Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

Peter S. Hansen1,2, Kerrie A. Buhagiar1,2, Benjamin Y. Kong3, Ronald J. Clarke3, David F. Gray1, and Helge H. Rasmussen1,2

1 Department of Cardiology, Royal North Shore Hospital, St. Leonards, NSW 2065; and 2 Department of Medicine and 3 School of Chemistry, University of Sydney, Sydney 2006, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To examine effects of cytosolic Na+, K+, and Cs+ on the voltage dependence of the Na+-K+ pump, we measured Na+-K+ pump current (Ip) of ventricular myocytes voltage-clamped at potentials (Vm) from -100 to +60 mV. Superfusates were designed to eliminate voltage dependence at extracellular pump sites. The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip) of 80 mM and a K+ concentration from 0 to 80 mM or with solutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When [Na]pip was 80 mM, K+ in pipette solutions had a voltage-dependent inhibitory effect on Ip and induced a negative slope of the Ip-Vm relationship. Cs+ in pipette solutions had an effect on Ip qualitatively similar to that of K+. Increases in Ip with increases in [Na]pip were voltage dependent. The dielectric coefficient derived from [Na]pip-Ip relationships at the different test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+ free.

access channels; ion transport; membrane dielectric; sodium/potassium-exchanging adenosinetriphosphatase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTERACTION of Na+ and K+ with extracellular sites of the membrane Na+-K+ pump has been studied in considerable detail. However, cytosolic pump sites are difficult to study in intact cells, and interactions of Na+ and K+ with these sites remain poorly understood (27). Studies on isolated Na+-K+-ATPase reconstituted in lipid membranes or vesicles indicated that binding of Na+ to the pump in its E1 conformational state in the Na+ translocation pathway is dependent on membrane voltage (5, 9, 25, 34). The largest effect of voltage dependence occurs at relatively low, rate-limiting concentrations of cytosolic Na+ near levels encountered in vivo. If similar voltage dependence occurs in intact cells, it might be an important target for cellular regulation of the pump and might be of particular importance in cells with large sustained changes in membrane potential such as cardiac myocytes (9).

Voltage-dependent binding of intracellular Na+ in the pump's Na+ translocation pathway is expected to give rise to a positive slope in the relationship between electrogenic Na+-K+ pump current (Ip) and membrane voltage (Vm). Nakao and Gadsby (23) studied the Ip-Vm relationships of internally perfused voltage-clamped single guinea pig ventricular myocytes with the whole cell patch-clamp technique. Experimental conditions were designed to eliminate voltage dependence arising from interaction of Na+ and K+ with the pump at extracellular sites, and wide-tipped patch pipettes were used to achieve good control of the intracellular Na+ concentration. Ip-Vm relationships, determined with four different Na+ concentrations ([Na]pip) in patch pipette solutions, had a shallow positive slope. However, an increase in [Na]pip caused an increase in Ip by the same factor at all Vm tested. A voltage-dependent increase in Ip with an increase in [Na]pip would have been expected if activation of the pump by cytosolic Na+ is voltage dependent. It was concluded that activation of the pump by cytosolic Na+ seems independent of membrane potential. The same conclusion was reached in a subsequent study on rat ventricular myocytes (30).

In agreement with the study of Nakao and Gadsby (23), we (10) also recorded a shallow positive slope of the Ip-Vm relationship of single cardiac myocytes under experimental conditions expected to eliminate voltage dependence arising at extracellular Na+-K+ pump sites. However, when myocytes were exposed to insulin under identical conditions, a negative slope was recorded (see Fig. 3, Ref. 10). The negative slope is difficult to explain within the framework of our current understanding of voltage-dependent steps in the Na+-K+ pump cycle.

A negative slope of the Ip-Vm relationship may be generated by voltage-dependent inhibition of the pump by a cytosolic cation. Although it is firmly established that cytosolic K+ inhibits the pump, such inhibition is not voltage dependent when studied in Na+-K+ pumps in isolated membrane fragments (1). However, the effect of cytosolic K+ on Na+-K+ pumps in situ in intact cells has not been systematically studied. We have examined the effect of cytosolic K+ on Ip at different membrane voltages. In an initial series of experiments we perfused the cytosolic compartment of ventricular myocytes with patch pipette solution containing Na+ in a high concentration expected to nearly saturate cytosolic binding at all Vm. Experimental conditions were designed to eliminate voltage-dependent steps at extracellular pump sites. Inclusion of K+ or the K+ congener Cs+ in pipette solutions inhibited Ip and induced a negative slope of the Ip-Vm relationship. The effect of an increase in the K+ pipette concentration ([K]pip) was voltage dependent. We also examined activation of the pump by cytosolic Na+. Because voltage-dependent inhibition by K+ might obscure voltage-dependent activation by Na+, we used a fixed [K]pip of 80 or 0 mM and variable levels of [Na]pip to determine Ip-Vm relationships.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ventricular myocytes were isolated from 52 male New Zealand White rabbits as described previously (10). They were stored at room temperature and used for experimentation on the day of isolation only.

Composition of superfusates. For measurement of Ip, myocytes were initially superfused with modified Tyrode solution warmed to 35 ± 0.5°C. The solution contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 0.44 NaH2PO4, 10 glucose, and 10 HEPES. It had a pH of 7.40 ± 0.01 at 35°C. When the whole cell configuration had been established, we switched to a superfusate that was nominally Ca2+ free and contained 0.2 mM Cd2+ and 2 mM Ba2+. To eliminate voltage dependence arising at extracellular Na+-K+ pump sites, this superfusate contained K+ in concentrations at or above physiological levels and was Na+ free or contained Na+ in a low concentration (27). It contained 15 mM K+ and was Na+ free in most experiments. We used 140 mM N-methyl-D-glucamine chloride (NMG-Cl) to maintain osmotic balance and ionic strength of the superfusate. In some experiments we used a K+ concentration of 5.6 mM and a Na+ concentration of 1.5 mM. These solutions were used to allow comparisons of results with data published by others.

Composition of pipette filling solutions. Solutions with high fixed Na+ and variable K+ concentration contained (in mM) 79 Na glutamate, 1 NaH2PO4, 0-80 KOH, 5 HEPES, 2 MgATP, 5 EGTA, 20 tetraethylammonium chloride (TEA-Cl), 31-45 aspartic acid, and 0-66 tetramethylammonium hydroxide (TMA-OH). Solutions with high fixed K+ and variable Na+ concentration contained (in mM) 5-80 NaOH, 79 K glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 20 TEA-Cl, 0-66 TMA-OH, and 45-31 aspartic acid. K+-free solutions with variable Na+ concentrations contained (in mM) 0-99 NaOH, 0.1-1 NaH2PO4, 5 HEPES, 2 MgATP, 5 EGTA, 20 TEA-Cl, 66-145 TMA-OH, and 45 aspartic acid. Cs+-containing solution included (in mM) 79 Na glutamate, 1 NaH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 20 TEA-Cl, 70 CsCl, and 50 aspartic acid. All pipette filling solutions were titrated to a pH of 7.2 at 22°C with 1 M HCl. Osmolarities measured with a vapor pressure osmometer (model 5500 osmometer, Wescor, Logan, UT) varied by <10% between solutions. We nulled offset potentials by placing both ground electrode and patch pipette in pipette filling solution (24). Jpcalc (P. Barry, University of New South Wales, Sydney, Australia) was used to estimate junction potentials between ground electrode and superfusates. They were <3 mV for all solutions and were not corrected for. Patch pipettes had resistances of 0.8-1.1 MOmega before the whole cell configuration was established. The resistance of pipettes always increases with seal formation. The Ip-Vm relationships were not always reproducible between experiments performed with identical solutions when the series resistance exceeded 2.2 MOmega . We therefore only proceeded with experiments when the series resistance in the whole cell configuration was at or below this level. To reduce effects of interrabbit variability, we used pipette filling solutions of different compositions in experiments on cells from each rabbit.

Measurement of Ip. Ip was identified as the shift in holding current induced by Na+-K+ pump blockade with 100 µM ouabain. Ouabain was always superfused 8-10 min after the whole cell configuration had been established. Holding currents were recorded during voltage steps of 320-ms duration in 20-mV increments to test potentials from -100 to +60 mV. Each test potential was bracketed by a return to a holding potential of -40 mV for 2 s. Recordings were averaged from three applications of the voltage-clamp protocol and sampled at 5-ms intervals for the last 50 ms at each test potential. The holding current was taken as the mean value of the samples. Membrane currents were recorded with the single-electrode voltage-clamp mode of an Axoclamp-2B amplifier, and voltage-clamp protocols were generated with pCLAMP (Axon Instruments, Foster City, CA). Unless otherwise specified, Ip is reported normalized for membrane capacitance. The capacitance for myocytes in this study was 127 ± 30 pF (mean ± SD).

Statistical analysis. Results are expressed as means ± SE unless otherwise indicated. Statistical comparisons were made using the Mann-Whitney rank-sum test and linear regression. We considered a P value <0.05 significant. The Hill and Boltzmann equations were fitted to data with nonlinear regression.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of pipette K+ and Cs+ on membrane currents. The voltage dependence of the Na+-K+ pump in cardiac myocytes must be studied under conditions that markedly reduce nonpump membrane currents and virtually eliminate time-dependent currents. This can be achieved with appropriate compositions of superfusates and patch pipette solutions. Membrane K+ currents are blocked by including Ba2+ in the superfusate and TEA-Cl and Cs+ in pipette solutions. In an initial series of experiments, we examined whether using K+ rather than Cs+ in pipette solutions altered the membrane conductance. Myocytes were patch-clamped with a pipette filling solution that included 80 mM K+. The superfusate was nominally Na+ free and contained 15 mM K+. Figure 1 shows currents elicited in response to steps in command voltages from -100 to +60 mV. They were small, largely time independent, and similar to the currents elicited when pipette solutions were K+ free and contained 70 mM Cs+ as illustrated previously (10).


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Fig. 1.   Holding currents at test potentials from -100 to +60 mV before and after exposure of a myocyte to 100 µM ouabain. Traces shown represent the currents averaged from 3 applications of the voltage-clamp protocol. Superfusate contained 0 mM Na+ and 15 mM K+, and pipette solution contained 80 mM Na+ and 80 mM K+. Membrane capacitance of the myocyte was 120 pF.

The voltage dependence of steady-state membrane currents determined with the K+-containing filling solutions is summarized in Fig. 2. We also performed experiments with a pipette filling solution containing 70 mM Cs+. To allow comparison with a previous study (7), the superfusate contained 1.5 mM Na+ and 5.6 mM K+. A summary of the currents is included in Fig. 2. Currents of myocytes patch-clamped with Cs+-containing pipettes were shifted in the outward direction relative to the currents of myocytes patch-clamped with K+-containing pipettes. This may be accounted for by an inhibitory effect of the 80 mM K+ in pipette solutions on Ip that was greater than the inhibitory effect of 70 mM Cs+ (11). The slope conductance, however, was similar for experiments performed with K+- and Cs+-containing pipette solutions (1.84 ± 0.15 vs. 1.85 ± 0.14 pA/mV). The conductance was also virtually identical to that found previously in guinea pig myocytes patch-clamped with Cs+-containing pipette solutions (estimated from Refs. 7 and 23). We conclude that nonpump membrane currents in experiments performed with K+-containing pipettes are similar to the currents one can expect with the Cs+-containing pipette solutions commonly used when studying pump currents in cardiac myocytes.


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Fig. 2.   Effect of Cs+ and K+ on the slope conductance summarized from 8 and 10 myocytes, respectively. Pipette solutions contained 70 mM Cs+ () or 80 mM K+ (). Vm, membrane voltage. Iwhole cell, holding current before exposure of myocytes to ouabain.

Effect of pipette K+ on Ip-Vm relationships. To examine the effect of intracellular K+ on the voltage dependence of Ip, we determined the Ip-Vm relationship for myocytes patch-clamped with [K]pip ranging from 0 to 80 mM. An example of membrane currents before and after exposure to ouabain in an experiment using a [K]pip of 80 mM is shown in Fig. 1. A summary of Ip-Vm relationships determined with all levels of [K]pip is shown in Fig. 3A. A Mann-Whitney test indicated that the mean value of Ip pooled for measurements at all test potentials with pipette solutions containing only 2.5 mM K+ was significantly less than the corresponding mean value of Ip measured with K+-free solutions.


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Fig. 3.   A: effect of pipette K+ concentration ([K]pip) on Na+-K+ pump current (Ip)-Vm relationships. Superfusate was Na+ free and contained 15 mM K+, and pipette solutions contained 80 mM Na+ and variable [K]pip. n, No. of myocytes in each group. B: normalized Ip-Vm relationships for a [K]pip of 0 or 10 mM.

Figure 3A shows that there is a [K]pip-dependent decrease in Ip. Ip does not appear to decrease by the same factor at all test potentials. This can be further examined by normalizing Ip to the Ip recorded at a fixed membrane potential. If there is a proportional, voltage-independent change in Ip with a change in pipette cation concentration, the normalized Ip-Vm relationships should overlap (7). We normalized Ip to the Ip recorded at 0 mV for experiments performed with a [K]pip of 0 or 10 mM. The normalized Ip-Vm relationships are shown in Fig. 3B. The slopes of the normalized relationships at test potentials more positive than -40 mV were compared by linear regression. The slope determined with a [K]pip of 10 mM was significantly more negative than the slope determined with K+-free patch pipette solutions. There was also a significant difference when the normalized Ip-Vm relationships were compared with a Mann-Whitney test. These findings indicate that [K]pip-induced Na+-K+ pump inhibition is voltage dependent.

Figure 3A indicates that within the [K]pip range we could study, K+ inhibition of the pump current is not complete, i.e., saturating concentrations of K+ do not reduce Ip to zero. The incomplete inhibition induced by K+ suggests that cells may contain K+-sensitive and relatively K+-insensitive subpopulations of pumps (see DISCUSSION). To highlight the incomplete inhibition, we plotted Ip as a function of [K]pip in Fig. 4A at one of the test potentials (0 mV). To further characterize the inhibitory effect of [K]pip on Ip (Ii), we subtracted the mean Ip measured at each [K]pip from the mean Ip measured when pipette solutions were K+ free. This subtraction was performed at each Vm. A [K]pip-Ii relationship derived from data obtained at 0 mV is shown in Fig. 4B. The Hill equation has been fitted to the data.


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Fig. 4.   A: Pump current remaining after inhibition by [K]pip. Ip at a test potential of 0 mV has been plotted for different [K]pip values. B: [K]pip dependence of inhibition by [K]pip on Ip (Ii) at Vm = 0 mV. The Hill equation has been fitted (solid line) to experimental data (open circle ). Half-maximal inhibitory [K]pip (KK1/2) was 7.2 mM, and the Hill coefficient was 1.0. There was a statistically significant increase in the Hill coefficients with an increase in Vm from 0.7 to 1.5 over the range of Vm studied. C: simulated inhibitory effect of cytosolic K+ on pump rate. Pump rate was simulated for cytosolic K+ corresponding to the [K]pip values used experimentally. The Hill equation has been fitted to the simulated values. KK1/2 derived from the fit was 8.0 mM, and the Hill coefficient was 1.5. The maximum turnover number at zero [K]pip used for the simulations was 10 s-1 (at 24°C). D: voltage dependence of KK1/2 derived from [K]pip-Ii relationships at test potentials from -100 to +60 mV (open circle ). Solid line represents the best fit of a Boltzmann distribution to the Vm-KK1/2 relationship.

A K+-induced acceleration of the backward reaction E1 + 2K+ right-arrow E2(K+)2 is a possible mechanism for the [K]pip-dependent Ii. If fast competitive binding of K+ and Na+ at two sites on the E1 conformation and selective binding of Na+ at a third site are in equilibrium, the following equation can be derived to describe the dependence of the observed rate constant, kobs, on cytosolic Na+ and K+ concentrations
k<SUB>obs</SUB><IT>=k</IT><SUB>max</SUB> (1)

<IT> · </IT><FR><NU><IT>K</IT><SUP>2</SUP><SUB><IT>K</IT></SUB>[K<SUP>+</SUP>]<SUP>2</SUP></NU><DE><AR><R><C>(1 + <IT>K</IT><SUB>K</SUB>[K<SUP>+</SUP>])<SUP>2</SUP>+ 2<IT>K</IT><SUB>N</SUB>[Na<SUP>+</SUP>] + 2<IT>K</IT><SUB>N</SUB><IT>K</IT><SUB>K</SUB>[Na<SUP>+</SUP>][K<SUP>+</SUP>]</C></R><R><C>+ <IT>K</IT><SUP>2</SUP><SUB>N</SUB>[Na<SUP>+</SUP>]<SUP>2</SUP>+ <IT>K′</IT><SUB>N</SUB> <IT>K</IT><SUP>2</SUP><SUB>N</SUB>[Na<SUP>+</SUP>]<SUP>3</SUP></C></R></AR></DE></FR>
where kmax represents the maximum rate for the E1 + 2K+ right-arrow E2(K+)2 transition when two sites on E1 are fully occupied by K+ ions. KN and KK represent the microscopic association constants for Na+ and K+ ions for the two nonspecific binding sites on E1, and K'N represents the association constant for the third binding site specific for Na+. A kmax of 550 s-1 (4), a KK of 100 M-1 (31), a KN of 1.2 × 102 M-1 (16), and a K'N of 3.9 × 102 M-1 (16) were obtained from the literature. It is apparent from the equation that K+-induced activation of the backward reaction should slow down the overall forward reaction. To test this, we carried out numerical simulations by adapting differential rate Eqs. 8-11 of Lüpfert et al. (20) for saturating substrate conditions to nonsaturating conditions, and we extended the equations by including Eq. 1 given above to take into account the E1 + 2K+ right-arrow E2(K+)2 backward reaction and Na+/K+ competition for E1 binding sites. The simulated inhibitory effect of cytosolic K+ shown in Fig. 4C was consistent with the inhibition of the K+-sensitive Na+-K+ pump subpopulation by [K]pip shown in Fig. 4B.

To describe the effect of Vm on the [K]pip-induced pump inhibition, we fitted the Hill equation to the [K]pip-Ii relationships at all test potentials. The [K]pip values for half-maximal inhibitory effect (KK1/2) derived from the fits are shown in Fig. 4D. KK1/2 appears to be voltage dependent. A dielectric coefficient of 0.15 was derived from a fit of the Boltzmann equation to the values for KK1/2.

Interaction of cytosolic K+ with the pump within an access channel is one of the possible mechanisms for voltage dependence of KK1/2. Such interaction may give rise to ouabain-sensitive transient currents that can be detected in response to sudden changes in Vm when experimental conditions allow only a limited set of partial reactions of the pump cycle (22, 26). In an attempt to demonstrate transients, we used Na+-free superfusates containing 15 mM K+ and Na+-free patch pipette solutions containing 10-25 mM K+. We could not detect ouabain-sensitive current transients, even when reducing the [ATP]pip to 50-100 µM and the temperature to 22°C to prolong their duration. Although our experimental system may not have allowed the detection of transients of short duration or low amplitude, these experiments do not support the existence of an access channel.

Effect of pipette Cs+ on Ip-Vm relationships. Because Cs+ can act as a K+ congener by inhibiting the pump at intracellular sites (11), we examined the effect of Cs+ in pipette solutions on the Ip-Vm relationship. We determined Ip-Vm relationships in superfusates containing 1.5 mM Na+ and 5.6 mM K+ (adopted from Ref. 7). Pipette solutions were K+ and Cs+ free or contained 70 mM Cs+. The Ip-Vm relationships are summarized in Fig. 5A. We compared the mean of the Ip values at all test potentials with Cs+-containing or Cs+-free pipette solutions by using the Mann-Whitney rank-sum test. Mean Ip of myocytes patch-clamped with Cs+-containing pipette solutions was significantly less than mean Ip of myocytes patch-clamped with Cs+-free solutions. To examine whether the inhibitory effect of Cs+ on Ip is voltage dependent, we normalized the Ip-Vm relationships shown in Fig. 5A to the Ip measured at 0 mV. The normalized relationships are shown in Fig. 5B. We fitted a regression line to the Ip-Vm relationships determined with and without Cs+ in pipette solutions at test potentials more negative than -20 mV and a regression line to the relationships at potentials of -20 mV or more positive. The slopes determined with and without pipette Cs+ were significantly different in both voltage ranges. To avoid any assumption of linearity, we also compared Ip-Vm relationships in Fig. 5B with a Mann-Whitney rank-sum test. There was a significant difference between the normalized relationships in both voltage ranges compared. We conclude that cytosolic Cs+ inhibits Ip in a voltage-dependent fashion, i.e., such inhibition is not specific for K+.


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Fig. 5.   A: effect of pipette Cs+ concentration ([Cs]pip) on the Ip-Vm relationship. The pipette solutions contained 80 mM Na+ and Cs+ as indicated. n, No. of myocytes in each group. B: data from A normalized to the Ip recorded at 0 mV to illustrate the effect of pipette Cs+ on the slopes of the Ip-Vm relationship.

Effect of intracellular Na+ on Ip-Vm relationships. To examine the effect of cytosolic Na+ on the voltage dependence of the Na+-K+ pump, we performed experiments with different levels of [Na]pip. Patch pipettes contained K+ in a first series of experiments. We used a constant [K]pip of 80 mM to maintain the inhibitory effect of intracellular K+ on Ip constant. [Na]pip ranged from 5 to 80 mM. The superfusate was Na+ free and contained 15 mM K+. The Ip-Vm relationships determined with the different levels of [Na]pip are shown in Fig. 6A. There is a monotonic increase in Ip with an increase in [Na]pip at all test potentials. However, Ip does not appear to increase by the same factor at all test potentials. To further examine this, we normalized Ip-Vm relationships determined with a [Na]pip of 10 or 80 mM to the Ip recorded at 0 mV. The normalized relationships are shown in Fig. 6B. Linear regression demonstrated a statistically significant difference between the slopes. A Mann-Whitney rank-sum test also indicated a significant difference between the normalized Ip-Vm relationships.


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Fig. 6.   Effect of [Na]pip on Ip-Vm relationships. Pipette solutions contained 80 mM K+ and variable [Na]pip. n, No. of myocytes in each group. B: data from A normalized to Ip recorded at 0 mV when [Na]pip was 10 and 80 mM.

To further describe the voltage dependence of the interaction of cytosolic Na+ with the pump, we transformed the Ip-Vm relationships shown in Fig. 6A to [Na]pip-Ip relationships at different test potentials. The [Na]pip-Ip relationship at one test potential (0 mV) is shown in Fig. 7A. The Hill equation has been fitted to the data. The [Na]pip values for half-maximal stimulation (KNa1/2) derived from Hill equations fitted to [Na]pip-Ip relationships at all test potentials are shown in Fig. 7B. A dielectric coefficient of 0.15 was derived from a fit of the Boltzmann equation.


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Fig. 7.   A: [Na]pip dependence of Ip at Vm = 0 mV. The Hill equation has been fitted (solid line) to experimental data (open circle ). [K]pip was 80 mM. There was a statistically significant increase in the Hill coefficients with an increase in Vm from 1.5-2.0 over the range of Vm studied. B: voltage dependence of the [Na]pip inducing half-maximal Ip (KNa1/2) derived from [Na]pip-Ip relationships at test potentials from -100 to +60 mV (). Solid line represents the best fit of a Boltzmann distribution to the Vm-KNa1/2 relationship.

In a second series of experiments we examined the effect of [Na]pip on the Ip-Vm relationship with K+-free patch pipette solutions. We wished to detect [Na]pip-dependent activation of the Na+-K+ pump with high resolution at low levels of [Na]pip as well as to measure Ip with nearly maximal pump activation. We therefore clustered the [Na]pip values at relatively low levels and at high levels with a range from 0.1 to 100 mM. The superfusate was Na+ free and contained 15 mM K+. The Ip-Vm relationships determined with the different levels of [Na]pip are shown in Fig. 8A. There was a monotonic increase in Ip with an increase in [Na]pip at all test potentials. To examine whether the [Na]pip-dependent increase in Ip is voltage dependent, we normalized Ip-Vm relationships determined with a [Na]pip of 5 or 100 mM to the Ip recorded at 0 mV. The normalized relationships are shown in Fig. 8B. Linear regression demonstrated a statistically significant difference between the slopes. A Mann-Whitney rank-sum test also indicated a significant difference between the normalized Ip-Vm relationships.


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Fig. 8.   A: effect of [Na]pip on Ip-Vm relationships. Pipette solutions were K+ and Cs+ free and contained a variable [Na]pip. n, No. of myocytes in each group. B: data from A normalized to Ip recorded at 0 mV when [Na]pip was 5 or 100 mM.

We transformed the Ip-Vm relationships shown in Fig. 8A to [Na]pip-Ip relationships at different test potentials. The [Na]pip-Ip relationship at one test potential (0 mV) is shown in Fig. 9A. The Hill equation has been fitted to the data. The KNa1/2 values derived from Hill equations fitted to [Na]pip-Ip relationships at all test potentials are shown in Fig. 9B. A dielectric coefficient of 0.06 was derived from a fit of the Boltzmann equation.


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Fig. 9.   A: [Na]pip dependence of Ip at Vm = 0 mV. The Hill equation has been fittted (solid line) to experimental data (open circle ). [K]pip was 0 mM. Hill coefficients varied from 1.5 to 1.4 with an increase in Vm over the range studied. The variation of the Hill coefficient with voltage was not statistically significant. B: voltage dependence of KNa1/2 derived from [Na]pip-Ip relationships at test potentials from -100 to +60 mV (open circle ). Solid line represents the best fit of a Boltzmann distribution to the Vm-KNa1/2 relationship.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Specificity of ouabain-induced changes in membrane currents. The interaction of Na+ (13, 14, 22) and K+ (26, 28) with the Na+-K+ pump at extracellular sites is voltage dependent. Figure 3 indicates that a negative slope of the Ip-Vm relationship can occur under conditions expected to eliminate this voltage dependence. We identified Ip as the shift in membrane current induced by ouabain. Although ouabain is a highly specific inhibitor of the Na+-K+ pump, the inhibition can give rise to changes in membrane current not arising directly from electrogenic Na+/K+ exchange.

Pump inhibition reduces cellular consumption of ATP. This, in turn, might inactivate ATP-sensitive K+ channels (15). However, at test potentials positive to the equilibrium potential for K+ (EK), ouabain-induced shifts in currents arising from ATP-sensitive K+ channels and the Na+-K+ pump are in the same direction. This would result in an overestimation of pump currents and hence a positive slope of the Ip-Vm relationship. The slope was negative at test potentials positive to the EK calculated from K+ concentrations in pipette solutions and superfusate according to the Nernst equation (Fig. 3). In addition, inclusion of 20 mM TEA-Cl in all pipette solutions should have blocked ATP-sensitive K+ channels (15).

Ouabain might also affect Na+-activated K+ channels reported to exist in cardiac myocytes of some species (18) if pump inhibition results in an increase in the Na+ concentration at cytosolic sites despite perfusion of the intracellular compartment. However, a role of Na+-activated K+ channels in the mechanism generating the negative slope of the Ip-Vm relationship is unlikely because the slope was reproduced when Cs+ replaced K+ in pipette solutions (Fig. 5) and cytosolic Cs+ blocks the Na+-activated K+ channel (18).

Inhibition of Ip by cytosolic K+ and Cs+. Figure 4, A and B, suggests that K+-induced inhibition saturates at a level where inhibition is incomplete. The mechanism for the incomplete inhibition is not identified. We speculate that the inhibition of Ip that is identified is attributable to a subpopulation of pump units. The measured Ip is generated by all isoforms of the pump in all locations. It is conceivable there is a difference in the affinity of cytosolic K+ for different isoforms of the pump or that the same isoform in different microdomains of the sarcolemma have different functional properties. It is possible, for example, that pumps in the cardiac myocyte sarcolemmal caveolae (21) are affected by the lipid-rich milieu and/or proximity to kinases characterizing these important microdomains. Experimental constraints did not allow us to examine whether additional inhibition of Ip occurs at [K]pip >80 mM.

K+-induced inhibition of Ip might be due to an effect of K+ on a rate-limiting E2 right-arrow E1 transition. The transition is probably rate limiting even under physiological circumstances (20). It seems highly likely that the transition was rate limiting under the conditions of our experiments because an allosteric effect of extracellular Na+ causes the rate of transition to decrease with a decrease in the extracellular Na+ concentration (6, 17, 33). Our experiments were performed with Na+-free superfusates.

Inhibition of the E2 right-arrow E1 transition could be due to binding of cytosolic K+ to the E1 or E2 conformation. Inhibition by binding directly to the E2 conformation is unlikely because such inhibition is only quantitatively significant at cytosolic ATP concentrations much lower than used in this study (33). Inhibition by binding to the E1 conformation could be due to inhibition of the forward reaction or acceleration of the backward reaction.

Inhibition of the forward reaction could in principle occur if K+ binds to cytosolic Na+ binding sites and inhibits the ATP phosphorylation reaction (29). At the cytosolic concentrations of Na+ and ATP we used, the rate constant (at 24°C) for the phosphorylation reaction in the absence of K+ can be estimated from Kane et al. (16) to be ~175 s-1. If Eq. A8 of Kane et al. (16) is modified to include competition with K+ at two cytosolic Na+ binding sites and if one assumes that the microscopic binding constant for K+ is 100 M-1 (31), the rate constant for the phosphorylation reaction at 80 mM K+ decreases by 7% only. This is much less than the K+-induced decrease in Ip we observed.

We used a model to examine the hypothesis that cytosolic K+ inhibits the overall forward pump rate by binding to the E1 conformation and accelerating the backward reaction. The model was based on kinetics reported under conditions similar to those we used in our experiments. It was consistent with the K+-induced pump inhibition we observed, even at the lowest [K]pip, and there was good agreement between the [K]pip-Ii relationship and the simulated K+-induced pump inhibition (Fig. 4) in the concentration range we could examine experimentally. The similarity in the effects of 10 mM [K]pip and 70 mM [Cs]pip on Ip-Vm relationships (see Figs. 3 and 5) is also consistent with the hypothesis that pump inhibition is due to binding of K+ or the K+ congener to the E1 conformation of the pump because the affinity of the E1 conformation for K+ is ~10-fold higher than its affinity for Cs+ (29).

[K]pip-induced inhibition of Ip increased with a shift in membrane potential toward positive values (Fig. 4D). Inspection of Eq. 1 indicates this could be due to a voltage-dependent increase in the association constant of K+ for the E1 conformation of the pump (KK) or to an increase in the rate constant for the conformational transition E1(K+)2 right-arrow E2(K+)2 (kmax). Previous studies do not support the existence of such voltage dependence in isolated Na+-K+-ATPase (1).

The Na+-K+ pump is a membrane-embedded molecule, and membrane constituents make an important contribution to pump function. It is pertinent to the present study that the membrane environment is a particularly important determinant of cytosolic K+/Na+ antagonism (32) and that such antagonism is a prominent feature of Na+-K+-ATPase in the heart (31). To our knowledge, voltage dependence of Na+-K+- ATPase has not been reported for enzyme isolated from the heart and might be difficult to study. Unless a recovery equally representative of all isoforms and microdomains can be ensured, it is unlikely that Na+-K+-ATPase in isolated membrane fragments reflects properties of all Na+-K+ pumps in an intact cell. Pumps embedded in lipid-rich membrane microdomains may be of particular concern in this regard because in situ membrane lipids, believed to be important in the regulation of the Na+-K+ pump (32), are easily altered in isolation procedures. It is not known whether they contribute to voltage-dependent regulation. The sarcolemma and the T tubular system of myocytes provide a highly inhomogeneous environment and a complexity that might be avoided in studies on cells with a less complex membrane structure. However, a detailed study on the effect of cytosolic K+ on the voltage dependence of the pump in such cells has not been reported.

Figure 3 suggests that the voltage-dependent component of K+-induced pump inhibition diminishes at a high [K]pip, and voltage dependence of the inhibition may be of little importance at the even higher physiological levels of cytosolic K+. The main practical implication is that our study may explain findings in previous studies. The effect of variable concentrations of the K+ congener Cs+ in pipette solutions (23) on Ip-Vm relationships may have prevented the detection of voltage-dependent interaction of Na+ with cytosolic sites.

Voltage-dependent activation of pump by cytosolic Na+. Previous studies reported that Ip-Vm relationships of ventricular myocytes, normalized to the Ip recorded at a test potential of 20 mV (23) or a [Na]pip of 85 mM (30), have similar slopes for different [Na]pip values. This suggests that pump activation by intracellular Na+ is independent of Vm. However, a decrease in [Cs]pip was used to maintain osmotic balance with increases in [Na]pip. Our study suggests that a decrease in [Cs]pip shifts the slope of the Ip-Vm relationship in a positive direction (Fig. 5) and offsets effects of increases in [Na]pip on the slope in the opposite direction. Without such effects of Cs+, Ip-Vm relationships in the previous studies (23, 30) might have been similar to the Ip-Vm relationships shown in Figs. 6 and 8 in the present study. A change in the normalized relationships with a change in [Na]pip is consistent with voltage-dependent binding of Na+ at a pump site facing the cytoplasmic side of the Na+-K+ pump.

Barmashenko et al. (3) also reported findings attributed to voltage-dependent binding of cytosolic Na+ to the Na+-K+ pump in intact cells. They measured Ip of voltage-clamped guinea pig ventricular myocytes suspended in Na+-free superfusates and perfused with K+- and K+ congener-free pipette solutions. A dielectric coefficient of ~0.08 was derived for the voltage dependence of the interaction of Na+ with cytosolic sites. We derived similar dielectric coefficients. However, discrepancies in important details between the two studies should be noted.

The Ip measured by Barmashenko et al. (3) at a test potential of 0 mV saturated at ~0.75 pA/pF, whereas we measured a much higher Ip of ~4.5 pA/pF at the same test potential (Fig. 8). Barmashenko et al. (3) attributed the low maximal Ip to the use of Cs+ rather than K+ to activate the pump at extracellular sites. Their patch pipettes had initial resistances of 2-4 MOmega . The access resistance is expected to have been approximately twofold higher. In our hands a resistance at such levels rather than the composition of solutions limits maximal pump rates. Regardless of the mechanism, any rate limitation not determined by the composition of pipette solutions must influence values for KNa1/2 of [Na]pip-Ip relationships and the dielectric coefficients that are derived.

We derived the dielectric coefficients from fits of Hill equations to the relationships between [Na]pip and Ip. The values we report were based on a fitting procedure that allowed the Hill coefficient to float. The dielectric coefficients we derived were sensitive to details of the fitting procedure. When we used Hill coefficients fixed at estimated mean values, we obtained different numerical values for the dielectric coefficients. However, they were within the 0.06-0.15 range of those derived with a floating Hill coefficient.

The numerical values of our dielectric coefficients are less than the dielectric coefficient of ~0.25 derived from measurements of charge movement of the electrogenic step with the pump restricted to a partial reaction in isolated membrane fragments (12). Our analysis is based on the overall forward pump rate and reflects interaction of Na+ with all three cytosolic sites, including the two believed to be located outside the electrical field of the membrane. Such analysis does not allow a distinction between the sites (25) and may have underestimated the dielectric coefficient of the electrogenic partial reaction. Our values are in reasonable agreement with those derived from Hill plots of pump rates measured in noncellular systems (5, 9, 25) and support the qualitative conclusion that the interaction of cytosolic Na+ with the Na+-K+ pump is voltage dependent. Our study does not justify conclusions about quantitative details of this interaction.

Kinetic equivalence of changes in substrate concentration and Vm in an access channel predicts a shift of Ip-Vm relationships along the voltage axis without a change in maximal current or shape of the relationship with a change in [Na]pip (28). Such a shift was not observed in this study (Figs. 5 and 6) and may be difficult to demonstrate for a shallow access channel. In addition, the conformational change from E1P to E2P is voltage dependent and we cannot rule out a contribution from it to the shape of the Ip-Vm relationships and hence a deviation from the prediction of an access channel model. The rate constant in one study is such that the E1P to E2P conformational change may be rate limiting (34), whereas much higher rate constants in two other studies (2, 8) suggest that it is unlikely that the conformational change contributes to the shape of the Ip-Vm relationship.

Lu et al. (19) found no charge movements related to binding of Na+ at cytoplasmic sites in giant membrane patches excised from cardiac myocytes. This implies that binding is independent of voltage or that voltage dependence is not a constant feature necessarily preserved in the excised membrane patches. A recent study (10) suggests the latter is the case. Exposure of cardiac myocytes to insulin shifted the Ip-Vm relationships in a manner consistent with elimination of voltage dependence of cytosolic Na+ binding. Effects of pharmacological blockers indicated involvement of cellular messengers and suggested effects on voltage dependence were mediated by dephosphorylation (10). The experiments of Lu et al. (19) were performed without ATP in cytosolic solutions. This may have facilitated dephosphorylation and hence reduced or eliminated voltage dependence of Na+ binding. These speculations are consistent with suggestions that voltage dependence of cytosolic Na+ binding under control of cellular messengers may be important in the regulation of Na+-K+ pump function (27).


    ACKNOWLEDGEMENTS

This study was supported by the North Shore Heart Research Foundation. P. S. Hansen received a Postgraduate Medical Research Scholarship from the National Heart Foundation of Australia, K. A. Buhagiar was supported by National Heart Foundation of Australia Grant G96S4589, and D. F. Gray received a National Health and Medical Research Council Postgraduate Research Scholarship. R. J. Clarke acknowledges support from the Australian Research Council.


    FOOTNOTES

Address for reprint requests and other correspondence: H. H. Rasmussen, Dept. of Cardiology, Royal North Shore Hospital, Pacific Highway, St. Leonards, NSW 2065, Australia (E-mail: helger{at}med.usyd.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.01343.2000

Received 19 December 2000; accepted in final form 24 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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