549

Journal of Phy8iology (1991), 434, pp. 549-560 With 7 fiture8 Printed in Great Britain

ROLE OF THE ANOMALOUS RECTIFIER IN DETERMINING MEMBRANE POTENTIALS OF MOUSE MUSCLE FIBRES AT LOW EXTRACELLULAR K+ BY J. SIEGENBEEK VAN HEUKELOM From the Cell Biophysics Workgroup, Department of Experimental Zoology, University of Amsterdam, Kruislaan 30, 1098 SM Amsterdam, The Netherlands

(Received 27 February 1990) SUMMARY

1. The membrane potential (Vm) of fibres of the extensor digitorum longus (EDL) of the mouse, measured at 35 °C and with extracellular potassium concentration (K+) 57 mM, was Vm =-76 mV. 2. Lowering K+ below 1 mm could lead to either a hyperpolarizing or a depolarizing response. When Vm was lower than - 75-5 mV in the control medium, a reduction of K+ to 0-76 mm led to a hyperpolarization of Vm (-95 0 + 07 mV, n = 40); otherwise a depolarization occurred (Vm =-47-2+ 1'1 mV, n = 21). 3. The difference in Vm responses did not correlate consistently with functional differences in cell types, as cells that originally hyperpolarized, could later depolarize. 4. The observed phenomena could be explained if the properties of the anomalous rectifier, AR (or inward-going rectifier), are considered to be similar to those observed in cardiac cells. 5. Apparently caesium acted as a competitive inhibitor; when the inhibition was strong enough the non-linear properties of the AR regeneratively amplified the depolarization to the full-blown depolarized state (Vm = -46'7 + 1-3 mV, n = 15). 6. Ouabain (10-4 M) reduced Vm (to -45 + 3 mV, n = 5) and reduced dramatically the selectivity of the cell membrane for potassium over sodium. These effects could be reversed readily by washing out the ouabain. 7. Adrenaline (2 /lM) added to the medium hyperpolarized Vm (AVm= -46 + 1-4 mV, n =9) and increased the changes induced by lowered K+ (from - 14-3 + 05 mV, n = 5 to - 180 + 08 mV, n = 9); the cells that originally depolarized when K+ was lowered could hyperpolarize after adrenaline addition. INTRODUCTION

The constituent of the potassium conductance gK that mainly determines the resting membrane potential (Vrest) in muscle cells (Standen & Stanfield, 1978; Kolb, 1990) and in heart cells in diastole (Carmeliet, Biermans, Callewaert & Vereecke, 1987; Carmeliet, 1989) is the anomalous rectifier (AR): 9K(AR)* The membrane channels which underlie the AR open when the membrane is hyperpolarized and the kinetics of this process can be approximated by a Boltzmann relationship in starfish MS 8308

550

J. SIEGENBEEK VAN HEUKELOM

egg cells (Hagiwara & Takahashi, 1974), heart cells (Carmeliet, 1989) and muscle cells (Standen & Stanfield, 1978). In addition to its voltage dependence, 9K(AR) also depends, according to a square-root relationship, on the extracellular potassium concentration (K+). This dependence on K+ and Vm allows the AR to exhibit a regenerative character when K+ is reduced. On reduction of K+ the membrane hyperpolarizes and consequently 9K(AR) increases. This increase in gK tends to enhance the hyperpolarization of Vm. At the same time, as K+ is decreased, YK(AR) should become smaller due to its dependence on K+. The exact balance between these two dependencies determines whether Vm will hyperpolarize or depolarize when K+ is decreased. When depolarization of Vm starts, it leads to a decrease in gK(AR) and consequently to a regenerative closure of the AR. This aspect of the regenerative switch-off of the AR and its physiological significance for heart cells is well established (Carmeliet, 1982; Carmeliet et al. 1987). In the sartorius muscle of the frog and the diaphragm muscle of the rat, however, K+ can be reduced to at least 0.1 mm (Hodgkin & Horowicz, 1959) or even to ('nominally') potassium-free media (Hinke, 1987; Kuba & Nohmi, 1987) without depolarization. Other skeletal muscle cells do show the expected dichotomy in recorded Vm values when K+ is reduced (M0lgaard, Stiirup-Johansen & Flatman, 1980; Seabrooke, Ward & White, 1988; this paper). The present study shows that the dichotomy can be measured in a single cell. Ouabain and adrenaline were used, because they influence the cellular K+ and Na+ homeostasis (Clausen & Everts, 1989). As caesium has been reported to block the AR (Isenberg, 1976), its influence at low K+ was also studied. A preliminary account of parts of this work has been given to the Physiological Society (Siegenbeek van Heukelom, 1990). METHODS

Preparation White Swiss mice (aged 6-18 weeks, weighing about 30 g, either sex) were killed by cervical dislocation. The extensor digitorum longus muscles (EDL) were isolated from the hindlegs and cleaned as much as possible of connective tissue, blood vessels and nerve fibres. One of the constituting muscle bundles was used for the experiments; care was taken not to touch the fibres during these procedures.

Measuring chamber and electronics The measuring chamber, made of Sylgard 184 (Dow Corning, MI, USA), had a volume of about 0-1 ml; the bathing fluid was continuously refreshed by perfusion (flow velocity 3 ml/min) at 35+1 °C. Solutions with K+ concentrations different from the control were made by equimolar replacement of NaCl by KCl or vice versa. The control solution contained (in mM): NaCl, 117-5; KCl, 5-7; NaHCO3, 25-0; NaH2PO4, 1-2; CaCl2, 2-5; MgSO4, 1-2; and glucose, 5-8; saturated with a humidified gas mixture composed of 95% O2 + 5 % C02; pH = 7-35-7*45. The preparation of the microelectrodes (filled with 3 M-KCl, tip resistance 20-80 MQ), and the use of electronics and recording apparatus are as described elsewhere (Albus, Bakker & Siegenbeek van Heukelom, 1983). In order to minimize the influences of electrode junction potentials, the membrane potential Vm was defined as the potential difference between the microelectrode in the cell and a microelectrode, fabricated in the same way (Zuidema, Dekker & Siegenbeek van Heukelom, 1985), that was placed outside the cell. All chemicals were analytically pure (Merck and Janssen Chimica); ouabain was bought from Merck (G-Strophanthin) and adrenaline (L-adrenaline, 99%) from Aldrich.

MEMBRANE POTENTIALS IN MUSCLE FIBRES AT LOW K+

551

Statistics The results are presented as mean + standard error of the mean (s.E.M.). In contrast to other reports (M0lgaard et al. 1980; Juel, 1986; Seabrooke et al. 1988), where averages of groups of shortlasting measurements are used, the present study compares the influence of the solution changes 0-76 mM-K+ 10 min

I

-60 mV

1 III

IV

V

VI

L

-80 mV I Fig. 1. After a continuous recording of Vm in a control solution (Vr"et =-76 mV) and in a low K+ medium (I, -86 mV; II, -46 mV) the electrode was retracted (III); the medium was set to control K+ and Vm was measured during short-lasting impalements (IV): Vrest =-734 + 1P7 mV, (n = 13). Then K+ was reduced to 0-76 mm (V) and hyperpolarized Vm (-88-3+2-3 mV, n = 16) as well as depolarized Vm (-51P0+0-8 mV, n = 23) values were found. After the return to the control solution (VI), Vrest became -74-0+1-1 mV (n = 26). Note that in the brief periods between the short-lasting impalements the electrode potential recorded outside the cells drifted slightly, a phenomenon that disappeared when the electrode was left at rest in the medium outside the cell for longer times. The dashed line corresponds to Vm = -75-5 mV. in one cell. Continuous measurement with the electrode in the same cell was carried out so that it is certain that the data come from a single cell. It allows the response of individual cells to be analysed. Figure 1 demonstrates that no significant differences in the data would be obtained, if the method of the authors mentioned above was used. Curves were fitted by a function consisting of a constant and one or two decaying exponential functions (see eqn (1)) to minimize the sum of the squared differences between the measured data and the function as well as the sum of the differences. In the figures, small switching artifacts, less than 4 mV and lasting less than 10 s, are not included. Data from Fig. 4 are tested with the sign test. RESULTS

Dependence of membrane potential on K+ Figures 1 and 2 illustrate that after a change is made in the medium composition from 5-7 to 0-76 mM-K+, Vm of the same cell can respond with a hyperpolarization followed by a depolarization. Long-term exposure to low K+ does not irreversibly change the cell behaviour, as the process of hyperpolarization followed by depolarization can be reset by exposure to the control solution. The response of the fibres is slow compared to those observed in cardiac preparations (Carmeliet, 1982; Carmeliet et al. 1987), perhaps because the fibres form an electronic cable or because

J. SIEGENBEEK VAN HEUKELOM

552

5.7 mM-K+

0 76 mM-K+

I

-60 mV -80 mV

Fig. 2. Reproducible hyperpolarization (-14 mV) as well as depolarization (+ 26 mV) in one cell with respect to control (Vr"et = -76 mV) due to repeated reduction of K+ from 5-7 to 0-76 mm. Dashed line corresponds to Vm = -75-5 mV.

-40 -50 -

I

I

-60 E

a

I

-70 -

E

-80 -

0-01

0.1

1

10

100

log Ko (mM)

Fig. 3. Averaged values of Vm as a function of K+ in the range 0-02 to 20 mM (semilogarithmically plotted). The depolarized Vm (*) values were averaged separately from the hyperpolarized Vm values (E). All data at one K+ value represent two or more (up to n = 114 at 5-7 mM) measurements. Error bars represent+ S.E.M.

the chloride conductance, being higher than the potassium conductance in muscle fibres (Hodgkin & Horowicz, 1959), delayed the response in the chloride-containing solutions used here. The diffusional delay of the solution switches was estimated to be 20 s. The cable properties also preclude a space clamping of the fibres, which is commonly done in studies on heart cells. The data illustrated in Fig. 3 show that at K+ < 1 mm, Vm can be hyperpolarized as well as depolarized with respect to Vrest. Frequently in one cell both possibilities occurred (cf. Fig. 2) but a depolarized Vm switching spontaneously back to a hyperpolarization was never observed (however, see the effects of adrenaline below). The way Vm responded to a protocol of continuous repeated switching from control

MEMBRANE POTENTIALS IN MUSCLE FIBRES AT LOW Ko+ 553 K+ to low K+ and back showed that after some time Vm in low K+ started to depolarize instead of hyperpolarizing, whereas Vrest only depolarized by less than 1 mV. This time was found to vary from 0 to more than 110 min, an observation that, together with the data shown in Figs 1 and 2, suggests that the bistable behaviour -20-

-75.5 mV

n=3

n= 18 X

0

-40-

X3

-60E E

-80 *

-100

-120

|

n=37

-90

-70

-80

n=3 -60

Vrest (mV) Fig. 4. When Vrest -75-5 mV Vm hyperpolarized at 0-76 mM-K' (with three exceptions); otherwise it depolarized (with three exceptions). Data (n = 61) are from forty-eight fibres of thirty-two preparations from twenty-nine animals.

is an intrinsic property of the cells being impaled. It is not very likely that spontaneous degradation of the cells caused the depolarization because the switch from hyperpolarization to depolarization could be reset and, in some experiments (n = 5), with the protocol of repeated switching to low KI, a depolarizing response to the switch was followed by a hyperpolarization. Whether low K+ would induce hyperpolarization or depolarization could be predicted from the initial resting Vm = Vrest as shown in Fig. 4: a distinct separation line was Vrest = -75-5 mV (sign test: P < 0 01). The average of all hyperpolarized Vm values was - 95 0 + 0 7 mV (n = 40) and the average depolarized Vm was -47 2+1-1 mV (n = 21). The influence of caesium Cs+ was exchanged equimolarly for all K+ in the low K+ situation: the results are shown in Fig. 5 and Table 1. Table 1 shows that Cs+-induced depolarization of Vm was 40 + 0 7 mV (n = 4) in the hyperpolarized and 3 0 + 0 4 mV (n = 9) in the depolarized situation. In these cases Cs+ behaves just like K+ but with lower apparent permeability. In fifteen cases the substitution of K+ by Cs+ in the hyperpolarized state led to an instantaneous depolarization (Fig. 5B). Thus the effectiveness of Cs+ appears to be dramatic in these cases, because as soon as Vm starts to depolarize and the AR starts to switch off, Vm fully depolarizes due to the intrinsic property of the AR to amplify regeneratively its own action.

554

J. SIEGENBEEK VAN HEUKELOM A 5.7

0.76 mM-Cs' 0O76mM-K+

B

0.76 mM-Cs+

mMLJ-'' 10 min 1 1%9 -

~

5-7 m

M

r-

10 min

r

S~

-60 mV -80 mV

-80 mV -100 mV

Fig. 5. After K+ was reduced from 5-7 to 076 mm and the cell hyperpolarized, Cs' replaced K+; two courses of Vm were observed: A, Cs' depolarizes the hyperpolarized Vm (due to low K+) by only 3 mV. B, Cs+ reversed Vm from hyperpolarized to depolarized; in the depolarized state the switch back to K+ hyperpolarized the membrane by 2 mV. TABLE 1. Effects of Cs+ and adrenaline on Vm A. Effect of Caesium VI(Cs+) n Vm(K+) AVm -973 + 30 -93.3+330 Hyperpolarized 4O0+±07 4 46-7+ 1-3 15 -478 + 1.5 -94-6+ 1-2 Switching response -507 +1-9 -47*7 +2-0 Depolarized 30+04 9 B. Effect of adrenaline on Vm in 5-7 mM-K+ (Vrest) n Vrest Vrest(Adr) ATVm -4-6 + 1-4 9 Adrenaline-induced -74-5 +0 9 -79-2 + 1-2 C. Effect of adrenaline on hyperpolarization of Vm in 0-76 mM-Ks+ &Vm n Vm(low K+) Vm(control Control (hyperpolarization) -75 9+110 -903 + 1-4 -14-3 + 0 5 5 Adrenaline -80.5 + 0 9 -98-5 + 1-6 -18-0+0-8 9 A, comparison of different actions of Cs+ substitution for low K+ (0-76 mM). The 'switching response' refers to the response of Vm to Cs+, switching from hyperpolarized to depolarized values with respect to Vm in 5-7 mM-K+. B, comparison of Vm = Vres,t with and without adrenaline (2 ,UM). C, comparison of Vm hyperpolarizations with and without adrenaline (2 /,M) induced by K+ changes (from 5-7 to 0-76 mM). All differential measurements (AVm) were obtained by comparing Vm data in the same recording.

Responses after conditioning of the cell A delayed equilibration of intracellular Cl- (ClI) retarding the response of Vm to low K+ could possibly be the reason that Vm started to depolarize. This possibility was investigated several times with the protocol of repeated medium switching in a fibre that depolarized in response to the switch to K+ = 076 mm. After switching back to control, the next switch to low K+ was preceded by a smaller, conditioning

MEMBRANE POTENTIALS IN MUSCLE FIBRES AT LOW K+

555

reduction of K+ to 1-9 mm which induced hyperpolarization. If delayed equilibration of ClT were the origin of the observed depolarization, the conditioning of the cell at K+ = 1-9 mm would allow ClI to equilibrate in two steps. Nevertheless, hyperpolarization did not occur when K+ = 0-76 mm even after the exposure times to K= 1-9 mm were as long as 25 min. 0 76 mm-K+ 8 55 mm-K+

-60 mV

m n

Ouabain

-

V-

-80 mVA

Fig. 6. Application of 1O-4 M-ouabain depolarizes Vm and diminishes the K+-induced potential changes. The interruptions in the recording represent short moments when the impalement failed. The two dashed lines connect the end values of Vm in 5-7 and 8-55 mMK+ respectively.

The influence of ouabain Application of ouabain (10-4 M) gives information about the interactions between the influences on Vm of the Na+-K+ pump and of the AR. These were assessed by continuously measuring Vm in one cell using K+ elevations to 8-55 mm (150 % of the control) instead of K+ reductions, to escape from the bistable behaviour of the AR. K+ was frequently switched from 5.7 mm to 8-55 mm and the change in Vm was taken as an indicator of the selectivity PK/PNa of the cell membrane. Figure 6 shows that the K+-induced potential changes decreased dramatically. They sometimes disappeared totally when ouabain was applied for longer periods than those shown in Fig. 6. At the end of the period of ouabain application the average change observed was 3-1 + 0-4 mV (n = 8). If the period of exposure to ouabain had been longer this value would have even been smaller. In contrast, the initial value was 9 3 + 05 mV (n = 8). This indicates that PK/pNa was severely reduced by ouabain application; a rough estimation (using the Goldman-Hodgkin-Katz equation applied to extracellular ion changes) shows that PK/pNa is reduced from 160 to 10. The most obvious conclusion from these recordings is that inhibition of the Na+-K+ pump is accompanied by a closure of the AR. On washing out ouabain, the selectivity returned. These experiments were severely hampered by the fact that the electrode rarely remained in the cell, possibly because of cell swelling. The time course of Vm during this application, in terms of exponentials, was analysed by drawing two exponential curves: one to connect the end values in

J. SIEGENBEEK VAN HEUKELO,!"

556

5-7 mM-K+ and another to connect the end values in 8-55 mM-K+ (see dashed lines in Fig. 6). Inspection of all recordings suggested that the curve for 5-7 mm fitted the equation: (1) Vm = Vm le-t/Tl + Vm 2 e-t/2 + constant. Frequently T2 was so large that the second part could be fitted with a straight line. Within 1-7+0-2 min (Tj; n = 8), a change of Vm with +7-4±+09 mV (Vm 1; n = 11) was found. The first 8-55 mM-Ks challenge after ouabain application still evoked a considerable depolarization: 67 +4% of the depolarization before ouabain (n = 8). The final value of Vm was -45+3 mV (n = 5). 0.76 mM-K+

5.7 mM-K+ 8.55

mM-K+ 10 min Adrenaline

-60 mV

-80 mV

Fig. 7. Recording in one cell showing that adrenaline (2 uM) hyperpolarizes V. in control conditions (here -6 mV) and increases the changes in Vm induced by low K+ (4 mV).

The influence of adrenaline Figure 7 shows a representative recording illustrating that Vrest hyperpolarized in the presence of 2 /sM-adrenaline and that the low K+-induced change was enhanced. Adrenaline addition could hyperpolarize a cell which before and after the addition was depolarized. Added shortly before the low K+ challenged was given, adrenaline could invert the depolarizing course of Vm into a hyperpolarizing one. Parts B and C of Table 1 compile all averaged results. DISCUSSION

The presence of the AR in the membrane of skeletal muscle fibres has been generally established with experiments using voltage clamp protocols (Adrian, 1972; Standen & Stanfield, 1978). The present paper demonstrates that in the EDL of the

MEMBRANE POTENTIALS IN MUSCLE FIBRES AT LOW K+ 557 mouse two stable Vm values in low K+ can be measured, that are similar to those observed in heart cells. Due to their cable-like structure the muscle fibres do not allow a whole-cell voltage clamp ('space-clamp'). Recently Sims & Dixon (1989) also reported a similar bistable Vm in osteoclasts due to the characteristics of the AR. Voltage clamp studies (most of them at K+ > 2 mm) show that 7K(AR) depends on Vm, whereas in unclamped cells Vm depends on the conductances. Therefore the use of the dependencies found with voltage clamp studies leads to circular equations; nevertheless they are used to explain the effects of low K+ on Vm qualitatively. Several quantitative analyses of 9K(AR) are available (Ciani, Krasne, Miyazaki & Hagiwara, 1978; Hille & Schwarz, 1978; Standen & Stanfield, 1978). The equation presented by Hagiwara's group (Hagiwara & Takahashi, 1974; Ciani et al. 1978) explicitly separates the dependence of 9K on K+ from that on Vm and is, therefore, used here to explain at the cellular level why regenerative depolarization occurs. The arguments can be simplified in the first approximation by assuming that at vrest chloride is equilibrated across the membrane (i.e. Ic, = 0), that 9Na is about 1 % of 9K and that the electrogenic influence of the Na+-K+ pump can be incorporated in the apparent selectivity of K+ over Na+ (Thomas, 1972). Application of Kirchhoff's current law gives, in first-order approximation, for steady-state Vm:

IK = -INa = gK(V.-EK)

YNa(ENa Vm), where IK and INa' gK and gNa' EK and ENa have their usual meanings. The produced by Hagiwara's groups (Ciani et al. 1978: eqn (37)), =

(2)

equation

B a/K+ YK(AR) =1 ±e(VhIv'

can be inserted to give:

B(Vm-EK)V/K+ 1

+e(AVm-AVh)/v =

9(

NaVm)

(3)

The value B is a calibration constant and contains the maximally obtainable 9K: K. The entities AVm and AVh represent membrane potentials with respect to EK: (Vm-EK) = AVm and (Vh-EK) = AVh. Vh is the value at which the voltage-dependent part of 9K is half-maximal and appears to be a constant for all K+ values (Hagiwara & Takahashi, 1974). The value v determines the steepness of the dependence Of 9K(AR) on Vm. In cardiac tissue and skeletal muscle (Hodgkin & Horowicz, 1959; Carmeliet et al. 1987; Standen & Stanfield, 1978), the opening occurs at more depolarized potentials with respect to EK. Equation (3) can be used to show that when K+ is decreased during the experiments Vm-EK must increase. As gNa is the background conductance of the membrane for sodium and (ENa - Vm) is large compared to the variations in Vm, gNa (ENa - Vm) may be approximated in the first order as a constant. When \/K+ is decreased, the other part of the left-hand side of eqn (3) should increase. This implies that Vm -EK must increase also and consequently also the denominator. As long as the differences are small, the sequence leads to a converging solution and a new steady state can be found. Apparently when K+ is decreased below 1 mm, and

558

J. SIEGENBEEK VAN HEUKELOM

originally Vrest > -75-5 mV, a new stable steady state is not found in the direction where Vm and EK both hyperpolarize. The influence of Cs+ can best be thought to be included in B, which contains SK. Cs' is known to be an impermeant blocking particle in single-file manner (cf. Hille & Schwarz, 1978) and this might account for the slightly reduced effect of Cs' at the depolarized potentials. However, an explanation of how this is effected kinetically is beyond the scope of this paper. The response time of 9K(AR) to electrical signals itself is fast enough to be considered instantaneous in this study. The results also show that delayed equilibration of ClT to a new Vm does not seriously influence the electrical responses of the fibre. The 33 % reduction of the K+-induced change in Vm shortly after ouabain application compares well with the reduction of the 'apparent PK.' to a fraction of 2/3 which is expected from the rheogenic contribution of the pump (Thomas, 1972; Zeuthen, 1981). The initial fast reduction in Vm after ouabain addition was not found by Seabrooke et al. (1988) in mouse EDL, probably because they did not measure continuously in one cell. The averaging of the data most probably obscured the rather fast reduction of Vm that is normally accepted as evidence for a rheogenic contribution of the pump. However, the Vm values for longer times after ouabain are comparable ( -50 mV). The authors concluded that the increase in PNa is the explanation for the loss of K+-Na+ selectivity and the depolarization, but this increase alone should be considerable if it is to explain why the K+-Na+ selectivity is totally lost. In addition, it is difficult to conceive how the rapid recovery of Vm might occur, after the potassium channels as well as the sodium channels were open for a considerable time, allowing an equilibration of the cytosol with the surrounding medium. Chemical equilibration of potassium and sodium is unlikely to be the sole origin, as washing out the ouabain restored Vm and the K+-Na+ selectivity within 30 min. Closure of the AR in the depolarized state has been demonstrated in Purkinje fibres (Carmeliet et al. 1987). The influence of adrenaline on Vm is a common observation and might occur either via the intracellular messenger cyclic AMP (Delbono & Kotsias, 1988), the calciumsensitive potassium conductance (IK(ca); Williams & Barnes, 1989), or an independent potassium channel similar to the one described by Lucero & Pappone (1990) for noradrenaline. The Na+-K+ pump creates the gradients for potassium and sodium on which the passive fluxes through the membrane drain. So the K+ gradient itself leads to a polarizing current through the AR and forms a buffer between both processes. If this current is needed to keep the AR open, it might be the reason why stimulation of the Na+-K+ pump by adrenaline enables cells to hyperpolarize further than normal. It can also explain why a hyperpolarization can switch over into a depolarization after a considerable delay (see Fig. 2) and why ouabain leads to the closure of the AR. The power delivered by the Na+-K+ pump during the experiments will also vary; due to its dependence on K+ (Soltoff & Mandel, 1984; Sejersted, 1988), and several of the cited kinetic values for K+ fall in the range where the present experiments were carried out. The possibility cannot be excluded that there is a more direct interaction between the AR and Na+-K+ pump, either by competition for common molecules (substrates or co-factors) such as Mg2+, Ca2+, or cyclic AMP, or within the membrane itself.

MEMBRANE POTENTIALS IN MUSCLE FIBRES AT LOW K+ 559 The results show that the observed variability in Vm does not necessarily stem from a clear-cut dichotomy in cell types, as suggested by M0lgaard et al. (1980). If more cell groups, each with their own characteristic properties, constitute the EDL (Griep, Pool, Lammeree, Wallinga-de Jonge, Seeder & Donselaar, 1980) dichotomy is not necessarily observed and the groups that easily remain hyperpolarized, like fibres in frog sartorius and rat diaphragm, are very small in the EDL. Variance in the presence of the AR has also been found in cardiac tissue (see review by Carmeliet et al. 1987). I thank A. Breed, R. Waldeck, G. Piras, G. Faas, H. Spelbrink, M. v. d. Bergh and S. v. Mechelen for their technical assistance and Drs W. J. Wadman and B. L. Roberts for critically reading the manuscript. This work was partly supported by the Netherlands Foundation for Biophysics with financial aid of the Netherlands Organization for the Advancement of Science

(NWO). REFERENCES

ADRIAN, R. H. (1972). Rectification in muscle membrane. Progress in Biophysics and Molecular Biology 19, 341-369. ALBUS, H., BAKKER, R. &-;SIEGENBEEK VAN HEUKELOM, J. (1983). Circuit analysis of membrane potential changes due to electrogenic sodium-dependent sugar transport in goldfish intestinal epithelium. Pflugers Archiv 398, 1-9. CARMELIET, E. (1982). Induction and removal of inward-going rectification in sheep cardiac purkinje fibres. Journal of Physiology 327, 285-308. CARMELIET, E. (1989). K+ channels in cardiac cells: mechanisms of activation, inactivation, rectification and K+ sensitivity. Pflugers Archiv 414, suppl 1, S88-92. CARMELIET, E., BIERMANS, G., CALLEWAERT, G. & VEREECKE, J. (1987). Potassium currents in cardiac cells. Experientia 43, 1175-1184. CIANI, S., KRASNE, S., MIYAZAKI, S & HAGIWARA, S. (1978). A model for anomalous rectification: electrochemical-potential-dependent gating of membrane channels. Journal of Membrane Biology 44, 103-134. CLAUSEN, T. & EVERTS, M. E. (1989). Regulation of the Na,K-pump in skeletal muscle. Kidney International 35, 1-13. DELBONO, 0. & KOTSIAS, B. A. (1988). Hyperpolarizing effect of aminophylline, theophylline, and cAMP in rat diaphragm fibers. Journal of Applied Physiology 64, 1893-1899. GRIEP, P. A. M., POOL, C. W., LAMMERE]E, G. C., WALLINGA-DE JONGE, W., SEEDER, T. & DONSELAAR, Y. C. (1980). Intramuscular and epimuscular microstimulation of single motor units. Neuroscience Letters 17, 191-196. HAGIWARA, S. & TAKAHASHI, K. (1974). The anomalous rectification and cation selectivity of the membrane of a starfish egg cell. Journal of Membrane Biology 18, 61-80. HILLE, B. & SCHWARZ, W. (1978). Potassium channels as multi-ion single-file pores. Journal of General Physiology 72, 409-442. HINKE, J. A. M. (1987). Transmembrane K+, Na+ and Cl- permeability and conductance changes in the frog sartorius fibres following ouabain and zero [K+]o treatment. Canadian Journal of Physiology and Pharmacology 65, 949-953. HODGKIN, A. L. & HOROWICZ, P. (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. Journal of Physiology 318, 127-160. ISENBERG, G. (1976). Cardiac Purkinje fibers: cesium as a tool to block inward rectifying potassium currents. Pflugers Archiv 365, 99-106. JUEL, C. (1986). Potassium and sodium shifts during in vitro isometric muscle contractions, and the time course of the ion-gradient recovery. Pfluigers Archiv 406, 458-463. KOLB, H.-A. (1990). Potassium channels in excitable and non-excitable cells. Reviews of Physiology, Biochemistry and Pharmacology 115, 51-91.

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