tthe Department of Physiology, University of Maryland School of Medicine, 660 W.

Transcription

1 J. Physiol. (1981), 317, pp With 9 text-figures Printed in Great Britain THE EFFECTS OF RUBIDIUM IONS AND MEMBRANE POTENTIAL ON THE INTRACELLULAR SODIUM ACTIVITY OF SHEEP PURKINJE FIBRES BY D. A. EISNER*, W. J. LEDERERt AND R. D. VAUGHAN-JONESt From the University Laboratory of Physiology, Parks Road, Oxford; tthe Department of Physiology, University of Maryland School of Medicine, 660 W. Redwood St, Baltimore, Maryland 21201, U.S.A.; and tthe University Department of Pharmacology, South Parks Road, Oxford (Received 28 November 1980) SUMMARY 1. Intracellular Na activity, aha, was measured in voltage-clamped sheep cardiac Purkinke fibres. 2. Increasing [Rb]0 from 0 to 4 mm in K-free solutions (at a fixed membrane potential) decreased aina. Further increases of [Rb]0 (up to 20 mm) had little or no effect. 3. Following exposure to Rb-free, K-free solution, the addition of a test concentration of Rb produced an exponential decrease of ana. The rate constant of decay of aka increased with increasing [Rb]o over the measured range (0-20 mm). 4. The accompanying electrogenic Na pump current transient decayed with the same rate constant as aia over the range of [Rb]o examined. During this decay the electrogenic Na pump current was a linear function of ana. Increasing [Rb]o increased the steepness of the dependence of the electrogenic current on ana. 5. A constant fraction of the net Na efflux was electrogenic. This fraction was not affected by varying [Rb]o over the range 0-20 mm. 6. Using a simple model, it is shown that the dependence of steady-state aka on [Rb]o is half-saturated by less than 1 mm-[rblo. The rate constant of decay of aika and the slope of the relationship between electrogenic Na pump current and aka are, however, better fitted with a lower affinity for Rb (K0.5 = 4 mm-[rb]o). 7. Depolarizing the membrane potential with the voltage clamp decreased aka; hyperpolarization increased it. These effects persisted in the presence of 10-5 M- strophanthidin. An effect of membrane potential on the net passive Na influx can account for the observations. 8. The effects of membrane potential on the net passive Na influx were examined by measuring the maximum rate of rise of aka at different holding potentials after inhibiting the Na-K pump in a K-free, Rb-free solution. Depolarization decreased the Na influx. 9. Using the constant field equation, the net passive Na influx was used to estimate the apparent Na permeability coefficient, PNa. This was between 0-8 x 10-8 and 1-5 x 10-8 cm sec-'. * Present address: Department of Physiology, University College London, Gower St, London.

2 190 D. A. EISNER, W. J. LEDERER AND R. D. VAUGHAN-JONES INTRODUCTION In the previous paper we examined the relationship between the intracellular Na activity, aba, and the electrogenic Na pump current in sheep cardiac Purkinje fibres under conditions where the Na-K pump was stimulated by an elevation of internal Na. The electrogenic Na pump current was found to be linearly related to ana. Furthermore the amount of charge extruded by the pump was a constant fraction of the total extrusion of Na. In this paper we have measured a'a in order to examine the external (K) activation site of the Na-K pump. The relationship between a'a and [K]0 is complicated by the fact that external K influences the membrane potential. In the snail neurone at least, changes of membrane potential have significant effects on aka (Thomas, 1972). It is therefore important to be able to separate effects of external K mediated by the Na-K pump from those secondary to changes of membrane potential. In previous studies with Purkinje fibres this has not been possible (Ellis, 1977; Deitmer & Ellis, 1978b) since the membrane potential was not controlled, but in the present work it was held constant with a voltage-clamp circuit. We have investigated the effects ofadding various [Rb]o to re-activate the Na-K pump (Rb is very similar to K in its ability to activate the Na-K pump: Eisner, Lederer & Vaughan-Jones, 1981). Upon adding external Rb, the Na-K pump activity can be assessed either from the rate of fall of aika plus the accompanying electrogenic pump current (cf. Deitmer & Ellis, 1978b; Eisner & Lederer, 1980a) or from the final equilibrium level of ana. We find that these two measures give different values of the Ko.5 for Rb activation of the pump. We also find that the fraction of Na extrusion that is electrogenic is independent of [Rb]o. We then examine the effects on aka of changing the membrane potential. Depolarization produces a significant decrease of aka which is shown to be caused mainly by a reduction in the passive Na influx. This confirms that if the membrane potential is not controlled then, when it changes, it will contribute to the effects of external K on ana. Preliminary reports of some of these results have appeared (Eisner, Vaughan-Jones, 1980; Vaughan-Jones, Lederer & Eisner, 1981). METHODS Lederer & The experimental methods employed in this paper are identical to those described in the previous paper (Eisner et al. 1981). Fig. 1 RESULTS shows an experiment designed to investigate the effects of various concentrations of external Rb on both aka and the electrogenic Na pump current. It has already been shown that K and Rb are very similar in their ability to activate the Na-K pump (Eisner & Lederer, 1980b; Eisner et al. 1981). Apart from the initial period in 4 mm-[k]0 shown at the beginning of Fig. 1, the control solution contained 10 mm-[rb]. and was K-free. The procedure adopted was firstly to raise aka by removing external Rb for 5 min periods. This was followed by the addition of a test concentration ( mm) of [Rb]0 which re-activated the Na-K pump producing a recovery of ana. The cycle was then completed by returning to the control solution

3 Rb AND MEMBRANE POTENTIAL ON aka 191 (10 mm-[rbl]). The membrane potential was held at -70 mv and, during Na-K pump re-activation, there was an outward, electrogenic Na pump current transient (Eisner et al. 1981). The greater the [Rb]o used to re-activate the Na-K pump, the larger was the peak size of this current transient and the faster was the subsequent decline of both the current and ana. For example in Fig. 1, the fall of both aka and the electrogenic current is much faster with 10 mm-[rb]o than with 2 or 1 mm-[rb]0. A 4Or- 10 min m C a) a E 12 E E 4[K], B 40 - C~~~~~~~~~~~ a, 40 t 18r -E 12 - E E Q~~0 0 Fig. 1. A. the effects of various [Rb lo on alwa and current. In each panel the top trace shows the current and the middle ada. Changes of [Rb]o are denoted in the lower trace. The bottom panel (B) is a continuation of the top record. Apart from a few minutes at the beginning (as indicated), [K]o was zero throughout the experiment. The control solution contained 10 mm-[rb]o. The holding potential was -70 mv. [Rb]o was reduced to zero for periods of 5 min. then a test concentration of Rb was added to re-activate the Na-K pump. After current and a1a had reached a steady state the control solution was re-applied. This protocol was repeated for the various [Rb]o shown. The steps in current and ana following recovery from the first exposure to 0 [Rb]o show a test of potential uniformity (see text). The membrane potential was stepped to various levels requiring the currents shown. The effects on aa are small compared with the changes in the rest of the experiment. Arrow I denotes the effects of perturbations in the flow of superfusing solution. Also shown in Fig. 1 is a test of the voltage uniformity of the preparation. This is shown immediately after the first re-activation with 10 mm-[rb]0. After a series of brief current pulses the membrane potential was depolarized to -49mV and -39 mv respectively for about 1 minm requiring the current pulses shown on the upper trace in Fig. 1 A. The difference in potential between the voltage and Na+-sensitive electrodes gives an estimate of the voltage non-uniformity along the

4 192 D. A. EISNER, W. J. LEDERER AND R. D. VA UGHAN-JONES Purkinje fibre. This is 1-7 mv for the larger current pulse (45 na) as measured from the corresponding change in the ai a trace. The largest electrogenic Na pump current transient measured in the experiment was 40 na at its peak (the re-activation with 10 mm-[rb]0) and the accompanying fall of aia represents a potential change of the Na signal of 18 mv. Consequently inaccuracy, if any, in the reading of aia caused by voltage non-uniformity will be less than 10%. will be less than 100%. Fig. 2 A shows semi-logarithmic plots of the decline of the electrogenic Na pump current (filled symbols) and aia (open symbols) following Na-K pump re-activation by various concentrations of Rb. The plots for different [Rb]o have been arbitrarily moved along the time axis to avoid overlap. Both current and aka fell exponentially and it is clear that a higher [Rb]0 produced a faster rate of decline of both parameters. This is in agreement with the results of Deitmer & Ellis (1978b) who found that in non-voltage-clamped Purkinje fibres, increasing [K]0 increased the rate constant of decay of aa. Fig. 2 A also demonstrates that the decay of current is parallel to that of aka. This is re-emphasized in Table 1, which shows results from the same experiment. Apart from some discrepancy in 4 mm-[rb]0 the half-time of decay of aka is very similar to that of current. The previous paper (Eisner et al. 1981) showed that this was true over a range of ana ( mm). The present result now extends this observation to include a range of [Rb]o (the complete range in four experiments was mm). Fig. 2 B shows the electrogenic Na pump current as a function ofaia after re-activating the Na-K pump with various [Rb]o. In the previous paper (Eisner et al. 1981) we showed that this current (AI) is a linear function of ana. The data in Fig. 2B now demonstrate that the slope of this relationship increases as [Rb]0 is increased. This is to be expected if Rb activates the Na-K pump since, at any given ana, the pump rate will be greater at a higher [Rb]o. It is to be noted in Fig. 2B that the relationship in 10 mm-[rb]o is not initially linear since the first two points deviate from the line. However, these correspond only to the first 15 sec of decay of the electrogenic current transient: during the remainder of the recovery (3-4 min) the relationship is reasonably linear. Such behaviour was only occasionally noted in other experiments. Fig. 1 also shows the steady-state level of aka reached in the different [Rb]o. It is evident that there is a large decrease in the steady-state level of aka as [Rb]o is increased from 0 5 to 10 mm (arrow c). A smaller decrease of aka is produced by increasing [Rb]0 from 2 to 10 mm (arrow b) but virtually no change of aka is seen on increasing [Rb]0 from 4 to 10 mm (arrow a). This might suggest that the Na-K pump is saturated by 4 mm-[rb]0. This observation, however, contrasts with the effects of [Rb]o on the rate constant of decay of aka and on the slope of the relationship between Al and aika. These parameters continued to increase substantially as [Rb]o was increased from 4 to 10 mm (Fig. 2A and B, and see Table 1), suggesting that the dependence of the Na-K pump on external Rb is not saturated even at 10 mm-[rb]0. This apparent paradox is difficult to account for in terms of any one simple explanation. To examine the problem further one needs a model for the relationship between the degree of activation of the external site of the Na-K pump and the three measured parameters: rate constant of decay of aka; slope of AI versus aia relationship; steady-state ana. Such a model has already been presented (Eisner & Lederer, 1980a) and we show below that it does not predict the observed paradox.

5 81 o 4 Rb AND MEMBRANE POTENTIAL ON asa 193 E z *5- s Il Time (min) I B < I ana (mm) Fig. 2. Analysis of the effects of various [Rbb. on the decay of aia and the electrogenic Na pump current. A, semi-logarithmic plot of decay of aia and current. aia and current from Fig. 1 are plotted at their levels above the steady-state value (AaNaa open symbols; AI, filled symbols). Different symbols correspond to different [Rbbo: 10 mm (@. 0); 4 mm (V V); 2 mm (EL U); 1 mm (A. A). The records for the various [RbJo have been arbitrarily shifted along the time axis to avoid overlap. For a given concentration. however, current and aka have been kept in register. The lines have been fitted by a least-squares method to all the points except those in the first 15 see after the peak of the electrogenic current. B, the dependence of the electrogenic Na pump current on aba. The data from Fig. I have been re-plotted to show Al as a function of a' for different [Rblo. as indicated :117

6 194 D. A. EISNER, W. J. LEDERER AND R. D. VA IUGHAN-JONES The apparent affinity of the Na-K pump for external Rb We will initially ignore all the possible Na extrusion mechanisms apart from the Na-K pump and will assume that the Na efflux across the cell membrane is equal to f(rbo) a1a where f(rb). depends on the degree of activation of the Na-K pump by external Rb. The electrogenic Na-K pump current is therefore given by: I = F r aa f(rb)o, where F is the Faraday and r the fraction of Na which is extruded electrogenically. TABLE 1. The effects of varying concentrations of Rb on Ada (data from experiment shown in Fig. 1) [Rb]lo tn.5 ti N ata Aa'a QC Qe/Aai d(ai)/(da~a) (mm) (see) (sec) (mm) (mm) (f(') (tcmmm1) (na mm-') Columns show from left to right: (i) [Rb]o; (ii) half-time of fall of a'a on re-activating the Na pump: (iii) half-time of fall of electrogenic Na pump current; (iv) steady-state a'na; (v) total change of aka on re-activating Na pump with test concentration of Rb following a 5 min exposure to Rb-free solution; (vi) area under the electrogenic Na pump current transient; (vii) Qe/Aa~a, which is a measure of the electrogenic fraction of Na extrusion; (viii) slope of the graph of electrogenic Na pump current transient versus ana. (From Fig. 2B). Therefore the slope of the relationship between Al and aia is given by: Slope = d(ai)/daka = F r f(rbo). (1) ana is given by: - dana/dt = J-f(Rbo) ana, where V is the preparation's intracellular volume, J the Na influx and y the intracellular Na activity coefficient. Therefore, on re-activating the Na-K pump, following exposure to an Rb-free solution, the rate constant, k, of decay of a'a is given by: k = yf(rbo)/v. (2) The steady-state level of aka is given by: (ana)t = = J/f(Rbo) or: 1/(aa)t x = f(rbo)/j. (3) Therefore eqns. (1), (2) and (3) show that f(rb.) should be proportional to the following parameters: 1/(aNa)t-..,; d(ai)dana; k (and therefore also 1/tNa). Fig. 3 shows a Lineweaver-Burk plot where the reciprocals of these parameters (i.e. (aka)t.00, daia/d(ai) and t, all obtained from Fig. 1) have been plotted as a function of 1/[Rb]o. The slope and to.5 data are reasonably well described by a single straight line, so that if one assumes Michaelis-Menten kinetics for the transport of Rb by the

7 Rb AND MEMBRANE POTENTIAL ON aia 195 Na-K pump, then the apparent affinity constant for external Rb (K0.5) is about 2 mm. However, the steady-state aka data in Fig. 3 are fitted by a very different line with a K0.5 of 08 mm. This difference in KO.5 emphasizes the discrepancy between steady-state and kinetic data. Fig. 4 shows results collected from four different experiments. The filled symbols show steady-state a'a as a function of [Rb]o. The values of aka have been normalized CU._Z~~~~~~~~~~~~~~~~~~~~~z. 0~~~~~~~~~ o ~~~~ ~~~~~~~~~~~~~~0-25 < C 25 -E 0 0* ([Rb]0)-' (mm-') Fig. 3. The dependence of various measures of Na pump activity on [Rb lo. Data from Figs. I and 2. The different parameters are plotted as a function of ([Rb]0)-. The left-hand ordinate shows the to.5 of decay of ai5 on re-activating the Na-K pump with a given [Rb 1o (0). The centre ordinate shows the steady-state level of alna (*). The right-hand ordinate shows the reciprocal of the gradient of the lines in Fig. 2 B for the relationship between AI and aia (V) to the value in 10 mm-[rb]o in that particular experiment. It is clear that the data are described by a K0,5 of less than 1 mm-[rb]o. The open symbols show the dependence of d(al)/dana and t/t05 on [Rb]o. In any given experiment the value of each parameter has been again normalized to the value in 10 mm-[rb]o. The two parameters have then been averaged. The points are fitted by a rectangular hyperbola (Ko.5 = 4 mm-[rblo). Again, this is a lower affinity than that required to fit the steady-state aka data. Possible explanations of this discrepancy will be considered in the Discussion. The above model is clearly oversimplified. First, if it is assumed that more than one K ion must bind to the external face of a Na-K pump unit for transport to occur (e.g. Baker, Blaustein, Keynes,.Manil, Shaw & Steinhardt, 1969) then it may be more appropriate to fit a sigmoid rather than a hyperbolic curve to the data shown in Fig. 4. This, however, would have a negligible effect upon the present estimates of K0.5. Of greater importance is the possibility that under certain conditions mechanisms other than the Na-K pump may affect ana. That these mechanisms are significant is shown by the fact that when the Na-K pump is totally inhibited by strophanthidin, aka never rises above mm (Deitmer & Ellis, 1978a, b). It can, however, be shown that the discrepancy between the steady-state and kinetic measurements cannot be resolved by making a simple allowance for the other Na extrusion mechanisms. 72-

8 196 P. A. EISNER, W. J. LEDERER AND R. D. VAUGHAN-JONES If we assume that these other processes are linearly related to a'a, an extra term must be added to f(rbo) in eqns. (2) and (3). This will increase the rate constant and decrease steady-state ana, thus changing the apparent dependence of both these parameters on [RbJo. Nevertheless, they should still both depend on [Rb]o in the same manner. Therefore the marked difference in apparent affinity for external Rb is not simply a result of neglecting other Na extrusion mechanisms. The presence of a large non-na-k pump component may explain the failure of a prediction made on the basis of a simple model which ignores this factor. Gadsby (1980) has shown that the simple model described above predicts that, following removal of external K to inhibit the Na-K pump, ana should double after a time given by the time constant of decay of the electrogenic Na pump current on re-activating the Na-K pump following exposure to K-free solution. It is, however, obvious in most of our experiments that aia rises much more slowly than expected on this analysis. The mean ana in 10 mm-[rb]o is 6-7 mm and the mean rate of rise of ala 1 1 mm min-. Therefore ar4a doubles in 0 [Rb]0 in about 6 min. This is several times longer than the time constant of the electrogenic Na pump current in 10 mm-[rb]o (mean value 1-6 min). This can be accounted for by the effects of other Na extrusion mechanisms since these will increase the rate of decay of ana following Na-K pump re-activation whilst slowing the rise of aia on Na-K pump inhibition. 15 ~ o X V ~~~~~~~~~~~~~~~~~~N M ~~~~~~~~~~~ 0 I Q.5/ C~~~~~~~~~~~~~- -_z 1_ TII I [Rb]0 (mm) Fig. 4. Collected data from four experiments showing the dependence of various measures of Na pump activity on [Rb]o. The (0) show the mean value of both (to.5)-1 for the decay of aka and the slope of the relationship between AI and a' a. In each experiment the value of each parameter has been divided by the value in 10 mm-[rblo in that experiment. The (@) show the steady-state value of a~a divided by the value of a'a in lomm-[rb10. Error bars show +S. E. M. The effects of [Rb]. on the coupling ratio of the Nla pump As explained in the previous paper (Eisner et al. 1981), when the Na-K pump is re-activated by adding back external Rb, the area under the electrogenic Na pump current transient (i.e. the time integral of the current) gives an estimate of the amount of Na extruded electrogenically during the recovery. The ratio of this area to the total change of aka that occurs during the re-activation is proportional to the fraction of total Na extrusion that is electrogenic. In the previous paper (Eisner et al. 1981) we pointed out that although there are problems with estimating the exact value of this fraction, relative changes should easily be detectable. Table 1 shows that the larger is [Rb]o, the greater are both the area and the change of aia. It also shows that the

9 Rb AND MEMBRANE POTENTIAL ON aia 197 ratio of the area to the change of ana is reasonably constant with respect to changes of [Rb]0. Pooled data from four experiments are shown in Fig. 5. The ordinate shows the value of the ratio expressed as a percentage of the mean in that experiment. The different symbols refer to different experiments. There is no obvious systematic dependence of this ratio on [Rb]0, suggesting that the electrogenic fraction of Na extrusion is essentially independent of [Rb]0. This extends the observations made in the previous paper (Eisner et al. 1981) that the fraction is independent of ana. 125 A _ A as75- A A.z [Rb]o (mm) Fig. 5. The effects of various [RbI0 on the electrogenie fraction of Na extrusion. The ordinate shows the ratio of the area under the electrogenic Na pump current divided by the change of ala (luring Na-K pump re-activation (Aaia). For each experiment the values of this ratio are expressed as a percentage of the mean value in that experiment. Different symbols represent different fibres. The effects of membrane potential on ana In the present experiments we have controlled the membrane potential with a voltage clamp. Thus the effects of re-activation of the Na-K pump with various levels ofexternal Rb are not complicated by changes of membrane potential. Previous work, however, has not used this technique and in these earlier experiments changes of membrane potential will have altered the electrochemical gradient for Na and so possibly the level of aika. In this section we have therefore investigated the effects on aa of altering the membrane potential in a controlled manner using the voltage clamp. Fig. 6A shows an experiment where the fibre was in a solution of 4 mm-[k]0 and the membrane potential held at -70 mv. Under these conditions aka was 4 mm. The membrane potential was then depolarized to -20 mv and this produced a fall of aka to about 3 mm with a t0.5 of 140 sec. On repolarization a'a recovered. Fig. 6B (from a different fibre) shows the effects of a larger potential step (from -30 to -110 mv). A substantial increase of aka was produced which was again reversed by depolarizing the membrane potential (in this case to -50 mv). In three preparations we have found that hyperpolarization always produces an increase of a'a and depolarization a fall. Does potential non-uniformity interfere with measurement of aka? As discussed in the Methods section of the previous paper (Eisner et al. 1981), the potential and

10 198 D. A. EISNER, W. J. LEDERER AND R. D. VA UGHAN-JONES Na+-sensitive electrodes are not in the same place and may therefore experience different membrane potentials. The difference in potential will depend on the amount of current injected to polarize the preparation. It is therefore possible that part of the apparent change of aika produced by changing the membrane potential could be an artifact of current passage. This possibility is, however, excluded by the current trace in Fig. 6A. The voltage non-uniformity should increase with the magnitude of the polarizing current (Kass, Siegelbaum & Tsien, 1979; Eisner et al. 1981). A large instantaneous outward shift of current is seen on depolarization. However, apart from a capacitive effect, no change is seen on the aka trace. Furthermore, when aka begins to decline, the amount of injected current is falling. Thus the change of aia is not related to the injected current and is therefore not an artifact of potential non-uniformity. A o~ ~~i~ 1/ min <20- l B c K 8 5 min E~~ ~~~~~~~~~ 12- ~ E ~ ~ ~ ~ E k Fig. 6. Effects of membrane potential on steady-state level of aba. A, effects of depolarization. Traces show: top, current; middle, aba; bottom, membrane potential (Em). The holding potential was -70 mv. The membrane potential was then depolarized to -20 mv for 9.3 min using the voltage clamp; following this it was returned to -70 mv. The superfusing solution contained 4 mm-[k]o. B, effects of hyperpolarization. Traces show: top, aiba; bottom, membrane potential. The membrane potential was initially -30 mv and was then hyperpolarized to -110 mv for 7-5 min. Finally it was returned to -50 mv. aj a is falling at the beginning of the trace, following a previous hyperpolarization (not shown). The superfusing solution contained 4 mm-[rb]o. How does a change of membrane potential affect aia? It is likely that changes of membrane potential alter the net passive Na influx. In this case, Na extrusion mechanisms such as the Na-K pump will establish a new steady-state aka to balance the altered influx. However, it is also possible that membrane potential affects, either directly or indirectly, the rate of Na-K pumping. A direct effect is thought to be unlikely since the electrogenic Na pump current appears to be independent of the membrane potential in the range -16 mv to

11 Rb AND MEMBRANE POTENTIAL ON aka mv (Eisner & Lederer, 1980a). Nevertheless an indirect effect is still possible. For example, depolarization results in a substantial accumulation of K ions in the restricted extracellular spaces of the Purkinje fibre (Baumgarten & Isenberg, 1977). This might then increase the activity of the Na-K pump and thus decrease aka. There is good reason for suspecting that this is not the main cause. In Fig. 6A the fibre was bathed in 4 mm-[k]0 and depolarization produced a decrease of ana. It has already been shown (Fig. 4) that increasing [Rb]0 above 4 mm has no significant effect on ana. Since Rb and K have similar effects on the Na-K pump in the Purkinje fibre (Eisner & Lederer, 1980b; Eisner et al. 1981), then an increase of Na-K pumping caused by a rise of extracellular K cannot account for the fall of ana. Furthermore min E E_70] LLJ Strophanthidin (10- M) Fig. 7. The effects of strophanthidin on the changes of ana produced by variations of membrane potential. Upper trace, aj a; lower trace, membrane potential (Em). The preparation had been exposed to 10- M-strophanthidin for 15 min before the start of the Figure. The solution contained 4 mm-[k]0, 0 [Rb]o. The holding potential was -20 mv. Then the holding potential was changed to -70 mv for O min. Following this the membrane potential was returned to -20 mv. After recovery at -20 mv another (11 min) hyperpolarization was applied. After recovery at -20 mv, strophanthidin was washed off. The later part of the recovery has been omitted from the Figure. After recovery at -20 mv, two hyperpolarizing pulses to -70 mv were applied. we find a similar dependence of aka on membrane potential when the Na-K pump is inhibited by strophanthidin (10-5 M). Under these conditions aka rises to a plateau level which is determined by a balance between the inward leak of Na and the non-na-k pump Na extrusion systems such as Na-Ca exchange (Deitmer & Ellis, 1978b; Ellis & Deitmer, 1978). The fibre shown in Fig. 7 was exposed to strophanthidin (10-5 M) at a holding potential of -20 mv. This produced an increase of aka to about 18 mm. Hyperpolarizing the membrane potential to -70 mv for 9 min reversibly increased ana by about 4 and 6 mm. After washing off strophanthidin ana recovered to 7-8 mm and a similar hyperpolarizing step now increased aka by 1-7 mm. Therefore changes of membrane potential still affect aka in the presence of 10-5 M-strophanthidin. An effect of membrane potential on the net Na influx rather than an effect on Na-K pumping thus seems a plausible explanation for the effects of membrane potential on aka. It is interesting that the potential dependence of aka is more

12 200 D. A. EiSNER, W. J. LEDERER AND R. D. VAUGHAN-JONES pronounced after Na-K pump inhibition. Presumably the mechanism that regulates aka under these conditions is less effective than the Na-K pump at extruding Na, so that altering the passive Na influx has a larger effect on ana. In order to investigate further the effects of membrane potential we have attempted to measure more directly its effect on the Na influx. Deitmer & Ellis (1978b, 1980) obtained estimates of this influx by measuring the maximum rate of rise of aia after inhibiting the Na-K pump with cardioactive steroids such as strophanthidin. It should be noted that, in the presence of Na extrusion mechanisms other than the Na-K pump, the rate of rise of aka only gives a measure of the leak influx minus these other Na extrusion mechanisms. For convenience we have usually inhibited the Na-K pump with K-free, Rb-free solutions. This will not completely inhibit the Na-K pump (Ellis, 1977) but should min E~ El-z10 A*' ' mv Em -57 mv 0 [Rb]0 0 [Rb]0 Fig. 8. Effect of membrane potential (Em) on rise of ana produced by inhibiting the Na-K pump. The control solution was 4 mm-[rb]0. [K]O was zero throughout the experiment. [Rb]o was reduced to zero for 10 min periods as shown. This was done first at -57 mv. After adding back 4 mm-[rb]0, the holding potential was depolarized to -20 mv. The fall of aja on depolarization has been omitted from the record. [Rb]0 was then lowered to zero for 10 min. nevertheless give a reasonable measure of the Na influx. Fig. 8 shows the effects of Rb-free solutions at two different membrane potentials, -57 and -20 mv. The initial holding potential was -57 mv. In the absence of external Rb, aka rose with a maximum rate of 0 75 mm min-'. Subsequently removing external Rb at -20 mv produced a much slower rise of ana (0-32 mm min-'). Fig. 9 shows that, in three preparations, the maximum rate of rise of aka decreased with depolarization. In one experiment the effect was similar when the Na-K pump was inhibited either by 10-5 M-strophanthidin (V) or 0 [Rb]o (V). Hence, as predicted, the net Na influx is sensitive to membrane potential. It is interesting to consider whether the effect on the Na influx can be accounted for entirely by changes of the electrochemical driving force for Na entry or whether a change of apparent Na permeability on depolarization must also be invoked. Here

13 Rb AND MEMBRANE POTENTIAL ON aia 201 the results are less conclusive. We can calculate the apparent PNa from the constant field equation. assuming the passive Na efflux to be negligible: PNa= JNa exp(vf/rt)-1 [Na]o( VF/RT) where JNa is the net influx of Na, estimated from the rate of rise of Na in Rb-free, K-free solution. As explained above this assumes that the Na-K pump is totally inhibited in K-free, Rb-free solution and that factors other than the Na-K pump do not contribute to Na efflux. Fig. 9B shows the values of PNa calculated for the experiments shown in Fig. 9A. At a membrane potential of -10 to -20 mv, apparent PNa is close to 1 x 10-8 cm sec-1. Although this sometimes increases on hyperpolarization, the effect is far less pronounced and not as consistent as the effect on the rate of rise of ana (compare Fig. 9A and B). Using the constant field equation A B C~~~~ E E~~~~ 2 S -- V ~~~~~~~~~~~~~~~~E ~~~~~~~~~~~~~0xM z -60~~~~~~ -4V I..~~~~ Potential (mv) Potential (mv) Fig. 9. The effects of membrane potential on Na influx. A, effect of membrane potential on rate of rise of Na produced by Na-K pump inhibition. In each experiment the fibre was superfused with a K-free, Rb-free solution and the maximum rate of rise of ana measured (ordinate). This is plotted (filled symbols) as a function of holding potential (abscissa). Different symbols represent different fibres. The open triangles show data from the same experiment as the (V). in which a'a was increased by exposure to 10-5 M- strophanthidin. B. effect of membrane potential on apparent PNa PNa is calculated from the constant field equation (see text). Symbols refer to same experiments as in A. above, one might note that a hyperpolarization from -10 to -60 mv would be expected to increase the rate of rise of aia by a factor of 21 if PNa remained unchanged. Clearly more experiments are required before we can be really certain whether or not a significant change of steady-state permeability occurs on depolarization. In some of the present cases the changes of Na influx can be most easily accounted for simply by a change in the passive electrochemical driving force for Na. The effects of membrane potential on the rate of rise of a'a upon inhibiting the

14 202 D. A. EISNER, W. J. LEDERER AND R. D. VAUGHAN-JONES Na-K pump are not entirely what would be predicted from the effects of tetrodotoxin (TTX) on the steady-state current-voltage relationship (Attwell, Cohen, Eisner, Ohba & Ojeda, 1979). TTX has a maximal effect on the current, and therefore presumably on the Na influx, at about -50 mv. The effect decreases to zero at more negative and positive potentials, suggesting that the TTX-sensitive Na influx has a biphasic potential dependence. This may be obscured in our experiments if (as suggested by Deitmer & Ellis, 1980) the TTX-sensitive Na influx is less than half the total Na influx. Nevertheless a more systematic investigation will require measuring Na influx over a more closely spaced potential range. In summary, the passive Na influx is decreased by depolarization and this effect can explain the fall of steady-state ana (Fig. 6) since the cell's Na extrusion mechanisms will be able to reset aka at a lower level. Furthermore, the observation that depolarization decreases aka even in the presence of strophanthidin supports the suggestion (Deitmer & Ellis, 1978a) that mechanisms other than the Na-K pump can sometimes assist the regulation of ana. DISCUSSION In this paper we have shown that, following an exposure to K-free, Rb-free solution, the rate constant of recovery of both aka and the electrogenic Na pump current transient increase in parallel when [Rb]0 is increased. The effects on aka are similar to those reported by Deitmer & Ellis (1978b) using external K. This is to be expected since Rb and K are very similar in their effects on the Na-K pump (Eisner et al. 1981). Using the area under the electrogenic Na pump current transient as a measure of the amount of Na extruded electrogenically, and the accompanying change of aia to estimate the total extrusion of Na, we have shown that the fraction of Na which is extruded electrogenically is essentially independent of [Rb]o. Taking this fraction as an index of the coupling coefficient of the Na-K pump (Eisner et al. 1981) implies that the coupling of Na to Rb movements remains constant with respect to changes of [Rb]o. This complements the results of the previous paper where it was concluded that the coupling ratio of the Na-K pump was independent of ana. A coupling ratio independent of external K has also been reported recently for the squid giant axon (Abercrombie & De Weer, 1978). In contrast other reports have suggested that the coupling in various excitable cells changes under these conditions (Den Hertog (1973), mammalian C fibres; Lieberman (1979), squid axon; Glitsch, Grabowski & Thielen (1978), guinea-pig atrium). The apparent discrepancy can be resolved since these latter reports all assumed that steady-state aka was unaffected by changing the external activator cation levels. We have, however, shown both theoretically and experimentally that in the Purkinje fibre this is not the case (see also Ellis, 1977). In other words, when the total electrogenic extrusion is increased by increasing [K]o or [Rb]o, the total change of aka also increases, so that their ratio remains constant. Perhaps the most direct demonstration of the dependence of the Na-K pump on [Rb]0 comes from experiments such as that shown in Fig. 2B. Here the electrogenic Na pump current transient is plotted as a function ofana. The slope of the relationship between current and aka is increased by increasing [Rb]o, showing that, at a given level of aka, the Na-K pump is activated by increasing [Rb]0.

15 Rb AND MEMBRANE POTENTIAL ON a2na 203 The apparent affinity of the Na-K pump for external Rb We have studied the apparent affinity of the Na-K pump for external Rb in two ways: (1) using kinetic measurements (either the rate constant of fall of aka or the slope of the relationship between electrogenic pump current and ana) and (2) measurements of steady-state ana. The kinetic measurements suggest a comparatively low affinity (Ko.5 ca. 4 mm). This value agrees reasonably well with that obtained by Eisner & Lederer (1980a) from an analysis of the rate constant of decay of the electrogenic Na pump current (Ko.5 ca. 6-3 mm). Furthermore, a low affinity for external K obtained from kinetic measurements of aka has been reported by Deitmer & Ellis (1978b; K0.5 ca mm). Deitmer & Ellis, however, obtained their value by measuring the maximum rate ofdecay ofaka as a function of [K]o used to re-activate the Na-K pump. This is not equivalent to the method used in this paper (cf. Eisner & Lederer, 1980a), where the rate constant of decay is measured. When higher [Rb]0 are used the absolute magnitude of the decay of aka increases as well as the rate constant (Fig. 1). Therefore the maximum rate of decay will increase by more than the rate constant, giving a spuriously high K0.5. An estimate based on analysis of rate constants gives a value of 2-6 mm (J. W. Deitmer & D. Ellis, personal communication). These affinities are in general lower than those found in most other tissues where values of K0.5 for external K in the order of t mm or less are reported (e.g. Glynn, 1956). More importantly we find that the dependence of the steady-state aka on [Rb]o suggests a high affinity of the pump for external Rb (Ko.5 < 1 mm). It is interesting that examination of the steady-state ak5 data of Deitmer & Ellis (1978b, their Fig. 2) also suggests a K0.5 of less than 1 mm. A qualitatively similar observation has been made in the snail neurone (Thomas, 1972) where resting aka remains constant when external K is changed over the range 1-8 mm. Ellis (1977) has examined aka in Purkinje fibres using a wider range of [K]o (up to 30 mm) but in these cases the depolarizations produced by the higher [K]o may have affected the resting ana (see below). At present we cannot account for the discrepancy between the K0.5 values obtained from the steady-state and kinetic measurements. One possibility is that, during Na-K pump re-activation, the concentration of Rb in the restricted extracellular spaces may be reduced. This extracellular depletion will become less severe as the Na-K pump rate declines with the fall of aika. In this case the relationship between [Rb]o and steady-state aia will give a more accurate measure of K0.5 than will the kinetic analysis. It is interesting to note that in the canine Purkinje fibre, measurements of the rate constant of decay of the electrogenic Na pump current transient give a K0.5 of about 1 mm (Gadsby, 1980). This could be explained if extracellular depletion is less than in the sheep Purkinje fibre. Problems with extracellular K-depletion have also been suggested to account partly for the high K0.5 for the ouabain-sensitive 42K uptake in the smooth muscle taenia coli (Widdicombe, 1977). Although evidence of such depletion in sheep Purkinje fibres has been presented (Eisner & Lederer, 1980a), it is not known whether this is significant enough to slow substantially the recovery of aka. If so, then the linear relationship between AlI (the electrogenic Na pump current) and aka is hard to explain. The slope of this relationship is sensitive to [Rb]0 (Fig. 2B) and should increase as the amount of depletion declines during the time

16 204 D. A. EISNER, W. J. LEDERER AND R. D. VAUGHAN-JONES course of a re-activation. In general no such bending of the Al versus aka relationship is seen. Other explanations for the difference between steady-state and kinetic estimates of K0.5 should also be considered. We have assumed that [Rb]o only affects aka via the Na-K pump. Our experiments were performed under voltage-clamp conditions to remove effects secondary to changes of membrane potential. It is, however, possible that external Rb or K could affect other Na fluxes not related to the Na-K pump. For example, a specific effect of Rb or K on the Na influx would affect the steady-state aia without any effect on the kinetic measurements (cf. eqns. (2) and (3)), providing a possible explanation for the discrepancy between the different estimates of K0.5. The effects of membrane potential on ana We have shown that deplolarization decreases the steady-state level of aka and that hyperpolarization increases ana. These effects are similar to those described in the snail neurone by Thomas (1972). Furthermore, in agreement with his results, we find that hyperpolarization increased the rate of rise of aka following Na pump inhibition. It seems likely that changing the membrane potential affects aka by altering the driving force for Na entry into the cell. The fact that aka is potential-sensitive means that care must be taken when interpreting the effects of changing external levels of Rb or K if these are accompanied by significant changes of membrane potential. For example (as pointed out by Ellis, 1977) the fall of a'a seen on increasing [K]0 from 10 up to mm (Ellis, 1977), could be due to the subsequent depolarization rather than an effect of [K]0 on the NE-K pump. In our experiments, when the membrane potential was kept constant, increasing [Rb]0 from 4 to 20 mm had little effect upon ak a (Fig. 4). It is finally worth noting the importance of the effects of membrane potential on aka in conventional voltage-clamp experiments in which the holding potential is changed. In view of the size of this effect on aka we suggest that, in any experiments where changes of aka could be important, the holding potential should be maintained constant. Alternatively, allowance must be made for possible changes of ana. We are grateful to D. Ellis and P. W. Flatman for comments on the manuscript. We thank D. Noble and A. J. Spindler for valuable discussion. The work was supported by a grant from the British Heart Foundation to D. Noble. This work was done during the tenure of a British-American research fellowship of the American Heart Association and the British Heart Foundation and part of this work has been supported by NIH grant HL (W. J. Lederer). R. D. Vaughan-Jones is an M.R.C. Senior Fellow. D. A. Eisner was an M.R.C. scholar. We thank R. A. Rixons Ltd, and especially Mr R. Shock, for supplying sheep hearts. REFERENCES ABERCROMBIE, R. F. & DE WEER, P. (1978). Electric current generated by squid giant axon sodium pump: external K and internal ADP effects. Am. J. Physiol. 235, C63-C68. ATTWELL, D., COHEN, I., EISNER, D., OHBA, M. & OJEDA, C. (1979). The steady-state TTX-sensitive ('window') sodium current in cardiac Purkinje fibres Pflugers Arch. 379, BAKER, P. F., BLAUSTEIN, M. P., KEYNES, R. D., MANIL, J., SHAW, T. I. & STEINHARDT, R. A. (1969). The ouabain sensitive fluxes of sodium and potassium in squid giant axons. J. Physiot. 200,

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