PENTAGON Na,K]-ATPase of Human Erythrocytes

PENTAGON Na,K]-ATPase of Human Erythrocytes

Previous experiments from this laboratory indicated that [Na,K]-ATPase of human erythrocytes might be susceptible to electric perturbation and might be one of the sites of electroporation when an electric field of the order of kV/cm is used to induce pores in human red cells [23

A membrane conductance which could be blocked by ouabain was detected. We then attempted to active this enzyme using a field which would generate a transmembrane potential of the order of 10 mV. Human red cells in an isotonic suspension were exposed to applied alternating electric fields of strengths up to 50 V/cm (peak-to-peak) and of frequencies between 1 Hz and 20 MHz. The transport of Na+ and Rb+ (or K+) into and out of the cytoplasm was measured by radioactive tracers and compared with control samples in which no ac field was imposed. The activity which was inhibited by ouabain was taken to be due to the catalytic action of the [Na,K]-ATPase. Other inhibitors of the enzyme such as oligomycin, vanadate and oubagenin also inhibited the electric field provoked activity. In contrast, inhibitors to anion transport and Na+/Na+ exchange have no appreciable effects except at concentrations 10-1000-fold higher than that required to inhibit these activities. The results of these experiments are summarized below [24-27].

(1) At 4 (degrees) C, the maximum ac stimulated activity (i.e. at optimum field strength and optimum frequency) was 15-20 ions/pump for the Rb+ uptake and 20-30 ions/pump for the Na+ efflux. The ratio of Na+ pump activity and Rb+ pump activity was roughly 3/2, although it was not strictly maintained for any one red cell sample.

(2) There was an optimum field strength of 20 V/cm (peak-to-peak) for activating both the Rb+ and the Na+ pumps. The maximum perturbation to the red cell transmembrane electric potential induced by this field is estimated to be 12 mV (i.e. +/-6 mV) by using the Maxwell relation. This value corresponds to a peak-to-peak electric field of 24 kV/cm if the thickness of the hydrophobic layer of the red cell membrane is assumed to be 5 nm.

(3) The optimum frequency for the Rb+ pump was 1.0 kHz and for the Na+ pump 1.0 MHz when a 20 V/cm ac field was used. The reason for this wide separation of optimum frequencies is not clear. It is, however, recognized that in the

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action potential of neurons, the rise of the Na+ current is 100 times faster than the rise of the K+ current.

(4) The Rb+ uptake and the Na+ efflux activities detected here were active transport against the respective ion concentration gradients. In the case of the Rb+ uptake, the cytoplasmic [Rb+] was 27 mM and the extracellular [Rb+] was 10-12.5 mM. If the electric field stimulated a passive permeation, it would also have stimulated the efflux of Rb+. The efflux was not effected by the electric field up to 60 V/cm. In the case of the Na+ efflux, the cytoplasmic [Na+] was 6-10 mM amd the extracellular [Na+] was 140-150 mM, and the electric field stimulated only the efflux but not the uptake.

(5) The Michaelis-Menten constant, K(m), of internal Na+ for Rb+ uptake and Na+ efflux was 8 mM and for the external Rb+, 1.5 mM, consistent with an ATP dependent pumping activity of the [Na,K]-ATPase.

(6) ATP depleted red cells (down to 10 uM) still retained nearly the same level of voltage stimulated activity, suggesting that ATP hydrolysis was not required. However, it is not known whether other nucleotide or phosphate ligands are essential.

(7) Although most experiments were carried out around 4 (degrees) C, where the basal activity (i.e. ATP hydrolysis activity) was negligible, experiments done at 25 (degrees)

{Fig. 4. Frequency windows for the electric activation of the Na+ pump and the Rb+ pump (or K+ pump) of human [Na,K]-ATPase. (A) Human erythrocytes in an isotonic saline were exposed to an ac field of 20 V/cm and 4 (degrees) C. The ouabain sensitive Na+ efflux (O) and Rb+ uptake (delta) are plotted against the frequency of the applied field. In the same experiment, no ouabain sensitive Na+ uptake and Rb+ efflux were induced. The cytoplasmic concentration of Na+ was 6 mM and that of Rb+, 27 mM (pre-loaded), and the external concentration of Na+ was 140 mM and of Rb+, 10 mM. Yet the ac field stimulated transport of both ions against there respective concentration gradient. No consumption of ATP was detected. (B) The Electroconformational Coupling (ECC) model was used to simulate the above results. By adjusting the rate constants, asymmetry factor and the gating charge, it is possible to reproduce the main feature of the frequency dependence curves of (A). No attempt was made to fit the experimental results numerically. All rate constants used in the computation were below the diffusion controlled limit. Details of the computations and adjustment of parameters will be presented.}

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showed enhanced pump activity due to the ac stimulation. However, at 37 (deg) C, where the basal activity was at its maximum, no further stimulation by the ac field was detected. (98.6F)

{Fig. 5. ATP synthesis in beef heart submitochondrial particles stimulated by pulsed electric fields. (A) Submitochondrial particles from beef heart were suspended in a medium containing 5 mM HEPES, 2.5 mM potassium phosphate, (32)P(i) tracer, 2.5 mM ADP, 1 mM MgCl(2), 1 mg/ml BSA, 2 mM NaCN, 0.25 M sucrose, 20 mM glucose, pH 7.0, with a varying concentration of dithiothretol (DTT). The sample was exposed to five pulses of a 25 kV/cm exponentially decaying electric field (time constant of 100 us). The ATP formed was trapped with a hexokinase system (each sample contained also 71.4 U/ml of hexokinase), and analyzed for yield. At the peak, the ATP yield was approximately 5 molecules of ATP per enzyme per pulse. The initial temperature of the sample was 15 (deg) C and at no time did the temperature exceed 25 (deg) C. A control sample was kept at 25 (deg) C and the count was subtracted from the data. (B) Submitochondrial particles were preincubated for the time shown on the abscissa in a 0.25 M sucrose, 50 mM potassium phosphate buffer before transferring to the medium given in (A) for a low amplitude ac stimulation (60 V/cm at 30 Hz). Incubation caused the dissociation of the natural inhibitor peptide and an increase in ATP yield (). A control sample kept at 15 (deg) C showed no ATP synthesis (o). See text for details.}

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These results established firmly that the electric field stimulated active transports of Na+, K+ and Rb+ were due to the activation of [Na,K]-ATPase, and that the energy required for driving the endergonic reaction was derived from the applied ac field. Figure 4A shows the frequency dependence of the Na+ pump and the Rb+ pump using a 20 V/cm ac field.

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