is not a direct consequence of channel closure as inhibition induced by quinidine and quinine is followed upon the removal of the drug only by the recovery of K⁺ _ATP channel activity and not by post-inhibitory activation. If ATP is applied to the exposed internal surface of a membrane patch when all of its K⁺ _ATP channel have run down subsequent removal of the ATP causes their activation. The magnitude and duration of the reactivation of K⁺ _ATP channels is shown to depend upon both the concentration of ATP and the length of time for which the membrane is exposed to ATP. We therefore have a paradoxical situation in that K⁺ channels which are inhibited by intracellular ATP require intracellular ATP to retain the ability to open.     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions

Isolated skin of the clawed frogXenopus laevis was mounted in an Ussing-chamber. The transcellular sodiumcurrent (I _Na) was identified either as amiloride-blockable (10^–3 mol/l) short-circuit current (I _SC), or by correctingI _SC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd²⁺ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd²⁺-concentration of 2,6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd²⁺ could not be seen when 10^–3 mol/l amiloride was placed in the outer, Na⁺-containing solution, nor when Na⁺ was replaced by Tris. This suggests that Cd²⁺ stimulatesI _Na. Cd²⁺ intefered with the Na⁺-current self-inhibition, and therefore with the saturation ofI _Na by increasing the apparent Michaelis constant (K _Na) of this process. The I _Na recline after stepping up mucosal [Na⁺] was much reduced in presence of Cd²⁺. Ca²⁺-ions on the mucosal side had an identical effect to Cd²⁺, and 10 mmol/l Ca²⁺ increaseI _Na by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca²⁺. The mechanism ofI _Na-stimulation by Ca²⁺ did not seem to differ from that of Cd²⁺. Thus, although of low Na⁺-transport capacity,Xenopus skin appears to be as good a model for Na⁺-transporting epithelia asRanidae skin, with the exception of the calcium effect which, so far, has not been reported forRanidae.     

Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (a _Na ⁱ ) and intracellular pH (pH_i) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. WhenRana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na₂SO₄ buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor, ethoxzolamide (10^–4M) blocks net H⁺ ion excretion and Na absorption, producing a depolarization of 25–30 mV of the apical membrane, potential (mc). We demonstrate the these changes are accompanied by a fall ina _Na ⁱ from 6.2±0.5 mmol/l to 3.4±0.6 mmol/l and an increase in pH_i from 7.20±0.03 to 7.38±0.08 (n=12 skins). Voltage clamping mc to its control value in the presence of ethoxzolamide restoreda _Na ⁱ but the pH_i remained alkaline. Furthermore, the fall ina _Na ⁱ produced by ethoxzolamide could be mimicked by voltage clamping mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na⁺ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H⁺ pump at the apical membrane.Addition of amiloride (10^–5 mol/l) or substitution of external Na⁺ by Mg²⁺ or K⁺ caused a hyperpolarization of mc and a fall ina _Na ⁱ . These effects were accompanied by an inhibition of H⁺ excretion and a fall in pH_i of 0.14 ±0.08 units (n=6 skins). However, when the effect, of Na⁺ transport inhibition on mc was prevented by imposing a voltage clamp no effects of amiloride on H⁺ excretion or pH_i were observed. Moreover, the effect of amiloride on pH_i could be reproduced in control skins by voltage clamping mc to –100 mV. The metabolic inhibitors vanadate (1 mmol/l) and di-cyclo hexyl carbodiimide (5×10^–5 mol/l) inhibited H⁺ excretion and decreased pH_i from 7.28±0.08 to 7.02±0.06 and from 7.30±0.06 to 7.12±0.05 (n=6 skins), respectively.These results indicate that an apical membrane H⁺ ATPase plays a role in regulating pH_i and the mechanism is sensitive to membrane potential.