Activation of electrogenic Rb+ transport of (Na,K)-ATPase by an electric field.

Previous study shows that human erythrocytes when exposed, in an isotonic suspension, to an electric field that generated 6-15 mV of transmembrane potential induced a Rb+ uptake that was sensitive to ouabain, a potent inhibitor of (Na,K)-ATPase ( Serpersu , E. H., and Tsong , T. Y. (1983) J. Membr . Biol. 74, 191-201). Here we present evidence that this uptake indeed involved the activity of (Na,K)-ATPase. Transport of Rb+, K+, and Na+ were carefully monitored during the voltage stimulation. It is shown that the electric field stimulated only the ouabain-sensitive influx of Rb+, and this uptake was against a chemical concentration gradient. The rate of the stimulated Rb+ uptake was measured under different intracellular Na+ and extracellular Rb+ concentrations. The Km for the stimulated Rb+ uptake was, respectively, 7 mM for the intracellular Na+ and 1.7 mM for the extracellular Rb+, consistent with the values for the red cell (Na,K)-ATPase. Yet, the voltage-sensitive Rb+ uptake did not depend on the intracellular ATP level. Neither did the voltage stimulation cause an elevation of ATP concentration in the red blood cells as was observed in mitochondrial and chloroplast ATP synthetase systems under higher electric field conditions. Since only Rb+ uptake was stimulated by the voltage, it follows then that the Na+ and the K+ pumping activities of the (Na,K)-ATPase could be decoupled, and the K+ pumping activity may derive from the electrogenic component of the enzyme action. In the present case, the applied electric field could polarize the membrane to provide membrane potential required for the electrogenic transport of Rb+. Data also show that vanadate at 180 microM completely inhibited the ATP-dependent Na+ and Rb+ pumping activities of the enzyme, but only reduced the voltage-stimulated Rb+ uptake to 50% level. This represents the first systematic study of the activation of a transport ATPase by an externally applied electric field.

Activation of Electrogenic Rb" Transport of (Na,K)-ATPase by an Electric Field* (Received for publication, December 30, 1983) Engin H. Serpersu  Previous study shows that human erythrocytes when exposed, in an isotonic suspension, to an electric field that generated 6-15 m V of transmembrane potential induced a Rb+ uptake that was sensitive to ouabain, a potent inhibitor of (Na,K)-ATPase (Serpersu,  Here we present evidence that this uptake indeed involved the activity of (Na,K)-ATPase. Transport of Rb+, K+, and Na' were carefully monitored during the voltage stimulation. It is shown that the electric field stimulated only the ouabain-sensitive influx of Rb+, and this uptake was against a chemical concentration gradient. The rate of the stimulated Rb' uptake was measured under different intracellular Na+ and extracellular Rb+ concentrations. The K,,, for the stimulated Rb+ uptake was, respectively, 7 m~ for the intracellular Na+ and 1.7 mM for the extracellular Rb+, consistent with the values for the red cell (Na,K)-ATPase. Yet, the voltage-sensitive Rb' uptake did not depend on the intracellular ATP level. Neither did the voltage stimulation cause an elevation of ATP concentration in the red blood cells as was observed in mitochondrial and chloroplast ATP synthetase systems under higher electric field conditions. Since only Rb' uptake was stimulated by the voltage, it follows then that the Na+ and the K+ pumping activities of the (Na,K)-ATPase could be decoupled, and the K+ pumping activity may derive from the electrogenic component of the enzyme action. In the present case, the applied electric field could polarize the membrane to provide membrane potential required for the electrogenic transport of Rb+. Data also show that vanadate at 180 PM completely inhibited the ATP-dependent Na' and Rb+ pumping activities of the enzyme, but only reduced the voltage-stimulated Rb+ uptake to 50% level. This represents the first systematic study of the activation of a transport ATPase by an externally applied electric field.

and Tian YOW TsongS
(Na,K)-ATPases' hydrolyze ATP to transport Na' and K+ against their respective chemical concentration gradient across a cell membrane, thus, maintaining the balance of * This work was supported by National Institutes of Health Grant GM 28795. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
f To whom all correspondence should be addressed. The abbreviations used are: (Na,K)-ATPase, sodium and potassium ions-stimulated adenosine triphosphatase; pCMBS, p-chloromercuribenzenesulfonic acid; RBC, red blood cells (human erythrocyte in this work). these two ions in many cell and tissue types (1,2). Under normal conditions, the stoichiometry of the transport in the red blood cell is such that for each ATP consumed, 3Na+ are transported out of the cell in exchange of 2K+ (3) and this activity is specifically inhibited by ouabain (4). The enzyme is asymmetrically oriented in cell membranes, and its affinity to Na' and K+ is very different on each side ( 5 , 6 ) . Depending on cation composition in the two sides, the Na' pump can switch to different modes, e.g. the Na+-Na' exchange mode (7), the K+-K' exchange mode (€9, or the uncoupled Na' efflux mode (9), and each mode has a different nucleotide (or Pi) requirement. Except for the normal 3Na+-2K+ exchange mode, all the other modes are nonelectrogenic, i.e. the exchange of ions is electrically neutral. The normal mode of the (Na,K)-ATPase, on the other hand, is electrogenic. This asymmetric pumping of Na+ and K' leads to the hyperpolarization of membranes (10,11). The significance of the membrane polarization, however, remains unclear.
Recent work from many laboratories have shown that it is possible to impose a membrane potential on a cell or organelle by exposing the cell or organelle to an external electric field (12)(13)(14)(15). Such applications of an electric field to cell suspensions have been shown to perforate cell membranes, cause changes in membrane conductance (16-18), or activate membrane-bound ATPases (19,20). This later effect of the electric field should be useful for the elucidation of the enzyme mechanism and should be of particular interest to biological chemists. Previously, we reported that an AC field in the range of 20 V/cm induced an ouabain-sensitive Rb' uptake by erythrocytes (21). Although the result suggested that (Na,K)-ATPase might be involved, evidence was preliminary and was not compelling. The present study has been undertaken to elucidate kinetic details of the voltage-induced Rb+ transport and to provide further evidence that, indeed, the uptake was mediated by the (Na,K)-ATPase. The artificially imposed membrane potential is shown to activate the electrogenic transport activity of the enzyme, and this activation did not alter cellular ATP level. Neither did the voltage stimulation, at the range that activated the K+ pump, activate the Na+ pump or trigger a synthesis of ATP by the enzyme. The result also suggests that the Na+ and the K+ pumping activity of the enzyme can be decoupled, i.e. they can function as two independent pumps.

EXPERIMENTAL PROCEDURES
Materiak-**Na+ and BBRb' were obtained from Amersham Cop. Ouabain and luciferin/luciferase were from Sigma. Liquiscint was supplied by Yellow Spring Instruments. Other chemicals were of the analytical grade.
Experimental Setup-Details of the experimental setup have been described elsewhere (21). Briefly, the device consists of a cylindrical Plexiglas chamber of 150-pl capacity. At the two sides of the chamber 7155 are two platinized platinum electrodes supported by brass holders. Through each brass holder is a circulating water that maintains the cell suspension in constant temperature.
Human red blood cells suspended in various isotonic solutions were placed in the Plexiglas chamber and an AC field of 20 V/cm at 1 kHz was then applied to the cell suspension for a designated period of time. After which, the sample was drawn for determination of ionic composition by radioactivity assay.
L o a d i n g of Ions into RBC-Two procedures were used. In the first method, washed red blood cells were resuspended, at 5% hematocrit, in 150 mM KC1, 1 mM MgClz, 10 mM Tris/HC1 buffer at pH 7.4, containing 20 pg/ml of chloramphenicol. pCMBS was added to the suspension at a concentration of 0.02 mM, and the suspension was kept at 4 "C. The loading solution was changed once after 5-6 h. The next day the cells were collected by centrifugation and washed once with the loading solution minus pCMBS. The cells were transferred to a recovery medium containing 2 mM adenine, 3 mM inosine, 4 mM cysteine, 10 mM glucose, 1 mM MgC12, 150 mM KCl, and 10 mM Tris/ HCI at pH 7.4 (22), and incubated in a shaker bath at 37 "C for 1 h.
Afterward, the cells were spun and washed three times with an isotonic KC1 solution, and Na' content of cells determined. When incorporation of '*Na+ was desired, both the loading and the recovery media contained "Na' at 2-4 cpm/pmol of specific activity. For the ion flux experiment, these cells were washed three times with a medium of designated ion compositions and resuspended in the same medium to 12-17% hematocrit.
In the second method, the cells were incubated for 24 h at 4 "C, at 5% hematocrit, in a solution containing 0-150 mM NaCl, 20 mM Tris/HCl, pH 7.4, 20 pg/ml of chloramphenicol, and an appropriate concentration of KC1 to maintain the isotonicity. At the end of the incubation period, the cells were then washed and the internal Na+ content was determined by flame photometry, using Li' as internal standard. These cells were then used for the ion flux experiment. In some cases, leakage of Na' into the cells during the incubation was determined by using "Na' tracer. pCMBS treatment of red cells did not alter the ratio of the ouabain-sensitive Na' efflux to Rb+ influx (the ratios were, respectively, 1.56 and 1.69 for the control and the pCMBS-treated and recovered samples). However, voltage-stimulated Rb' uptake with pCMBS-treated samples showed a greater variation (30-40%). Thus, we have chosen to alter Na' content of red cell samples by passive diffusion described above.
Cation Uptake Measurements-The experiments followed essentially the procedure described earlier (21). Briefly, cells were suspended, at 12-17% hematocrit, in a standard medium of 140 mM NaCI, 10 mM RbCl with =Rb+ tracer, 1 mM MgCIZ, 20 mM Tris/HCl, pH 7.4, or in a medium of designated composition (see tables), with or without 50 p~ ouabain. After withdrawing zero time aliquots, 150 111 of each suspension was placed in a stimulation chamber while the rest of the suspensions were kept at the same temperature as the stimulated samples. An AC field at 20 V/cm, 1 kHz was then applied to the ouabain-containing sample and the sample without ouabain for 60 min. At the end, triplicate samples from each suspension were washed twice with a standard medium containing no =Rb' tracer. The =Rb+ content of samples were then determined as described (21). Also, the hematocrit index of each sample was determined in duplicates. The specific activity of Rb' was usually 15-20 cpm/pmol. The result of the cell uptake of Rb' is expressed as attomoles/erythrocyte/ h (attomoles/RBC-h), assuming 93 pm3 red cell volume (17) and also that 50% of the cell volume is intracellular space (1 amol/RBC-h = 0.0108 mmol/liter of cells/h). In experiments varying the Rb+ concentration of stimulating medium, Na' concentration was adjusted so that the sum of two ions was 150 mM. The ATP driven Rb' uptake activity was also measured by radioactivity assay to compare with the K,,, of the external K' concentration on the ATP-dependent Rb+ transport activity. The change in the intracellular concentrations of Na+ and Rb' during the stimulation was -0.3 f 0. 15 and 0.6 f 0.20 mM, respectively, as calculated from Na' and Rb' tracer assay.
For experiments measuring the vanadate inhibition of ATPase activity, cells were preincubated with 180 p M vanadate at room temperature in the standard medium, at a hematocrit of 40%. After 40 min, the suspension was diluted with a medium containing ffiRb+ to a desired ionic composition and the Rb' uptake experiment was performed as described above.
Depletion and Regeneration of ATP in Red Blood Cells-Red blood cells were suspended, at 5% hematocrit, in a 140 mM NaCI, 10 mM KC1, 1 mM MgClz, 20 mM Tris/HCl, pH 7.4, solution containing 20 pg/ml of chloramphenicol, and incubated at 26 "C for 20 h. Thereafter, the cells were further kept at 37 "C for 90 min, and then washed once with the medium. ATP content of the cells was determined, and the cells were used for cation uptake experiments immediately. Another portion of the ATP-depleted cells were suspended, at 10% hematocrit, in a recovery medium containing 2 mM adenine, 3 mM inosine, 10 mM glucose, 1 mM M&lz, 140 mM NaCI, 10 mM KC], 20 mM Tris/HCl, pH 7.4. After recovery at 37 "C for 1 h in a shaker bath, the cells were washed twice with 140 mM NaCI, 10 mM KCl, 20 mM Tris/HCl, pH 7.4. ATP content of the cells was then determined and cells were used in the cation uptake experiment.
For the determination of ATP concentration, the luciferin/luciferase bioluminescence method was used (24). Fresh luciferin/luciferase mixture was prepared at 20 mg/ml, and 100 pl of this solution was added to 900 111 of 0.1 M Tris/HC1, pH 7.4, buffer in a cuvette in a LKB Wallac 1250 luminometer. An initial luminescence level was recorded. Luminescence level of aliquots of the perchloric acid extracts of RBC samples appropriately diluted with 0.1 M Tris/HCl, pH 7.4, was measured. The value was then converted to ATP concentration with a calibration curve obtained with ATP standard solutions which also contained the same concentration of perchlorate.

Voltage-induced
Rb' Uptak-Previous study (21) established that the AC field in the range of 10-30 V/cm, 0.1-100 kHz induced a Rb' uptake that was inhibited by ouabain. Data in Table   I indicate that this uptake was an active transport against the chemical concentration gradient. In experiments 1-4, the cellular concentration of Rb+ was 25-28 mM and the extracellular concentration was 10-12.5 mM. Yet a 20 V/cm, 1 kHz AC field stimulated the uptake of Rb' (15-25 amol/RBC-h, depending on temperatures) that was completely suppressed by 50 p~ of ouabain. Data in Table I also show that the substitution of Rb' with K' in the cytoplasm did not alter the result (see experiments 2, 4, and 5). Since this was always the case, in many subsequent experiments the preloading of Rb' was omitted.
To demonstrate that the voltage-induced Rb' uptake was not due to a modification of the passive permeability of the membranes by the voltage we have monitored both efflux and influx of Rb', K+, and Na', and the result is shown in Table  11. Two conditions were used. In experiment 1 (4 "C), the cells contained 27.5 mM of Rb+, and the voltage stimulation induced only the ouabain-sensitive uptake, but not the efflux of Rb+. Not even the ouabain-insensitive leakage of Rb' was stimulated by the AC field (compare values for cells unstimulated in the presence of 50 p~ ouabain and cells stimulated with an electric field in the presence of 50 p~ ouabain). Na' movement in either direction was insensitive to the AC stimulation. Results in experiment 2 confirm the above observation. The higher stimulated Rb' uptake in this case was due to the higher temperature (26 "C) in which the experiment was done.
Voltage-induced Rb' Transport Depended on Internal Nu+ Concentrations-The ATP dependent, (Na,K)-ATPase-catalyzed Rb' transport is known to depend on intracellular Na+ concentration. The rate increases as the internal concentration of Na' increases, leaving the apparent affinity for Rb' in the external site of the Na' pump unaltered (25)(26)(27). If the voltage-stimulated Rb' uptake is mediated by the same enzyme the rate of the uptake should also depend on the internal Na' concentration. RBC samples with different internal Na' concentrations were prepared and stimulated by the AC field at 20 V/cm and 1 kHz for 1 h. The Rb' uptake of these samples was determined by radioactivity assay. Fig. 1A shows the results obtained at 26 "C.
The total Rb' uptake in the absence of voltage stimulation as a function of internal Na+ concentration of the cells is given in the filled circles. When ouabain was present in the medium, the uptake dropped. The difference between the two TABLE I Voltage-stimulated Rb uptake in human erythrocytes is an active transport Rb loading was done either by the passive diffusion method (see "Experimental Procedures") or by using the high voltage loading method (21). After loading, the cells were washed (3 X) with a solution of proper ionic compositions (indicated in the column, Extracellular ion concentration). The extracellular solution was adjusted isotonic with sucrose. After washing, the cells were suspended to a hematocrit of 12-17% with the same solution and Rb uptake was performed. The voltage stimulation was done with an AC field of 20 V/cm at 1 kHz for 60 min. USO, SO, US, and S denote, respectively, unstimulated cells with 50 .uM ouabain, stimulated cells with 50 pM ouabain, unstimulated cells, and stimulated cells. Changes in the intracellular ionic concentration were less than 0.5 mM for Na, K, and Rb during the Rb uptake experiment. Each data is the mean of 6-27 measurements, and standard deviation is indicated in the parenthesis. (  curves represents Rb' transport catalyzed by the normal (Na,K)-ATPase activity at 26 "C. This activity, as shown in Fig. 1B (open circles), depended on the internal Na+ concentration with a K,,, of 6 mM. When AC stimulation was applied, the Rb+ uptake increased further (curve with open squares in Fig. 1A). The difference between the Rb' uptakes of the stimulated and the unstimulated samples is shown in the curve with the filled squares in Fig. 1B. The K,  The above result demonstrates that the presence of Na' in the cytoplasmic side of the RBC was essential for the (Na,K)-ATPase to respond to the voltage stimulation. Variation in the external Na+ concentration (between 2.5 and 60 mM) did not affect the rate of the voltage-stimulated Rb' uptake (data . The cells were then spun and resuspended in the standard assay medium (see "Experimental Procedures") at 12-17% hematocrit with 15-20 cpm/pmol of %Rb' specific activity, with and without 50 phi ouabain. 20-el aliquots from each suspension were withdrawn and washed twice with cold nonradioactive standard medium, then 150 91 from each group was placed in an AC stimulation chamber (21). 20 V/cm at 1 kHz AC field was applied to both chambers for 60 min at 26 "C. The remainder of the two suspensions were also kept at the same temperatures (unstimulated samples). At the end of the stimulation period, 2O-el triplicates from each sample were washed twice as mentioned above. The =Rb' content of the cells was then determined (21). Two other aliquots from each sample were also drawn into capillary tubes and hematocrit index of each sample determined. A, the total Rb+ uptake in stimulated (El), unstimulated is given. The two data points in the filled circles were obtained with samples (voltage stimulated) in which Na+ content was depleted with pCMBS method. Each data point represents mean of three separate experiments run in triplicate. Standard deviation from the mean is shown. is given. The data point in the fiLled circle was obtained with a stimulated sample in which cellular Na+ content was depleted by pCMBS method. The data in the open triangle was obtained with the same sample but without voltage stimulation. Each data point represents two separate experiments run in triplicate. The standard deviation from the mean is given. not shown). This is consistent with the notion that the action of Na' on this enzyme is membrane side specific (28).
The same experiment was also done at 6 "C. At this temperature the activity of (Na,K)-ATPase was relatively low and the ouabain-sensitive Rb+ uptake without voltage stimulation was low (Fig. 2, A and B ) . Voltage stimulation of RBC more than tripled the ouabain-sensitive Rb+ uptake by the RBC samples. The K,,, values of the (Na,K)-ATPase activity and the voltage-stimulated Rb' uptake are again within ex-perimental uncertainty identical, i.e. 8 mM for the internal Na+ concentration (Fig. 2, A and B).  Fig. 3 is replotted in the expanded scale to emphasize the cooperative nature of the Rb' uptake. A, both the plots of the Rb' uptake for the ouabain sensitive part of the normal uptake (0) and the extra uptake due to voltage stimulation (0) show sigmoid dependence on the external Rb' concentration. B, the reciprocal plots indicate that more than one Rb' binding sites were involved in the normal, ouabain-sensitive (0) and the voltage-stimulated (0) Rb' uptake. The maximum rate at 26 "C for the normal (Na,K)-ATPase catalyzed and the voltage-stimulated Rb' uptake was, respectively, 52.6 and 23.5 amol/RBC-h. U means Rb' uptake in amol/RBC-h. part of Rb' uptake (open circles), i.e. the transport due to the normal (Na,K)-ATPase activity a t 26 "C, and the net voltage stimulated part of Rb' uptake (filled squares) are further shown in Fig. 3B. Both curves are similar in shape and they both reach maximum rates around 5 mM extracellular Rb' concentration. Data points at Rb' concentrations below 5 mM are replotted in an expanded scale in Fig. 4A in order to indicate the sigmoid character of the curves. This result suggests that in the process of the voltage-stimulated Rb' transport more than one Rb' binding sites were involved, similar to the Rb' pumping activity of (Na,K)-ATPase (24, 29). In fact, when the data are plotted in the reciprocal form as in Fig. 4B both curves deviate from linearity. In this plot, ouabain-sensitive influx data for unstimulated sample was extracted from the slopes of the time dependence curves with different extracellular Rb' concentrations (data not shown), and the maximum rates obtained were, respectively, 52.6 and 23.5 amol/RBC-h (or 0.57 and 0.25 mmol/liter of cells/h) for the (Na,K)-ATPase-catalyzed and the voltage-stimulated activities. For all the Rb' concentration used, the Rb' uptake by the RRC was linear within the time range studied.
The Hill coefficients obtained from data shown in Figs. 3 and 4 are 1.84 and 1.66 for the ouabain-sensitive, unstimulated Rb' uptake and the voltage-stimulated uptake, respectively. From the same figure, K,,, values for extracellular Rb' concentration are extracted to be 0.7 and 1.7 mM, respectively, for the unstimulated and the stimulated Rb' transport.
Effect of Vanadate-The above result clearly indicates that the voltage-stimulated Rb' uptake involved the activity of the (Na,K)-ATPase. Since the voltage stimulation failed to activate Na' efflux (Table 11) it would be interesting to examine the effect of ATPase inhibitors on the stimulated Rb' uptake.

Rb uptake of human erythrocytes under various conditions
Rb uptake experiments were performed as described in the legend to Fig. 1. The symbols used are explained in Table I. ND means not determined. The data in the last three columns are ouabain-sensitive basal activity (US-USO), total ouabain sensitive activity (S-SO), and net voltage-stimulated activity (S-US). The values obtained this way are slightly different from values of the difference between the total activity and the basal activity. Each data point represents 6-27 measurements; mean value and standard deviation (in parenthesis) are given. ATP depletion and regeneration were done as described under "Experimental Procedures." The ATP level for experiment 2 at 6 "C was 16.9 (1.8) and 17.0 (1.3) pM, respectively, for cells before and after the voltage stimulation. A control sample gave 16.9 (1.8) and 15.6 (2.2) p d before and after incubation at similar condition but without voltage treatment. The experiment 5 was done with a red cell sample from an individual with a higher basal (Na, K)-ATPase activity. ( (30, 31). Red blood cell samples were treated with 180 f i~ vanadate at room temperature for 40 min. In a control experiment, this treatment completely abolished the ouabain-sensitive Na' efflux and Rb' uptake by the RBC. Yet the RBC still exhibited 50% of the voltage-stimulated Rb' uptake activity when treated with an external electric field of 20 V/cm at 1 kHz (Table 111). More interestingly, the voltage-stimulated Rb' uptake under these conditions was completely suppressed by the presence of 50 PM of ouabain in the external medium (Table 111).
Effect of Intracellular ATP Concentration-Up to this point all experiments were done with red blood cells of normal ATP level (0.6-1.0 mM). As mentioned, voltage stimulations have been found to trigger synthesis of ATP in mitochondria, chloroplasts, and reconstituted ATP synthetase systems (19,20). It is, thus, important to check whether the AC stimulation caused an increased ATP consumption, or conversely an elevation of ATP level in the RBC samples. RBC samples were metabolically depleted as described under "Experimental Procedures." The ATP level of these cells dropped from 0.6-1.0 mM to 10-20 PM, so did the normal ATP-dependent Rb' pumping activity of the cell: at 26 "C, it decreased from 39.6 to 19.9 amol/RBC-h (Table 111). The voltage-stimulated Rb+ uptake, on the other hand, was not affected (22.4 amol/RBCh for normal and 22.8 amol/RBC-h for the ATP-depleted cells). The measurement of cellular ATP concentration of the ATP-depleted cells revealed no difference before and after the electric field stimulation (see legend to Table 111). The ATPdepleted cells were incubated in recovery medium ("Experimental Procedures"), and the ATP level in these cells was restored to 216 WM. This return of cellular ATP concentration also restored the normal ATPase activity to 87% its initial value (Table 111). Again, there was no effect on the voltagestimulated Rb' uptake. Erythrocytes contain high levels of adenylate kinase and some phosphate hydrolyzing activities that may obscure ATP hydrolysis activity of (Na,K)-ATPase. Since changes in cellular ATP concentration by greater than one order of magnitude did not alter the voltage-induced Rb' uptake, it is unlikely that the voltage treatment stimulated the ATP hydrolysis activity of the (Na,K)-ATPase. A low affinity ATP binding site is believed to regulate conversion of enzyme between the phosphorylated and the dephosphorylated forms (32, 33). The result presented here indicates that this low affinity site was not directly involved in the voltage-stimulated Rb+ transport activity.
Another interesting observation was the correlation between the basal level of the (Na,K)-pump and the voltagestimulated Rb' uptake activity of blood samples from different individuals. The RBC sample from an individual that exhibited a ouabain-sensitive Rb' pumping activity of 79.9 amol/RBC-h compared to the individual of 39.6 amol/RBCh in Table 111 also exhibited twice the voltage-stimulated Rb+ uptake activity (39.7 amol/RBC-h compared to 22.4 amol/ RBC-h). It is not known whether the RBC sample of this individual contained higher enzyme concentration or similar enzyme concentration but with higher specific activity. In any case, the voltage-stimulated, ouabain-sensitive Rb' pumping activity described here increased with increasing basal level of (Na,K)-pump.

DlSCUSSION
Voltage-stimulated Rb' Uptake Was Due to the Induced Membrane Potential-It is now recognized that when a cell or an organelle in suspension is exposed to an electric field an electrical potential is generated across the membrane. The magnitude and the sign of the induced membrane potential depend on the position of interest on the membrane surface. For a spherical membrane vesicle, the induced membrane potential, A 3 , is described by, A* = 1.5 aE cos 8 in which a, E, and 8 are, respectively, the radius of the vesicle, the electric field strength in volts/cm, and the angle between the field vector and the radial at the point of interest on the membrane surface (13,15,20). The maximum potential experienced by a molecule on the membrane surface is k1.5 aE and it occurs at 0 =180" and 0". This is 1.5 a/d (d being the thickness of the membrane) times more intense than that the molecule would experience in solution. For a molecule in the erythrocyte membrane this value is roughly 1000. Our data indicate that maximum stimulation of Rb+ uptake occurred at electric field strength of 20 V/cm, i.e. an induced transmembrane potential of 12 mV (21). Activation of (Na,K)-ATPase is shown to hyperpolarize erythrocyte membranes by this same magnitude (11). The previous study (21) also shows that electric fields below or above 20 V/cm reduced the effectiveness of the stimulation. For field strengths below 35 V/cm no stimulation of Rb' efflux or movement of Na' in either direction could be detected (see a complete set of data in Table 11). The constant level of ouabain-insensitive uptake (Tables I and 11) makes it less likely that the ouabain binding blocked the electrogenic pump and altered Rb' uptake indirectly by shifting the membrane potential. These observations indicate that there was no induction of passive permeation pores at this range of voltage. Neither was there any irreversible damage done to the permeation barrier of the red cells as was reported using electric pulses in the kilovolts/cm-range (12)(13)(14)(15)(16)(17)(18). Effects of joule heating as the source of the observed Rb' uptake was unequivocally ruled out by the fact that direct measurements of samples showed negligible temperature elevation (less than 0.5 "C) and that the stimulated uptake was dependent on the frequency of the applied AC field (21). The optimum frequency occurred a t 1 kHz when 20 V/cm-electric field was used.
The Voltage-stimulated Rb' Uptake Involved Activity of (Na,K)-ATPase-Data presented in this article as well as in the previous study (21) support the contention that the voltage-induced Rb' uptake involved the (Na,K)-ATPase. First, the uptake was against a concentration gradient, indicating that active transport occurred (Table I). Since the uptake was completely inhibited by ouabain, the most likely candidate is (Na,K)-ATPase. Second, the rate of uptake depended on the internal Na' concentration and the external K' or Rb' concentration in the same manner (similar K, values) as the ATP driven ion translocation through the (Na,K)-ATPase. Third, the Rb' uptake was consistent with ATP driven Rb' movement. Since Rb'/K' efflux and Na' transport in either direction did not occur, an unrelated activity was unlikely involved (Table 11). Finally, erythrocyte samples from an individual with higher basal level of (Na,K)-pump activity also exhibited higher values of AC-stimulated Rb' uptake.
The stimulated Rb' influx required the presence of intracellular Na+. The necessity for Na ion, without being transported during stimulation may involve the strong inhibitory effect of Na' on K'-K' exchange mode of (Na,K)-ATPase (8). Since K'-K' exchange is not an electrogenic process, intracellular Na' could hold the enzyme in its electrogenic Na'-K' transport mode. How did the electric field trigger the K' pump without simultaneously activating the Na' pump of the (Na,K)-ATPase? We suggested (21) that an electric fieldinduced membrane potential could trigger a conformational change of the enzyme, allowing the bound Rb (or K) ions to be translocated into the red cell. We also suggested that since only K' or Rb' influx was stimulated by the electric field, the enzyme must, in effect, functioned as a K' rectifier. K' or Rb' ion could then be driven across the membrane by the membrane potential which either is generated by the efflux of Na ions (11) or, in the present case, induced by the applied electric field. Data in Table I11 indicate that under identical solvent conditions the rate of the stimulated uptake increased 6-7-fold as the temperature was raised from 6 to 26 "C. The activation energy of the process is approximately 15 kcal/mol. This magnitude of activation process is consistent with a conformational change of a protein. It has been postulated that during the hydrolysis of ATP the catalyzing ions become occluded within the enzyme and are subsequently released after a slow conformational change (32). Indeed, occlusion following a transport of Rb+ and Ca2+ has been demonstrated for (Na,K)-ATPase and Ca2+-ATPase, respectively (34,35).
The action of vanadate on the stimulated Rb' uptake seemed complicated. The vanadate treatment, which inhibited the ouabain-sensitive Rb' uptake and Na' efflux in the control cells, did inhibit 50% of the stimulated uptake, which was completely suppressed by ouabain. Vanadate has been proposed as a transitional state analogue for phosphate hydrolysis because of its ability to exist in a stable trigonal bypyramidal structure (36). V-0 bond unlike P-0 bond forms and breaks rapidly (37). If vanadate forms a complex with an aspartate group (like phosphate), then its stability on the native protein is due to its surroundings rather than to its covalent bondper se (38). Conformational changes within the enzyme upon application of electric field may release vanadate allowing translocation of the already bound Rb' .
All modes of the (Na,K)-ATPase require nucleotides. In spite of the 50% decrease in basal (Na,K)-pump activity when the ATP level dropped from millimolar to 20 p~, no change in the stimulated Rb' uptake was found (Table 11). When the cells were regenerated, they regained 87% of their original basal activity, while stimulated uptake was unchanged (Table  11). Determination of intracellular ATP before and after the stimulation did not show significant differences. These results do not directly rule out the requirement for ATP hydrolysis (or synthesis), since, as mentioned, the cells have high levels of adenylate kinase activity. However, unchanged stimulated Rb+uptake after more than an order of magnitude change in ATP concentration suggests that ATP hydrolysis was not required. Moreover, enzyme turnover that consumed ATP would have stimulated also the Na' efflux. This clearly was not the case. Since the number of the enzyme was the same in the ATP-depleted and the -undepleted cells, the constant rate of stimulated uptake implies that it was due to the number of enzymes affected by the field instead of their state of activity. However, it is not clear whether nucleotide binding to a high affinity site is required for the observed Rb' uptake.
These findings can be represented by a simple reaction scheme.

\ ,
Membrane potential E*(Rb+j In this scheme, the membrane potential induces a conformational change of the (Na,K)-ATPase to E* form. This may be analogous to E2 form of the enzyme in the Albers-Post scheme (39), which has a higher affinity for (Rb+),. Then the outside Rb+ combines with this form of the enzyme and is consequently transported inside. The internal Na' accelerates the conversion of the enzyme to the original conformation, either before or after the release of Rb+. Thus, the application of an electric field to a red cell suspension activates the Rb' pumping activity of (Na,K)-ATPase, which may represent the membrane potential sensitive part of the Na+-K' exchange cycle.