Electrophysiology of the sodium-potassium-ATPase in cardiac cells.

Like several other ion transporters, the Na(+)-K(+) pump of animal cells is electrogenic. The pump generates the pump current I(p). Under physiological conditions, I(p) is an outward current. It can be measured by electrophysiological methods. These methods permit the study of characteristics of the Na(+)-K(+) pump in its physiological environment, i.e., in the cell membrane. The cell membrane, across which a potential gradient exists, separates the cytosol and extracellular medium, which have distinctly different ionic compositions. The introduction of the patch-clamp techniques and the enzymatic isolation of cells have facilitated the investigation of I(p) in single cardiac myocytes. This review summarizes and discusses the results obtained from I(p) measurements in isolated cardiac cells. These results offer new exciting insights into the voltage and ionic dependence of the Na(+)-K(+) pump activity, its effect on membrane potential, and its modulation by hormones, transmitters, and drugs. They are fundamental for our current understanding of Na(+)-K(+) pumping in electrically excitable cells.

Differences in the protein-kinase-A-dependent regulation of CFTR Cl- channels and Na+-K+ pumps in guinea-pig ventricular myocytes.

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.

The current produced by the E779A mutant rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells.

The current (I(p)) generated by the wild-type or the glutamate (E) 779 alanine (A) mutant of the rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells was studied at 35 degrees C by means of whole-cell recording in Na(+)-free and Na(+)-containing solution. Glutamate 779 is located in the fifth transmembrane domain of the alpha-subunit of the Na(+)/K(+)-ATPase. Compared with the wild-type, the E779A mutant exhibited an apparent K(+)(o)-affinity decreased by a factor of 3-4 both in Na(+)-free and in Na(+)-containing media. The competition of Na(+)(o) and K(+)(o) for cation binding sites of the pump remained unchanged. Similarly, in Na(+)-free solution the shape of the I(p)-V curves for various external K(+)-concentrations ([K(+)](o)) was essentially the same. However, in Na(+)-containing solutions the shape of I(p)-V curves from cells expressing the mutant of the rat alpha1-subunit clearly differed from the shape observed in cells expressing the wild-type, but voltage dependence of the pump current persisted. A prominent Na(+)(o)-activated, electrogenic Na(+)-transport mediated by the pump, displaying little voltage dependence in the potential range tested (-80 to +60 mV), was present in the cells expressing the E779A mutant pump. The data suggest that exchanging E779 for A in the rat Na(+)/K(+) pump alpha1-subunit causes a modest decrease in the apparent K(+)(o) affinity and a profound, Na(+)(o)-dependent alteration in the electrogenicity of the mutant pump expressed in HEK 293 cells.

Activation of the cAMP-protein kinase A pathway facilitates Na+ translocation by the Na+-K+ pump in guinea-pig ventricular myocytes.

1. The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea-pig ventricular myocytes by means of whole-cell voltage clamp at 30 C. 2. In external solution containing 144 mM Na+ (Na+o) and 10 mM K+ (K+o), steady-state Na+-K+ pump current (Ip) activated by 5 mM pipette Na+ (Na+pip) at -20 mV was reversibly augmented by forskolin (4 microM) to 133 +/- 4 % of the control current (n = 15). The forskolin analogue 1, 9-dideoxyforskolin (10 microM), which does not activate adenylyl cyclases, did not increase Ip (n = 2). Application of the protein kinase A (PKA) inhibitor H-89 (10 microM) in the continued presence of forskolin reversed the forskolin-induced elevation of Ip (n = 3). 3. The forskolin effect on Ip persisted in the presence of 50 mM Na+pip which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased Ip to 142 +/- 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0.013 nM (n = 5) and to 138 +/- 4 % of the control Ip when free [Ca2+]pip was 15 nM (n = 9). 4. In Na+-free external solution, Ip activated by 50 mM Na+pip and 1.5 mM K+o was likewise increased by forskolin but to a lesser extent than in Na+-containing medium (116 +/- 3 % of control, n = 10). 5. In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q-V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0.5) and the maximum amount of movable charge (DeltaQmax). Forskolin (2-10 microM) shifted V0.5 to more negative values while DeltaQmax was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 microM; n = 2) and abolished by a peptide inhibitor of PKA (PKI, 10 microM; n = 5). 6. We conclude that activation of the cAMP-PKA pathway in guinea-pig ventricular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart.

Depolarization increases the apparent affinity of the Na+-K+ pump to cytoplasmic Na+ in isolated guinea-pig ventricular myocytes.

1. In order to investigate the possible effect of membrane potential on cytoplasmic Na+ binding to the Na+-K+ pump, we studied Na+-K+ pump current-voltage relationships in single guinea-pig ventricular myocytes whole-cell voltage clamped with pipette solutions containing various concentrations of Na+ ([Na+]pip) and either tetraethylammonium (TEA+) or N-methyl-D-glucamine (NMDG+) as the main cation. The experiments were conducted at 30 C under conditions designed to abolish the known voltage dependence of other steps in the pump cycle, i.e. in Na+-free external media containing 20 mM Cs+. 2. Na+-K+ pump current (Ip) was absent in cells dialysed with Na+-free pipette solutions and was almost voltage independent at 50 mM Na+pip (potential range: -100 to +40 mV). By contrast, the activation of Ip by 0.5-5 mM Na+pip was clearly voltage sensitive and increased with depolarization, independently of the main intracellular cation species. 3. The apparent affinity of the Na+-K+ pump for cytoplasmic Na+ increased monotonically with depolarization. The [Na+]pip required for half-maximal Ip activation (K0.5 value) amounted to 5.6 mM at -100 mV and to 2.2 mM at +40 mV. 4. The results suggest that cytoplasmic Na+ binding and/or a subsequent partial reaction in the pump cycle prior to Na+ release is voltage dependent. From the voltage dependence of the K0.5 values the dielectric coefficient for intracellular Na+ binding/translocation was calculated to be approximately 0.08. The voltage-dependent mechanism might add to the activation of the cardiac Na+-K+ pump during cardiac excitation.

High affinity regulation of cardiac Cl- and Ca2+ conductances by (13R)-spiroforskolin.

The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (I(Cl(cAMP))) and the atrial L-type calcium current (I(Ca,L)) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30 degrees C and 20-22 degrees C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated I(Cl(CAMP)) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC50 value) amounted to 9.6x10(-11) M and was lower than the EC50 value for forskolin (2.4x10(-8) M). (13R)-spiroforskolin evoked a smaller maximal I(Cl(cAMP)) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated I(Cl(cAMP)) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased I(Cl(cAMP)) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial I(Ca,L) (13R)-spiroforskolin displayed a smaller activation and a lower EC50 value (5.8x10(-10) M) than forskolin (EC50 value: 3.7x10(-7) M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase.

Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations.

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na+/K+-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na+/K+ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na+ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to approximately 5 x 10(-7) M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na+/K+ pump inhibition (K'D value) were calculated to be approximately 10(-6) M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K'D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K'D value for 22-allyl-digitoxigenin was even larger and amounted to approximately 4 x 10(-5) M. The K'D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (approximately 9 x 10(2) s(-1) M[-1]) was smaller than the association rate constant of digitoxigenin (approximately 2 x 10(4) s(-1) M[-1]), whereas the apparent dissociation rate constants of both compounds were similar (approximately 4 x 10(-3) s[-1]). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (approximately 3 x 10(3) s(-1) M[-1]) and a larger dissociation rate constant (approximately 8 x 10(-2) M[-1]). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na+/K+ pump by affinity labeling.

Activation of the Na+/K+ pump current by intra- and extracellular Li ions in single guinea-pig cardiac cells.

Li+ is the only ion that can replace the physiological intra- and extracellular activator cations of the Na+/K+ pump. In order to study this singular property of Li+ in some detail, the activation of the Na+/K+ pump current (Ip) by intra- and extracellular Li+ (Li+; Li[o]+) was measured in isolated guinea-pig ventricular myocytes by means of whole cell recording at 34 degrees C and a holding potential of -20 mV. Ip was identified as current blocked by dihydro-ouabain. Half-maximal Ip activation occurred at 23 mM Li(o)+ (K0.5 value) in cells containing Na+ (50 or 100 mM) and at 73 mM Li(o)+ in myocytes containing Li+ (100 mM). The K0.5 value of Ip activation by Li(o)+ increased with depolarisation, suggesting the transfer of 0.2 of an elementary charge across the electric field of the sacrolemma during Li(o)+-binding. An intracellular Li+ concentration of 36 mM caused half-maximal Ip activation in cells superfused with Na+- and Li+-free media containing 1 mM K+. In Na+-free solutions. the Ip-V curve displayed a positive slope at negative membrane potentials. A negative slope at positive potentials was observed in Li+-containing media. It is concluded that Li+ is less efficacious and potent than the physiological pump activator cations. The shape of the Ip-V curves in Na+-free solutions supports the view that the cardiac Na+/K+ pump contains a channel-like structure and suggests that there are voltage-sensitive steps in the pump cycle, apart from the binding of external cations.

Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells.

The Na/K pump current I(p) of single HEK293 cells either untransfected (endogenous I(p)) or transfected with the alpha1 subunit of the rat Na/K pump (exogenous I(p)) was investigated in Na-containing solution by means of whole-cell recording at 30 degrees C. The endogenous I(p) was irreversibly blocked by 10(-4) M ouabain or 2 x 10(-4) M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF(-1) at 0 mV and 5.4 mM K(o). It was half maximally activated at 1.5 mM K(o) and increased linearly with depolarization over the entire voltage range studied (-80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant alpha1 subunit of the rat Na/K pump showed an I(p) in the presence of 10(-4) M ouabain and 2 x 10(-4) M DHO, respectively. This exogenous I(p) was reversibly blocked by 10(-2) M ouabain. Half maximal activation of the exogenous I(p) occurred at 1.7 mM K(o). Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I(p) of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I(p) in HEK293 cells is generated by the alpha1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.

Cardiac Na+ pump current-voltage relationships at various transmembrane gradients of the pumped cations.

Thermodynamic considerations predict changes of the Na+ pump current (Ip)-voltage (V) relationship of animal cells upon variations of the electrochemical gradients against which cations must be pumped. Experimental data in support of the predictions are sparse. Therefore, the effect on the Ip-V relationship of various electrochemical gradients for pumped Na+ and Cs+ was studied at constant deltaGATP (approximately -39kJ/mol in cardioballs from sheep Purkinje fibres. Control of the subsarcolemmal ionic concentrations during whole-cell recording was ensured by activation of Ip below its half maximal activity or by measuring the initial Ip following reactivation of the Na+/K+ pump. With gradients close to physiological conditions Ip was outward over the entire voltage range and the Ip-V relationship showed a maximum near zero potential. Steepening the ionic gradients diminished the Ip amplitude and outward pump current was no longer detectable between -65 mV and -110 mV. Flattened ionic gradients increased the Ip amplitude and shifted apparently the reversal potential Erev to more negative values. These changes are in line with theoretical considerations. The measured Ip-V relationships were fitted by curves computed on the basis of a simplified Post-Albers scheme of Na+/Cs+ pumping. The increased Ip amplitude at flat ionic gradients was due to a decrease of [Cs+]o for half maximal Ip activation. The maximal Ip amplitude remained unaffected

Change of Na+ pump current reversal potential in sheep cardiac Purkinje cells with varying free energy of ATP hydrolysis.

1. The Na(+)-K+ pump current, Ip, of cardioballs from isolated sheep cardiac Purkinje cells was measured at 30-34 degrees C by means of whole-cell recording. 2. Under physiological conditions Ip is an outward current. Experimental conditions which cause a less negative free energy of intracellular ATP hydrolysis (delta GATP) and steeper sarcolemmal gradients for the pumped Na+ and Cs+ ions evoked an Ip in the inward direction over a wide range of membrane potentials. The reversal of the Ip direction was reversible. 3. The inwardly directed Ip increased with increasingly negative membrane potentials and amounted to -0.13 +/- 0.03 microA cm-2 (mean +/- S.E.M.; n = 6) at -95 mV. 4. The reversal potential (Erev) of Ip was studied as a function of delta GATP at constant sarcolemmal gradients of the pumped cations. 5. In order to vary delta GATP the cell interior was dialysed with patch pipette solutions containing 10 mM ATP and different concentrations of ADP and inorganic phosphate. The media were composed to produce delta GATP levels of about -58, -49 and -39 kJ mol-1. 6. A less negative delta GATP shifted Erev to more positive membrane potentials. From measurements of Ip as a function of membrane potential Erev was estimated to be -195, -115 and -60 mV at delta GATP levels of approximately -58, -49 and -39 kJ mol-1, respectively. The calculated Erev amounted to -224 mV at delta GATP approximately -58 kJ mol-1, -126 mV at delta GATP approximately 49 kJ mol-1 and -24 mV at delta GATP approximately -39 kJ mol-1. 7. Possible reasons for the discrepancy between estimated and calculated Erev values are discussed. 8. Shifting delta GATP to less negative values not only altered Erev but also diminished Ip at each membrane potential tested. The maximal Ip (Ip,max), which can be activated by external Cs+ (Cs+o), decreased under these conditions, whereas [Cs+]o causing half-maximal Ip activation remained unchanged. Similarly, the voltage dependence of Ip activation by Cs+o was unaffected. 9. It is concluded that Erev of Ip varies with delta GATP at constant sarcolemmal gradients of the pumped cations. This agrees with thermodynamic considerations.

The antagonistic effect of K+o and dihydro-ouabain on the Na+ pump current of single rat and guinea-pig cardiac cells.

1. The antagonistic effect of extracellular potassium ions (K+o) and dihydro-ouabain (DHO) on the Na(+)-K+ pump current (Ip) was studied in isolated ventricular cells. 2. The myocytes were isolated from rats and guinea-pigs, two species with different sensitivity towards cardiac glycosides. Ip measurements were performed at 32-34 degrees C by means of whole-cell recording. The membrane potential was held at -20 mV throughout. 3. The DHO concentration ([DHO]) required for half-maximal Ip inhibition (apparent KD value, KD') amounted to 2.4 x 10(-3) and 1.4 x 10(-5) M for rat and guinea-pig myocytes, respectively, at 5.4 mM K+o. 4. The data suggest one-to-one binding of DHO to the Na(+)-K+ pump and a smaller association rate constant, as well as a larger dissociation rate constant, for binding of DHO in the rat cells. 5. Ip activation by K+o was nearly identical in myocytes of both species and was measured to be half-maximal at approximately 1 mM K+o. Half-maximal Ip activation by K+o remained essentially unchanged, but Ip decreased in media containing [DHO] near the respective KD' at 5.4 mM K+o. 6. The concentration-response curve of Ip inhibition by DHO was shifted to higher [DHO] at higher [K+]o. KD' increased correspondingly. The slope of the curve was unaffected. 7. Ip and KD' displayed a similar dependence on [K+]o. 8. KD' was larger in Na(+)-free than in Na(+)-containing media under conditions in which the activation of Ip by K+o was nearly the same. 9. It is concluded that the antagonism between K+o and DHO, with regard to the activation of Ip, is non-competitive. A possible mechanism of the antagonism is discussed. The mechanism implies binding of K+o and DHO to different conformational states of the Na(+)-K+ pump which are temporarily exposed to the external face of the sarcolemma in the pump cycle. The DHO-bound states do not participate in the generation of Ip.

The effect of cardiac glycosides on the Na+ pump current-voltage relationship of isolated rat and guinea-pig heart cells.

1. Whole-cell recording from isolated rat and guinea-pig ventricular myocytes revealed a change of the cardiac Na+ pump current (Ip)-voltage (V) relationship by cardiac glycosides, specific inhibitors of the Na(+)-K+ pump. 2. Dihydro-ouabain (DHO) diminished Ip in rat ventricular cells at 0 mV in a concentration-dependent manner. 3. The concentration-response curve of Ip inhibition caused by DHO was shifted to higher [DHO] at higher extracellular K+ concentrations ([K+]o) or at more negative membrane potentials. 4. In rat myocytes, DHO immediately flattened the normalized cardiac Ip-V curve and evoked or enhanced a region of negative slope. 5. Ouabain, at concentrations which caused a comparable inhibition of Ip, exerted DHO-like effects on the Ip-V relationship of rat ventricular myocytes. However, the effects developed more slowly. 6. A slowly developing alteration of the Ip-V curve was also observed upon application of DHO to guinea-pig ventricular cells. The range of [DHO] used was about 100-fold lower than that applied to rat ventricular cells, but was equally effective for Ip inhibition. 7. Increasing the K+ concentration of DHO-containing media affected the existing equilibrium of DHO binding to the cardiac Na(+)-K+ pump. A new equilibrium was reached within about 3 s in rat ventricular myocytes, but only within about 50 s in guinea-pig ventricular cells under the experimental conditions chosen. 8. It is concluded that the changes of the cardiac Ip-V curve induced by cardiac glycosides are mediated by voltage-dependent variations of the local [K+]o at the K+ binding sites of the Na(+)-K+ pump in an 'access channel'. The variations were estimated by means of the Boltzmann equation. The estimations agreed with those derived from the measured DHO binding to the Na(+)-K+ pump at various [K+]o. A new equilibrium of glycoside binding to the pump is established at the altered [K+]o. The time necessary to reach the new binding equilibrium varies with the cardioactive steroid, its concentration and the glycoside sensitivity of the cardiac cells.

Na+ pump current-voltage relationships of rabbit cardiac Purkinje cells in Na(+)-free solution.

1. The Na+ pump current (Ip) of isolated, single rabbit cardiac Purkinje cells in Na(+)-free solution was measured at 32-34 degrees C by means of whole-cell recording. 2. The Ip amplitude was studied as a function of clamp potential (Vc) and external concentration of various monovalent cations known to activate the Na(+)-K+ pump. 3. Under conditions which strongly activated Ip the Ip-Vc curve of the cells displayed a positive slope at membrane potentials negative to -20 mV and little variation at more positive potentials. 4. The Ip-Vc relationship showed an extended region of negative slope at positive and negative potentials in solutions containing low concentrations of activator cations which caused little Ip activation. A positive slope of the Ip-Vc curve was occasionally observed at clamp potentials negative to -60 mV under these conditions. 5. The shape of the Ip-Vc relation was independent of the cation species used as external Ip activator. 6. At zero membrane potential half-maximum Ip activation (K0.5(Vc = 0 mV) occurred at 0.05 mM Tl+, 0.08 mM K+, 0.4 mM NH4+ and 1.5 mM Cs+. The Hill coefficient derived amounted to 0.9 for Tl+, 1.2 for K+, 1.04 for NH4+ and 1.5 for Cs+. 7. The concentrations of external activator cations required for half-maximum Ip activation increased with depolarization. The voltage dependence of the K0.5 values could be described by a single exponential function for clamp potentials positive to -40 mV. 8. The steepness of the function is determined by a factor alpha, indicating the apparent fraction of an elementary charge which moves in the electrical field across the sarcolemma when external monovalent cations bind to the Na(+)-K+ pump. 9. The alpha values were calculated to be 0.32 for Tl+, 0.24 for K+, 0.29 for NH4+ and 0.18 for Cs+. Possible interpretations of the alpha values are considered. 10. It is suggested that binding of external monovalent activator cations to the Na(+)-K+ pump (or a process related to the binding) is voltage dependent. This potential-dependent process determines mainly the shape of the Ip-Vc curve in cardiac Purkinje cells superfused with Na(+)-free media containing low concentrations (< K0.5(Vc = 0 mV)) of K+ or its congeners.

The Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP production.

The role of glycolysis and oxidative phosphorylation in providing the ATP for the cardiac Na+/K+ pump was studied in cardioballs from sheep Purkinje fibres. As an indicator of the pump activity, the pump current Ip was measured at -20 mV and 30-33 degrees C by means of whole-cell recording. During intracellular perfusion with a pipette solution containing 5 mM ATP and 15 mM glucose Ip reached a maximum within 8 min and declined to 50% of this value within 27 min after gaining access to the cell interior. Perfusion with an ATP- and glucose-free medium barely enhanced the Ip decline. Inhibition of the oxidative phosphorylation by carbonylcyanide m-chlorophenylhydrazone (CCCP, 2 microM or 20 microM) moderately accelerated the effect of the ATP- and glucose-free pipette solution. Addition of 2 mM iodoacetic acid (an inhibitor of glycolysis) to the latter medium further enhanced the Ip decrease with time. Inhibition of the glycolytic ATP synthesis by 2-deoxy-D-glucose (5 mM) caused a dramatic decline of Ip to half of its maximum within 7.3 min. Pyruvate (5 mM) and inorganic phosphate (2 mM) did not affect the fast Ip decline evoked by the ATP- and glucose-free, 2-deoxyglucose-containing medium, whereas 2 microM CCCP still hastened the fast Ip decrease slightly. This effect of complete metabolic inhibition was reversed by switching to an inhibitor-free pipette solution containing 15 mM ATP. It is concluded that the Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP synthesis.

The kinetics of the inhibition by dihydroouabain of the sodium pump current in single rabbit cardiac Purkinje cells.

The kinetics of the inhibition by dihydroouabain (DHO) of the Na pump current were studied in isolated rabbit cardiac Purkinje cells by means of whole-cell recording. A fast exchange of the extracellular solution at the membrane of the cell studied was performed via two multi-barrelled pipettes nearby. Judging from the steady-state inhibition of the Na pump current at various DHO concentrations half maximal inhibition occurred at 1 x 10(-5) mol/l DHO in a medium containing 2 mmol/l K+ and at 3.5 x 10(-5) mol/l DHO in a solution containing 10.8 mmol/l K+. Assuming a reversible one-to-one binding reaction, the time course of DHO binding and unbinding was analysed under a variety of conditions. It was shown that the antagonistic effect of K+ on the inhibition of the pump current was entirely due to a decrease of the association rate constant of DHO binding to the sodium pump at higher K+ concentrations. The association rate constant amounted to 1 x 10(4) (mol/l)-1.s-1 at 2 mmol/l K+ and to 2.8 x 10(3) (mol/l)-1.s-1 at 10.8 mmol/l K+. The dissociation rate constant of DHO unbinding remained unchanged (approximately 0.06 s-1). The equilibrium dissociation constant KD for the inhibition of the pump current by DHO decreased by a factor 3 to 5 if the Na+ concentration of the patch pipette solution was augmented from 5 to 50 mmol/l. Increasing DHO concentrations inhibited the pump current increasingly faster and to a larger extent. The KD values derived showed little variation with the concentration of DHO applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)-K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip-V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage-sensitive steps in the cardiac Na(+)-K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes of the subsarcolemmal Na+ concentration in internally perfused cardiac cells.

Transients of Na+/K+ pump and of Na+/Ca2+ exchange current occur during whole-cell recording from cardiac cells upon quick changes of active Na+ efflux. The transients reflect a temporary loss of control of the subsarcolemmal Na+ concentration. Even in the steady state the control is not complete is certain cells. Quantitative studies on ion transport by whole-cell recording are meaningful only if an adequate control of the submembranal ionic composition is demonstrated.