Potassium currents in the frog node of Ranvier

Proo. Biophys. molec. Biol. 1983. Vol. 42, pp. 1 20, 1983. Printed in Great Britain. All rights reserved.

0079 6107/83 $0.00+50 Copyright ~" 1983 Pergamon Press Ltd.

POTASSIUM CURRENTS IN THE FROG NODE OF RANVIER J. M. DUBOIS Laboratoire de Neurobiologie, Ecole Normale Supkrieure, 46 rue d" Ulm, 75005 Paris, France*

CONTENTS 1. INTRODUCTION

1

II. HODGKIN-HUXLEY-FRANKENHAEUSER MODEL

1

III. DELAYED ACTIVATION OF 1K

3

lV. BLOCKING AGENTS

3

1. Tetraethylammonium (TEA) 2. Cesium 3. 4-Aminopyridine (4-AP)

3 3 3

V. CHANGE IN K ELECTROCHEMICAL GRADIENT DURING K FLUXES

4

VI. INSTANTANEOUS CURRENT VOLTAGE RELATIONSHIPS

5

VII. FAST AND SLOW CURRENTS

6

VIII. TWO FAST CURRENTS

10

IX. MOTOR AND SENSORY FIBRES

12

X. CONCLUSION

12

XI. K CURRENTS IN OTHER PREPARATIONS

1. 2. 3. 4.

13

Mammalian Node o f Rant,let Squid Giant Axon Neurones Skeletal Muscle

13 14

15 16

Xll. CONCLUDING REMARKS ACKNOWLEDGEMENTS REFERENCES

16 17 17

I. INTRODUCTION Electrophysiological studies of cell membrane permeabilities have revealed the existence of a large variety of potassium currents whose properties and physiological roles are different. Until the last years, in contrast with muscles and neurones, the voltage dependent potassium permeability of the frog myelinated nerve fibres was considered to be governed by only one type of channel. However, recent studies on this preparation have revealed the existence of different and independent potassium currents likely flowing through different types of potassium channel (Ilyin et al., 1980; Dubois, 1981b, 1982b). The aim of this review is to summarise the different results in the literature concerning the potassium currents of frog node of Ranvier and to compare the properties of the nodal K channels to those of K channels in other preparations. II. H O D G K I N - H U X L E Y - F R A N K E N H A E U S E R

MODEL

Twenty years ago, Frankenhaeuser (1962a,b,c, 1963) described the change in K permeability of the node of Ranvier on the basis of the Hodgkin-Huxley model (1952). Under * Present address: JPB 42: I-A

L a b o r a t o i r e de P h y s i o l o g i e C o m p a r ~ e , B~.t 443 Universit~ Paris XI, 91405-Orsay Cedex 1

2

J . M . I)uBoJs

voltage clamp conditions, the delay outward current recorded during depolarizations in normal K solutions was interpreted as being mainly transported by K ions through specific pathways (later referred to as the K channels) whose opening and closing were controlled by time and voltage dependent gates. The change in K permeability was described by the eqn : P~=PK

(11

n" /~

where P~ is the K permeability, F'K the maximum K permeability: n, the activation variable (function of time and voltage): a. the number of subunits (later referred to as the gating particles) controlling the opening of one channel and k, the inactivation variable (function of time and voltage). Various values for the exponent a have been proposed, including 2 lFrankenhaeuser, 1963) and 4 (Dodge, 1963; Koppenh6fer, 1967: Hille, 1968). A value greater than unity is necessary to take account of the sigmoid activation of the K current during a step depolarization. During long lasting depolarizations, the activated K current decreases. This has been attributed to a slow inactivation (variable k). During a depolarization, k was found to decrease exponentially with time (Frankenhaeuser, 1963) or to be the sum of two exponential time declining components (k t and k2) (Schwarz and Vogel, 1971; Lewis, 1971 ). In this preparation, the fully activated current voltage relationship has not been considered to be linear in normal K solutions but curved upward at high positive voltages. The existence of an instantaneous rectification, relatively well fit by the Goldman-HodgkinKatz (Goldman, 1943; Hodgkin and Katz, 1949)constant field equation, has been assumed (Frankenhaeuser, 1962c). Upon repolarization in both normal and high K solutions, an inward tail current appears (Fig. 1 ). In high K solutions, this tail current was assumed to be mainly transported by K ions 2 5 nnO01K

10nA I

1onto'=

117rnM K

~ 40

1•

•. 40

r

I...........

J !

FI~;. 1. Traces of K current recorded in normal K-Ringer (left) and in isotonic K-Ringer (right) during and after 300 msec depolarizations to - 40 and + 40 mV. Na current was blocked by l r x (10 " ,4 ~. Linear capacity and leakage currents were compensated for automatically. Note the tail inward currents upon repolarization in both media. Holding potential: 90 inV. lemperature: 12 ('.

since at negative membrane potentials, the electrochemical gradient for K ions is inwardly directed. Consequently, repolarization after activation of the K permeability in high K solution induces an inward tail of K current whose time course reflects the shutting offofthe K permeability. In contrast, in normal K solutions the equilibrium potential for K ions, calculated on the basis of the Nernst equation, is - 9 7 mV at 20 C. Consequently repolarization to the normal resting potential ( - 70 mV) or to - 9 0 mV (Fig. 1 ) must induce an outward tail of K current. Based on this reasoning, the observed inward tail current in normal K solutions was denoted "Iv" by Frankenhaeuser and was assumed to be mainly carried by ions other than K. Moreover, it was observed (Frankenhaeuser, 1962b) that the inward tail current was reduced by the replacement of all the external NaCI by sucrose. This observation led Frankenhaeuser to conclude that Ip was mainly carried by Na. Based upon pharmacological and kinetical analyses of l Kand I,,, a different interpretation was given later tsee Section V and VI) according to which Iv was carried by K ions previously accumulated in a restricted perinodal space.

Potassium currents in the frog node of ranvier

3

II1. D E L A Y E D A C T I V A T I O N OFIK During a step depolarization, the activation of IK is sigmoid. In the frame of the HodgkinHuxley model, this property is related to the value of the exponent a in eqn (1) (different than 1) and indicates that a gating particles must be displaced to open one channel. However, when the depolarization is preceded by an hyperpolarization, the "delay" in activation oflK is increased and the exponent a must be increased. This effect, first observed for the squid axon by Cole and Moore (1960), is probably incompatible with the Hodgkin-Huxley model since it seems difficult to admit that the number of particles controlling the opening of one channel is modulated by changes in membrane potential. Whatever the origin of this effect, it indicates that either the Hodgkin-Huxley model fails to describe certain properties of K channels or that some parameters are uncontrolled in the experimental conditions. The Cole-Moore effect has been studied in myelinated nerve fibre (Palti et al., 1976; Ganot et al., 1978; Begenisich, 1979; Ilyin et al., 1980; Dubois, 1981a) both in normal K solutions and in high K solutions. The conclusion of these studies is that the opening of the channels may follow a multistep transition or (and) (a) there is more than one kind of K channel and (b) some events external to the membrane contribute to alter the time course of the current (Ganot et al., 1978). IV. B L O C K I N G A G E N T S Several agents have been found to block I~ in myelinated nerve fibre and in numerous other cells. Among them, three are particularly interesting since their effects are almost exclusively limited to I K and they are commonly used in most of the cells to characterize or eliminate a K current. These agents are tetraethylammonium, cesium and 4-aminopyridine. 1. Tetraethylammonium ( T E A ) This agent blocks I K in the node of Ranvier when applied from outside or from inside (Schmidt and St/impfli, 1966; Koppenh6fer, 1967; Hille, 1967; Koppenh6fer and Vogel, 1969; Mozhaeva and Naumov, 1972). In internal application by diffusion from the cut ends of the fibre (Koppenh6fer and Vogel, 1969) the blockade is enhanced by depolarizations which suggests that the receptor sites lie within the membrane and are only accessible to TEA molecules when the channels are open (Armstrong and Hille, 1972). In external application, TEA blocks the K current with a dissociation constant of 0.4-0.5 m g and the block is independent of voltage (Hille, 1967; Mozhaeva and Naumov, 1972). 2. Cesium Cesium ions (Cs) block I K both from the outside and inside. The block is voltagedependent and likely K concentration-dependent so that inward K current is increasingly blocked by external Cs as the voltage is made increasingly negative and the outward I K is increasingly blocked by internal Cs as the voltage is made increasingly positive (Dubois and Bergman, 1975c, 1977). The same result is found with internal Na (Bergman, 1970) and internal barium (Woll, 1982). It is assumed that the blocking ions bind to a site which is located at about a third of the electrical distance from the inner side of the membrane (Dubois and Bergman, 1977; Woll, 1982). Outward K current is not blocked by external Cs but on the contrary is slightly increased when Cs is added to a K free external solution (Dubois and Bergman, 1977). Moreover, inward K current is not blocked by internal Cs (Dubois and Bergman, 1975c). It is assumed that before reaching the blocking site, external Cs can take the place of K on a site controlling the gating of K channels (Dubois and Bergman, 1977). To explain the relief of block which is found by increasing [K]o, it is assumed that the blocking ions are knocked off their binding site (located in the K channel) by K ions coming from the other side of the membrane (Hille and Schwarz, 1978). 3.4-Aminopyridine (4-AP) 4-AP (and 3 -4 diaminopyridine) block I K and the effects depend on membrane potential

4

J . M . DUBOlS

(Ulbricht and Wagner, 1976: Ulbricht et at., 1982). The block is removed during depolarization so that strongly depolarizing pulses reveal an apparent slow activation of l K which gradually increases during a train of pulses. These observations suggest a relief from block during depolarization and a very slow restoration of block at the resting potential (Ulbricht and Wagner, 1976). It is concluded that the receptor for 4-AP lies within the membrane and that its affinity depends on the membrane potential so that a fraction of potassium channels becomes unblocked during long depolarizing pulses. In contrast to internal TEA, the opening of the K channel gate would not be prerequisite for 4-A P to bind to its receptor and following membrane depolarization, the positively charged molecule would tend to leave the channel according to the tramsmembrane fields (Pichon et al., 1982). V. C H A N G E

IN K ELECTROCHEMICAL DURING

GRADIENT

K FLUXES

Upon repolarization to the normal resting potential, there appears an inward tail current (Iv) which according to Franhenhaeuser (1962b) is transported by Na ions. However, Koppenh6fer (1967) and Koppenh6fer and Vogel (1969) showed that external TEA suppressed both the delayed K current and I v. Armstrong and Hille (1972) observed similar effects with other quaternary ammoniums applied internally. They suggested, at variance with Frankenhaeuser, that the inward I v could result from a change in the potassium equilibrium potential due to an accumulation of K ions in a perinodal space. Dubois and Bergman (1975a) have shown that, whereas neither tetrodotoxin nor replacement of external Na by Tris affected Iv, all the agents tested which affected the delayed K current altered Iv in the same way. Moreover, it was shown that, following depolarizations of various durations, the initial value of I v and its reversal potential changed simultaneously with the activation of the delayed outward current (Fig. 2). From these observations, it was concluded that Ip was carried by K ions accumulated in a perinodal space during the depolarizing pulse. Based on the three-compartment model proposed by Frankenhaeuser and Hodgkin (1956) to take account of the K accumulation observed in the squid axon a perinodal space thickness of 2900 ,~ and a K permeability for the barrier between the space and the bulk solution of 0.019 cm/sec were calculated for a specific node studied. Based on several determinations and using

,oo.[

If

,

- ~"

f

- ~

#

C ~

r-

i 5 msec I I

IK (r,A)

1

2

3

4

5

6

t (rnsec)

5

F](;. 2. K outward current and tail inward current recorded in normal K-Ringer plus T1"X at room temperature. Above: outward currents associated with depolarizing pulses of different duration to + 7 0 mV and inward tail currents recorded at - 1 2 0 mV. Holding potential: - 7 0 mV. Below: outward current (filled circles) at the end of depolarizing pulses to + 70 mV and initial value of inward tail current (open circles ) measu red at - 120 mV plotted against duration of depolarizing pulse (From Dubois and Bergman, 1975a).

Potassium currents in the frog node of ranvier

5

the three-compartment model, Moran et al. (1980) have calculated an apparent space thickness of 5900_+ 700 A and a permeability of the barrier of 0.015 _+0.001 cm/sec. As an alternative explanation for K accumulation, Moran et al. (1980) have shown that a model of restricted ionic diffusion in an unstirred aqueous layer adjacent to the axolemma was also consistent with the experimental data provided that the diffusion coefficient was 1.8 +0.2 × 10 -6 cm2/sec (i.e. 1/10 that of K ions in water) and the unstirred layer thickness was 1.4 -t- 0.1 /~m. While K accumulation does not seem questionable from electrophysiological studies (Dubois and Bergman, 1975a; Moran et al., 1980; Bergman et al., 1980; Dubois, 1981a; De Bruin, 1982), it requires further elucidations, including the anatomical substrate of the postulated diffusion barrier (see Ulbricht, 1981; Berthold and Rydmack, 1983). Whereas K accumulation, deduced from the occurrence of inward tail current at the normal resting potential, seems to be almost always present in nodes of Ranvier from Xenopus laevis, Rana esculenta and Rana ridibunda, it occurs little if at all in the node of Ranvier from northern Rana pipiens (Armstrong and Hille, 1972; Begenisich, 1979). Moreover, K accumulation is faster and more pronounced in sensory than in motor fibres from Rana esculenta (Palti et al., 1980). These observations suggest differences in the ultrastructure of the perinodal space of fibres from different species and of motor and sensory fibres from the same species. From this point of view, it is interesting to note that in spinal root nerve fibres of cat, the total nodal membrane area was found to be about 25~o larger in motor than in sensory fibres of equal calibre (Rydmark and Berthold, 1983). In normal K solutions (2.5 mM) the K concentration outside the membrane, calculated from the reversal potential of the instantaneous K current (see next paragraph) increases significantly during depolarizations. It may reach 20 30 mM (Dubois and Bergman, 1975a; Palti et al., 1980; Dubois, 1981a; De Bruin, 1982) and in some fibres can even become larger than the internal K concentration (assumed to be constant) (Dubois, 1981a). In isotonic K solutions, the K accumulation is minimized. However, large and long lasting K currents can still induce significant K accumulation (when the current is outward) or depletion of K ions from the perinodal space (when the current is inward) (Dubois, 198 la). Whereas only 4~,, of fibres (from Rana esculenta) do not exhibit significant K accumulation in normal external K solution (De Bruin, 1982), EK does not significantly change during depolarizations of various amplitudes for 40~,, of the fibres in isotonic K solutions (Dubois, 1981a). VI. I N S T A N T A N E O U S C U R R E N T - V O L T A G E R E L A T I O N S H I P S According to Frankenhaeuser (1982b), instantaneous K current does not increase linearly with voltage in normal K solution but exhibits a marked outward rectification which can be described by the constant field equation (Goldman, 1943). However, it must be noted that in the Frankenhaeuser's analysis, the inward tail current was considered to be carried by other ions than potassium. Consequently, the instantaneous K current was calculated from extrapolation to time of repolarization with the assumption that EK was constant and the K current in normal Ringer's solution changed with the same time course as it does in high K Ringer's solution. Assuming that after blockade of both the inactivating and non inactivating (Dubois and Bergman, 1975b) Na currents, the inward tail current "Ip" was essentially carried by K ions, roughly linear instantaneous 1 V curves were found in normal Ringer's solution (Fig. 3) (Dubois and Bergman, 1977; Palti et al., 1980; De Bruin, 1982) and in external solutions of various K concentrations (Dubois and Bergman, 1977; Attwell et al., 1980). Consequently, the conductance (gK), calculated from the K current measured at a given time and the actual value of E K (instantaneous reversal potential of the K current) can be used as a convenient expression to describe the properties of the K system. After blockade of Na currents and subtraction of linear leakage current, the steady state reversal potential of the remaining current recorded in external solutions of various K concentrations follows the Nernst prediction (Dubois and Bergman, 1975b). This indicates that, under these conditions, the delayed current is essentially carried by K ions. It must be noted that while the reversal potential of the steady state current recorded during

6

J.M. Dusols

5/j50 30

A ¢~

~20 E o

©

60 vp

(my) 6o

-I0

l

-20

-50

40 E >"

f

2O 0

i

I

0

IO

I

~

I

30

i 4O

tpp

i 5O

-20

(ms)

FIG. 3. Instantaneous I-V relationships in normal K-Ringer for different depolarizing prepulse durations (tpp). The points correspond to amplitude of zero-time-tail currents, Io, elicited when the membrane potential was stepped froma prepulse, Vpp of 100mV to various test pulses, Vp. Valuesof prepulse duration tpp (in msec) are given adjacent to each line. Inset: VKshift as a function of tpp. Symbols denote zero-cross over points of the instantaneous I V relationships. Na current was blocked by TTX. Potentials are given relative to the resting potential. Temperature: 15C (From Moran et al., 1980. Reproduced from the Biophysical Journal, 1980, 32, 939 954 by copyright permission of the Biophysical Society).

depolarizations of various amplitudes (steady state reversal potential) follows the Nernst prediction calculated from the K concentration in the external solution, the reversal potential of the instantaneous current recorded after a depolarization (instantaneous reversal potential) does not follow the Nernst prediction calculated from the K concentration in the external solution. This is simply due to the fact that during depolarizations near E K, I K is small or nil and does not induce a significant K accumulation. On the contrary, the instantaneous current is recorded after a depolarization which, in low K solutions, induces a marked accumulation and shifts E K toward positive values. As a consequence of changes in EK during depolarizations in low K media, quantitative studies of the properties of the K system cannot be directly undertaken from K current analyses but require corrections for changes in driving force (Palti et al., 1980; Dubois, 1981a; De Bruin, 1982) or should be made in experimental conditions (K rich solutions) where E K remains constant (Mozhaeva and Naumov, 1972; Ilyin et al., 1980; Dubois, 198 la,b, 1982b). For this reason, most of the recent results presented in the next paragraphs were first obtained in K rich media and then confirmed in normal Ringer's solutions. VII. FAST A N D S L O W C U R R E N T S The existence of several types of K channels (and thus of several distinct K currents) in the frog node of Ranvier was proposed by several authors on the basis of voltage clamp experiments (Schwarz and Vogel, 1971; Palti et al., 1976; Van den Berg et al., 1977" llyin et

Potassium currents in the frog node of ranvier

7

al., 1980; Krylov and Makovsky, 1978; Palti et al., 1980). Schwarz and Vogel (1971) showed that the steady state inactivation of I K remained incomplete even with large depolarizing pulses. Moreover, they showed that the inactivation of I Kdeveloped in two phases (fast and slow inactivations). On the basis of K current noise analyses, Van den Berg et al. (1977) showed that both the affinities for tetraethylammonium and the voltage-dependent properties of K channels were different after depolarization lasting either 400 msec or 60 sec. These observations suggest that I Xis composed of more than one component. However, as a general rule, the occurrence of several current components can be accounted for either by the existence of several types of channels or by a multi-step transition between the closed and open states of only one type of channel. In the frame of the first hypothesis (several types of channels), the different current components should be independent and it should be possible to alter the properties of one component without affecting the other ones. On the contrary, the second hypothesis (multiple states of one type of channel) implies that both components are coupled. Thus, alteration of one component should affect the other ones. Ilyin et al. (1980) showed that in isotonic K Ringer's solution, the tail of K current upon repolarization after conditioning depolarizations of various amplitudes and durations, can be decomposed into two exponential functions of time (IKIo r lKfas t and IK. or IKslo,~) (Fig. 4). Figure 4 presents I X tails recorded at E 2 = - 100 mV after conditioning depolarizations (El) of various amplitudes lasting 20 msec (a) and 500 msec (b). It can be seen that with increasing duration of El, Ix, (fast component) inactivates whereas IKn (slow component) does not (see also Dubois, 198 lb). From quantitative analyses of both IK~and IK~~under different experimental • --

(a)

(b)

IK

51 .......

E R -

=::='-c=

Eo

Pff~IKI -2.0I.-~

!

__

E2 IKI

-2.0 ,

I -1.0

-1.0

o

0

5

10 15 20

0

I

5 10 15 20

Time(sec) FIG. 4. Evidence for fast and slow K currents. Above: tail currents recorded in 128 mM K-Ringer plus 3.10 -4 mM TTX at E 2 = - 100 mV after a prepulse E t of 20 msec (a) and 500 msec (b) and various amplitudes ( - 6 0 , - 4 2 , - 19 and + 6 5 mV). Leakage current subtracted. Calibration current bar: 5. 10 9A. Below : semilogarithmic representation of the IK tails above and decomposition of the currents in two components IK~ and IK,. Note that the sole effect of a long conditioning prepulse consists of a reduction of the magnitude of the fast component IK~.Temperature: 15 18"C (from llyin et al., 19801.

8

J . M . Du~ols

conditions, Ilyin et al. (1980) concluded that the results could not be accounted for by a multistate model but were in favor of the existence of two different K channels corresponding to fast (I) and slow (ll) tail currents. More decisive arguments in favor of the existence of fast and slow K channels were provided by experiments in which the fast component of the tail current was suppressed whereas the slow component remained unchanged (Dubois, 198 lb). The best way to separate two currents is to pharmacologically block one component without affecting the other. For this purpose, 4-aminopyridine (4-AP) was found to be an efficient tool to separate fast and slow K currents. To avoid complications due to changes in driving force during depolarizations, the conductance was activated by conditioning depolarizing pulses to 0 mV in isotonic K solutions. In these conditions, the K current is nil and E~ is necessarily constant during the conditioning depolarizations. It has been shown that 4-AP (10 7 10 2 M) added to the external isotonic K solution specifically blocked the fast tail current (Fig. 5) with an apparent dissociation constant of 10-5 M, whereas the slow [.-A P] (M) _

lo-3

lO-5

n

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5r,~,l

-I

12 9

\ •

\\

\\

6 3 --;,'--t~ . . . .

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10 -5

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(M)

FIG. 5. Separations of fast and slow K currents by 4-aminopyridine. The tail current was recorded in 117 mM K at E = - 90 mV after a 100 msec conditioning depolarization to 0 mV. Na current was blocked by TTX. Capacity and leakage currents were compensated for automatically. Above: tail currents in the presence of 0, 10 -~and 10 3 M4-AP. Below: instantaneous extrapolated slow phase (filled circles) and instantaneous fast phase (open circles) calculated as the difference between the initial peak of the tail current and the instantaneous (extrapolated) slow phase of the tail current as a function of the logarithm of 4-AP concentration. The curve represents the inhibition of the fast phase assuming a one to one reaction between channels and 4-AP molecules with a dissociation constant of 10 5. Temperature: 12'C (From Dubois, 1981b).

component remained unaltered (in amplitude and time course) even with 4-AP concentrations (10 ~ 10 2 M) which completely blocked the fast component (Dubois, 1981b; 1982a). These results prove that the K current of the nodal membrane is composed of (at least) two components corresponding to two types of K channels, one (fast) which is sensitive to 4-AP and the other (slow) which is insensitive to 4-AP. As suggested by the results of Schwarz and Vogel (1971) and Lewis (1971), a fraction of K current (about 20%) fails to inactivate during large and long lasting depolarizations. Since the slow component of the instantaneous tail current represents about 20% of the total instantaneous tail current (Ilyin et al., 1980; Dubois, 1981b) and does not inactivate (Fig. 4 and Dubois, 1981b) it was tempting to suppose that the fast component could be fully inactivated. Consequently, it was assumed that the slow current could be separated from the total current by long lasting depolarizations which would inactivate the fast current (Dubois, 1981 b). Figure 6 represents the effects of conditioning depolarizations to 0 mV and of various

Potassium currents in the frog node of ranvier tVc (sec)

-S

9

|0

0.1

180

-! (nA)f

9 ~0.~ 1

t 2O

L

40

60

80

180

IVc (see)

FI(i. 6. Separation of fast and slow K currents by long lasting conditioningdepolarizations. The tail current was recorded in 117 mM-K at E - -90 mV after conditioning depolarizations to 0 mV of various durations. Na current was blockedby TTX. Capacityand leakagecurrents werecompensated for automatically. Above: tail current recorded after 100 msec, 10 sec and 3 min conditioning depolarizations (V~). Below:instantaneous values of the extrapolated slow phase (filled circles) and instantaneous fast phase (open circles)calculated as the differencebetween the initial peak of the tail current and the instantaneous (extrapolated) slow phase of the tail current versus the duration of the conditioning depolarization. Temperature: 12C. (From Dubois, 1981b).

durations upon the fast and slow components of the tail current. Whereas the slow tail current was constant for pulse durations from 2 to 180 sec, the fast current declined continuously during this period and was completely inactivated after 180 sec depolarization. This result is in agreement with the hypothesis proposed above and confirms the view that the K current is composed of two independent components. It is important to note the similarity between the results of Figs 5 and 6 in which the two components were separated by two different methods, pharmacologically in Fig. 5 and kinetically in Fig. 6. In the presence of 1 mM 4-AP, the block of the fast current was not removed by repetitive pulsing. In such conditions, the slow current could be studied separately. After blockade of the fast current by 4-AP, recordings of the remaining current revealed the presence of a slow K current in external solutions of various K concentrations including normal K Ringer's solution (Dubois, 198 lc). Studies of the properties of the slow current show that its activation is exponential. Consequently, the kinetic properties of the slow K channels can be described by a two state model, indicating that the channels are gated by only one particle (Dubois, 1981b). The time constant of slow conductance change varies with voltage in a bell shaped manner between about 10 and 20 msec at 15-18°C (Ilyin et at., 1980) or 20-100 msec at 12~C (Dubois, 1981b). The steady state activation-voltage curve of the slow conductance (gK~) is less steep than the corresponding curve of the fast conductance (gKf) and gK~ is activated at more negative voltages than gKf (Ilyin et al., 1980; Dubois, 1981b). Both the fast and the slow currents are blocked by Cs and TEA (Dubois, 1981b, c, 1982a). However, the slow current is less sensitive to external TEA than the fast one. This would explain the results of Van den Berg et al. (1977) showing a larger blocking effect of TEA after 400 msec of depolarization (for which both gKf and gK~ are activated) than after 60 sec of depolarization for which gKr is largely inactivated).

10

J.M. DUBOlS VIII. T W O FAST C U R R E N T S

At present, no pharmacological agent has been found to specifically block the slow K current. Consequently, the study of the fast current should be carried out from steady state currents recorded during short depolarizations activating a small proportion of slow channels, or from tail currents recorded in isotonic K solutions after subtraction of the slow current extrapolated to time zero of repolarization. Another way to obtain only the fast current is to subtract the current remaining in the presence of 4-AP from the total current recorded in the absence of 4-AP. For repolarization to a given voltage, the instantaneous fast current is directly proportional to the fast conductance at the end of the preceding conditioning depolarization. The conductance-voltage curves obtained from instantaneous fast currents recorded after conditioning depolarizations of various amplitudes systematically showed a bend near - 4 0 mV (Fig. 7, curve 1 ). Assuming that both the two limbs of the fast conductance voltage

GKf (,,s)

1

015

--90-~

O]

2 r

I

0.05

U--90

v

i

.

-

A

v

.

.

.

.

•

I

.

•

2

I

-50

FIG. 7. Evidence for the existence of two fast currents. The fast K conductance was calculated in 117 mM K frominstantaneous current (total instantaneous current minus instantaneous extrapolated slow tail current) recorded at 90 mV after activation of the conductanceby 200 msecconditioning depolarizations (Vc)of various amplitudes. Curve 1 was obtained with pulse protocol 1 and curve 2 was obtained with pulse protocol 2. In 2, each conditioningdepolarization was preceded by a pulse to 0 mV lasting 3.7 sec which completely inactivated the second limb of curve 1 and only partly decreased the first limb of curve 1. Temperature: 12'C. (From Dubois, 198lb).

curve and the two phases of the current inactivation (Schwarz and Vogel, 1971 ; Lewis, 1971 ; Dubois, 198 lb) corresponded to two independent components, it was hypothesized that long lasting conditioning depolarizations should completely suppress the conductance component which inactivates quickly while only partially reduce the component which inactivates slowly (Dubois, 1981b). The results presented Fig. 7 (curve 2) are in agreement with this hypothesis. The fast conductance-voltage curve obtained with long lasting conditioning pulses is composed of only one limb, corresponding to the first limb of the total curve reduced by a constant factor. It is concluded that the fast current is composed of two components: IKf~ which activates in the voltage range - 8 0 to - 4 0 mV and inactivates slowly (r ~ 45 sec at 0 mV and 12°C) and IKf2 which activates in the voltage range - 4 0 to 50 mV and inactivates quickly ( z ~ 2 sec at 0 mV and 12'C). It is important to note that whether lKf I and IKf 2 activate in different voltage ranges and inactivate with different time constants, their deactivation kinetics at - 9 0 mV are identical since, after depolarizations of various amplitudes activating either only IKf ~ o r IKf 1 and lKf2, the fast tail currents have similar time courses. The preceding conclusions were based on results obtained from tail currents recorded in isotonic K solutions. In normal K Ringer's solution, the inactivation develops in two phases (Schwarz and Vogel, 1971; Lewis, 1971) and after correction for K accumulation, the conductance-voltage curve presents a bend near - 40 mV (Palti et al., 1980; Dubois, 1981a). These observations confirm the existence of two fast inactivating currents. However, as pointed out before, the best way to separate two independent current components is to block

Potassium currents in the frog node of ranvier

11

one of them pharmacologically. F o r this purpose, capsaicine was found to be an efficient tool for separating the two fast K currents (Dubois, 1982b). Capsaicine is k n o w n to excite unmyelinated primary sensory neurones (Theriault et aL, 1979), to depolarize the intraspinal parts of the dorsal root fibres in rat (Ault et al., 1980) and to p r o l o n g the duration of action potentials in chick cultured sensory neurones (Godfraind et al., 198 I). W h e n added to the external solution bathing a node of Ranvier, capsaicine, at concentrations of about 10- 5 M, reversibly reduces the K current (recorded in normal K or isotonic K solutions) in a time dependent m a n n e r but only for large depolarizations (Fig. 8). Analyses of the steady state fast and slow conductance-voltage curves reveal that capsaicine (20 #M) suppresses the second control

Na-Ringer b

o

capsaicin

.......... - - -

f

C +70

-J............ r"

d-4o

.-~ .......... ~

_(r.......~...

J ............ •

I

K-Ringer

r

r-

.............S

.............

e +60

r-

Fic;. 8. Membrane currents recorded during and after depolarizations to various potentials (numbers)

in Na-or K-solutions both in the absence (left) and in the presence ofcapsaicin 20 I~M(a,b,c) and 10 # i (d,e). In Na-Ringer solution, the holding potential was -70 mV and the depolarizations were preceded by a 50 msec hyperpolarization to - 110 mV. In K-Ringer solution, the holding potential was - 90 mV. Symmetrical leakage and capacity currents were compensated for automatically. Note that in K-Ringer solutions, the fast tail current was noticeably reduced after the depolarization to +60 mV but was almost unchanged after the depolarization to -40 mV. Calibrations: 10 hA, 10 ms(a); 10 nA, 100 ms (b,c,d,e}. Temperature: l l C (from Dubois, 1982b).

limb (gKr2) of the fast conductance-voltage curve but does not significantly affect either the first limb (gKfl) of the fast conductance-voltage curve or the slow K c o n d u c t a n c e (Fig. 9). These results suggest that capsaicine blocks only one current c o m p o n e n t (IKf2). Furthermore, they provide pharmacological evidence for the existence within the nodal m e m b r a n e of two types of fast K channels. It must be noted that capsaicine does not affect significantly the duration of action potentials in the node Ranvier (Dubois, 1982b). This is p r o b a b l y due to the fact that capsaicine blocks IKf2 during depolarizations with a constant of several milliseconds. D u r i n g an action potential or a train of action potentials, the block would be insufficient to alter the action ootential duration.

12

J . M . I)UBOIS

A 0.2

£01

I

-50

I

I

0

50

V :rnV

B 01 005

,IP,]b'~,1~ ,~'[ -50

I 0

[ 50

V{mV)

FIG. 9. S e p a r a t i o n of fast-1 and fast-2 K currents by capsaicinc. Fast (A) and slow (B) c o n d u c t a n c e -

voltage curves in the absence (filled circles) and in the presenceof 20 tlM capsaicin (open circles). The slow and fast conductances were respectively calculated from instantaneous slow current (extrapolated to time of repolarization) and fast current (total instantaneous current minus extrapolated instantaneous slow current) recorded in K-Ringer solution at the end (/t" 200 ms depolarizations to various potentials (horizontal axis). Holding potential: 90 inV. remperature: 11 'C (From Dubois, 1982b). Studies of properties of fast currents are complicated by the presence of the slow current which, at present cannot be blocked specifically. During short depolarizing pulses (10 50 msec), it can be assumed that the fast current is minimally contaminated by the slow current. Consequently, the properties of the fast currents should be very similar to those of the total current (see first section). IX. M O T O R A N D S E N S O R Y F I B R E S Myelinated nerve fibres are separable into two distinct groups, sensory and motor. While sensory fibres fire repetitively, m o t o r fibres fire only once in response to a sustained stimulus. Furthermore, the initial rate of decline of the action potential is faster in sensory than in m o t o r fibres (Schmidt and Sfiimpfli, 1964). Differences between N a and K currents of the two types of fibres were reported earlier (see review by Neumcke, 1981 ). Concerning the K system, it appears that the conductance-voltage curve of sensory fibres compared to that of motor fibres is shifted toward positive voltages (Bergman and StLimpfli, 1966; Bretag and Stfimpfli, 1975). Moreover, the m a x i m u m conductance (Palti et al., 1980) and the unitary conductance (Neumcke et al., 1980) are larger in sensory than in motor fibres and the time constant of conductance activation as smaller in sensory than in m o t o r fibres (Palti et al., 1980). These differences were interpreted in terms of the existence of different K channels in the two types of fibres (Palti et at., 1980). Based on the analysis of three different K currents, it has been shown that the major difference lies in the proportion of fast-1 and fast-2 conductances (Dubois, 1981b). Figure 10 represents relative fast and slow conductance-voltage curves in a motor and a sensory fibre. The proportion ofgKf ~ versus the total conductance is about 50% in the m o t o r fibre and about 30% in the sensory fibre. From these results, it might appear that the difference in spike frequency adaptation of m o t o r and sensory fibres could be somehow related to the different proportion of gKrl and gKf2. However, other observations suggest that it is mostly due to differences in the N a system (Palti et al., 1980; Dubois, 1982a). x. C O N C L U S I O N Beginning with the work of Frankenhaeuser (1962a,b,c, 1963) which described the properties of the K permeability on the basis of the Hodgkin-Huxley model, it was assumed

Potassium currents in the frog node of ranvier

°°f°U

I,~. ~. ~.~.

o'~ s0

- 50

0

13

sensory

o~ 5o

50

-50

mV

0

50

mY

FI(i. 10. K + conductance-voltage relationships in a motor and sensory fibre. The relative fast {circles) and slow (squares) K ~ conductances were calculated from fast and slow K + currents recorded in isotonic K ~ Ringer at -90 mV after 150 msec depolarizing pulses of various amplitudes. Temperature: 12 15' (from Dubois, 1982a). that the population of K channels within the nodal m e m b r a n e was uniform. However, several observations which showed deviations from the predictions of the model indicated that the situation was m o r e complicated. The first cause of these complications is the existence of a m a r k e d accumulation of K ions in the perinodal space during K outflow. After correction for changes in driving force or under conditions (high K solutions) where the accumulation is minimized, it appears that the K current is c o m p o s e d of three independent c o m p o n e n t s (IKfl, IKr2 and IKs) with different activation and inactivation kinetics and different sensitivities to T E A and 4-AP. Curves depicting the steady state voltagedependencies of these c o m p o n e n t s are presented in Fig. 11. In the absence of specific blocking

-!0.5

I

-100

-50

0

50

i

(mY)

FI(;. 11. Steady state voltage dependence of K currents activation, s,, f~, and L , represent respectively the slow, fast-I and fast-2 steady state conductances relative to their maximum. Temperature: 12 C (From Dubois, 1981b). agents for IKf I and IKs, it seems difficult at present to undertake kinetic analysis of both IKf l and IKr2 activation. In spite of this lack of information, it would be of interest to investigate by mathematical reconstruction procedures the roles of the different K currents in electrical excitability. F r o m this point of view, it has been predicted (Krylov and Makovsky, 1978) and concluded from indirect observations (Dubois, 1982a) that the slow current would play an important role in the mechanism of spike frequency adaptation. XI. K C U R R E N T S

IN O T H E R

PREPARATIONS

Since the existence of different K currents has been shown in several preparations, it was of interest to c o m p a r e these currents with those of the frog node of Ranvier. F o r this purpose, we will briefly review the properties of K currents in some excitable membranes. This review will be limited to the purely voltage dependent outwardly rectifying currents and we will mainly stress the effects of blockers such as T E A and 4-AP.

1. Mammalian Node of Ranvier In contrast with the frog node of Ranvier, the delayed o u t w a r d current in m a m m a l i a n node of Ranvier is very small or even absent. This observation was first reported on peripheral rat

14

J.M. DuBols

fibres by Horackova et al. (1968) and was confirmed in peripheral fibres from the rat (Brismar, 1979, 1980), the rabbit (Chiu et al., 1979) and in central myelinated axons of the rat (Kocsis and Waxman, 1980). This suggests that the nodal membrane of mammalian fibres has few, if any, potassium channels. However, when the myelin around the node of Ranvier is acutely disrupted either following osmotic and enzymatic manipulations, stretching the fibre (Chiu and Ritchie, 1980, 1981), in alloxan diabetic rats (Brismar, 1979) or in rat nerve fibres demyelinated with diphteria toxin (Bostock et al., 1981), a delayed outward current appears which is blocked by TEA, Cs and 4-AP (Chiu and Ritchie, 1980, 1981; Sherratt et al., 1980: Bostock et al., 1981). Moreover, this current becomes inward at membrane potentials more negative than E K in high K solutions (Chiu and Ritchie, 1981) and its activation time constant is voltage dependent and similar in size to the time constant observed in the frog node of Ranvier. It is concluded that the delayed current in demyelinated nerve fibres is carried by potassium and thus K channels which are absent (or in low density ) in the nodal region are normally present in the paranodal region. It must be noted that the existence of K channels was also revealed in demyelinated internode of frog nerve fibres (Chiu and Ritchic, 1982). In contrast with the observations reported above on mammalian fibres, Binah and Palti (1981) reported K currents similar to those found in the frog node in normal rat nerve fibres. The origin of these currents is unclear. After demyelination, the appearance of K currents is concomitant with a large increase in the membrane capacity (Chiu and Ritchie, 1981). According to Binah and Palti, the node capacity in their preparation was similar to that of the frog node. This rules out the possibility that the myelin was lifted from the axolemma in their experiments. However it is possible that, after slight unsticking of the myelin around the nodal region, K channels are unmasked without change in the membrane capacity. Another explanation for the appearance of K currents in certain normal libtcs is that K channels are normally present in the nodal region of very large fibres as suggested by' Smith and Schauf (1981 ) who showed that, in the frog node, the ration g,K/gN, increased with the fibre diameter. The question which arises is whether in normal or demyelinated mammalian nerve fibres there exist several classes of K channels similar to those described in frog node. At present, it is difficult to answer this question since the effects of 4-AP were only tested on compound action potentials of demyelinated axons (Bostock et al., 1981). However, it has been shown that after an initial delay, the K current activation shows double exponential kinetics (Binah and Palti, 1981). Moreover, after internal application of TEA and Cs to a demyelinated nerve fibre, there remains a slow outward current (Fig. 12). These observations may suggest that a fraction of the delayed current flows through slow channels which (like those of frog node) should be less sensitive to TEA than fast ones.

2. Squid Giant A x o n

The properties of delayed current in squid axon are very similar to those of the frog node of Ranvier with the exception that TEA is only effective from the inside (Armstrong and Binstock, 1965). Internal Na (French and Wells, 1977) and external and internal Cs and Ba (Adelman and French, 1978; Eaton and Brodwick, 1980; Armstrong and Taylor, 19801 induce a voltage dependent block of I K. Increased [K]~ reduces the block by external Cs, external and internal Ba and increased [K]o reduces the block by internal Ba. It is assumed that blocking ions and K ions compete for a common site controlling the opening of the channels (Eaton and Brodwick, 1980; Armstrong and Taylor, 1980). Until now, the K current in squid axon was considered to consist of only one component. However, some indirect observations may suggest that their exists a slow current very similar to that described in the frog node. 4-AP blocks the K current and the blockade is voltage, time and frequency dependent (Yeh et al., 1976; Meves and Pichon, 1977). However, large concentrations (1-10 mM) of 4-AP do not completely block the delayed current (Meres and Pichon, 1977). Moreover, if the K current inactivates during long lasting depolarizations, the inactivation is incomplete (Ehrenstein and Gilbert, 1966). These observations may suggest that a non inactivating current, insensitive to 4-AP is also present in the squid axon.

Potassium currents in the frog node of ranvier

I

15

I 5 ms

Io nA o

[ I" 5m$

FK;. 12. Late outward current in a rabbit node after acute treatment to loosen the myelin. Na current was blocked by TTX. Above: Family of currents in response to depolarizations from a holding potential of - 8 0 mV to various test potentials in the range -72.5 mV to +62.5 mV in 15 mV increments. Below: the outward currents were partly blocked by changing 10 min before recordings the end pool solution from KC1 to 80 mM CsCI and 80 mM TEA CI. Leakage current corrected. Temperature: 24'C (From Chiu and Ritchie, 1980, Reprinted by permission from Nature, 284 pp 170 171 Copyright ~, 1980 Macmillan Journals Limited.

3. Neurones In neurones, the o u t w a r d K c u r r e n t can be d e c o m p o s e d into several c o m p o n e n t s differing in a c t i v a t i o n a n d i n a c t i v a t i o n kinetics, v o l t a g e d e p e n d e n c e a n d sensitivity to 4 - A P a n d T E A (see reviews by T h o m p s o n a n d Aldrich, 1980; D. J. A d a m s et al., 1980; P. R. A d a m s , 1982). D u r i n g a d e p o l a r i z i n g pulse from a h o l d i n g p o t e n t i a l m o r e negative t h a n a b o u t - 40 mV, a t r a n s i e n t o u t w a r d K current, t e r m e d the " A - c u r r e n t " ( H a g i w a r a et al., 1961 ; C o n n o r s a n d Stevens, 1971), rises r a p i d l y to p e a k a n d then decays e x p o n e n t i a l l y (z ~ 200-600 msec). Its rising p h a s e is s i g m o i d a n d can be fitted r e a s o n a b l y well b y an e x p o n e n t i a l raised to a p o w e r of 3 4 (Neher, 1971 ; T h o m p s o n , 1977) and whose time c o n s t a n t varies b e t w e e n 1 a n d 25 msec with changes in voltage. The c h a n g e s in A - c u r r e n t s t e a d y state a c t i v a t i o n a n d i n a c t i v a t i o n o c c u r respectively in the v o l t a g e ranges - 60 m V to a b o u t 0 m V a n d - 100 mV to - 40 mV. I A is slightly decrease b y external T E A which, at c o n c e n t r a t i o n of 100 mM, causes a 5 0 - 6 0 ~ r e d u c t i o n of the p e a k o u t w a r d current. 4 - A P b l o c k s A - c u r r e n t a n d it is m u c h m o r e effective t h a n T E A . A s s u m i n g t h a t one m o l e c u l e b i n d s to a single site, 4 - A P b l o c k s Ac h a n n e l s with an a p p a r e n t d i s s o c i a t i o n c o n s t a n t of 1.5 mM ( T h o m p s o n , 1977). At h o l d i n g voltages m o r e positive t h a n a b o u t - 4 0 mV, A - c u r r e n t is c o m p l e t e l y i n a c t i v a t e d and, following a v o l t a g e step, the r e m a i n i n g o u t w a r d c u r r e n t can be d e c o m p o s e d into t w o d e l a y e d c u r r e n t s : a p u r e v o l t a g e - d e p e n d e n t c u r r e n t ( K - c u r r e n t or late o u t w a r d c u r r e n t ) which is b l o c k e d by T E A with an a p p a r e n t d i s s o c i a t i o n c o n s t a n t of 6 8 mM ( T h o m s o n , 1977; H e r m a n n a n d G o r m a n , 1981b) a n d a C a - a c t i v a t e d c u r r e n t which can be b l o c k e d by calcium a n t a g o n i s t s o r internal E G T A . D u r i n g a d e p o l a r i z a t i o n , K - c u r r e n t rises relatively slowly to p e a k a n d then declines e x p o n e n t i a l l y (z ~ 1 3 sec) to a n o n zero s t e a d y state value. Its rising p h a s e is s i g m o i d a n d can be fitted by an e x p o n e n t i a l raised to a p o w e r of two. Its time c o n s t a n t c h a n g e s with v o l t a g e between l 0 msec a n d 40 msec. T h e s t e a d y state activation and inactivation of K - c u r r e n t change respectively over the voltage ranges - 30 mV to + 30 mV and - 90 mV to - 20 mV. K c u r r e n t in Tritonia n e u r o n e s is n o t affected b y 4 - A P at c o n c e n t r a t i o n s which b l o c k the t r a n s i e n t c u r r e n t ( T h o m p s o n , 1977). In Aplysia

16

J.M. Dut~ols

pacemaker neurones R-15 and L6, 4-AP blocks the delayed K current with an apparent dissociation constant of 0.8 mM at 0 mV (Hermann and G o r m a n , 198 la). In vertebrate neurones, another outward current (IM) has been described by Brown and Adams (1980). M-current is selectively inhibited by muscarinic agonists but only slightly decreased by external TEA. It activates exponentially with a time constant changing with voltage between about 50 msec and 100 msec and does not inactivate. Its steady state activation changes over the voltage range from about - 70 mV to about 20 inV. Finally, a much slower component of outward current (Ix) has been described in molluscan neurones by Patridge and Stevens (1976). I~ raises exponentially with a time constant which, changes between 0.5 sec and 2 sec with changes in voltage. Its steady state activation changes over the voltage range - 100 mV to about + 50 inV. I~ does not inactivate and it is not affected by external TEA. Like I ~ in frog node of Ranvier. Ix plays an important role in the mechanism of spike frequency adaptation. In some respects, these currents resemble those described in the frog node of Ranvier. Judging from the voltage ranges of activation, A and K-currents in neurones are respectively comparable to I~-~ and IKf2 in the node of Ranvier. However, while K-current and 1~ 2 inactivates with about the same time constant, the inactivation of I A is much faster than the inactivation of IKf ~ (about 100 times). Moreover, 1K activates more slowly than IK~2 (about 10 times). Finally, whereas IKr ~ and I~f2 are almost equally blocked by TEA and 4-AP, l,x is only slightly affected by TEA and I K is insensitive to 4-AP in Tritonia neurones but blocked by 4-AP in Aplysia neurones. I s and IM in neurones are comparable to Ix,, in myelinated nerve fibres. However, 1s activates more slowly than IK~ (about 10 times). Except that Ks-channels in the node of Ranvier are not affected by muscarine, they seem to be very similar to Mchannels in neurones with the following minor quantitative differences: the half activation voltage of IK~ appears to be some 10 20 mV more hyperpolarized than the corresponding voltage of I Mand the effective valency of the gating particles is 2 3 times smaller in S than in M channels.

4. Skeletal Muscle Two outward K currents have been described in frog sartorius muscle: a fast and a slow current (Adrian et al., 1970). The rising phase of the fast current can be described by an exponential raised to a power of four. Its activation time constant varies between about 5 msec and 50 msec with changes in voltage and its steady state activation changes over the voltage range - 8 0 mV to 0 mV. during a maintained depolarization, the fast current inactivates completely in some seconds. As in the node of Ranvier, the slow current can be separated from the total current by extrapolation of the slow component of the tail current arising upon repolarization. The slow current reaches a m a x i m u m (about 20°/~, of the total m a x i m u m total current) in about 3 sec and then declines to a steady level (about 30'~, of its maximum value) in about 10 sec. Both fast and slow K currents are partially blocked by TEA (Stanfield, 1970) but only the fast current is blocked by 4-AP (Duval and Leoty, 1980). From this point of view; it seems that K currents are very similar in the skeletal muscle and in the node of Ranvier. However, in skeletal muscle, there appears to exist only one type of fast current. Moreover, it has been noted that while both fast and slow currents are present in rat soleus muscle, the slow current is virtually absent in rat iliacus muscle (Duval and Leoty, 1980).

XII. C O N C L U D I N G

REMARKS

From the present review, it appears that the existence of both fast and slow K currents is a common feature of excitable membranes. Generally, fast currents inactivate during long lasting depolarizations and are blocked by 4-AP. In contrast, slow currents do not exhibit inactivation, are not affected by 4-AP and are less sensitive to TEA than fast currents. Moreover, while the rising phase of fast currents is sigmoid, slow currents activate exponentially. Fast currents are activated during a single action potential and they contribute to the repolarizing phase of the spike and the refractory period. Slow currents are

Potassium currents in the frog node of ranvier

17

a c t i v a t e d d u r i n g trains of spikes a n d p l a y a role in the m e c h a n i s m of spike frequency adaptation. In m o s t of the cells superfused with n o r m a l K solutions, o u t w a r d K currents induce an a c c u m u l a t i o n of K ions at the external face of the m e m b r a n e . T h e c o n s e q u e n c e s of this a c c u m u l a t i o n are i m p o r t a n t b o t h for the analysis of K c o n d u c t a n c e changes a n d for the activity of the cells. F o r the analysis of the c o n d u c t a n c e s changes, the p a r a m e t e r s must be c a l c u l a t e d ~ither in n o r m a l K s o l u t i o n s from the m e a s u r e d c u r r e n t a n d c o r r e c t e d ionic driving forces or directly from the m e a s u r e d c u r r e n t r e c o r d e d in high K solutions where the a c c u m u l a t i o n is minimized. It is well k n o w n that, in different p r e p a r a t i o n s , the K c o n d u c t a n c e is a function of the external K c o n c e n t r a t i o n ( D u b o i s a n d Bergman, 1977; Arhem, 1980; Dubois, 1981b; Swenson and A r m s t r o n g , 1981; Junge 1982). In consequence, the K a c c u m u l a t i o n s h o u l d t h e o r e t i c a l l y induce c o n d u c t a n c e changes. However, it has been recently suggested t h a t p r o l o n g e d a p p l i c a t i o n s of high [ K ] o (via c h a n g e s of K c o n c e n t r a t i o n in the b u l k s o l u t i o n ) induce a m o d i f i c a t i o n of K c o n d u c t a n c e p a r a m e t e r s whereas the effects of r a p i d c h a n g e s of [ K ] o (via the a c c u m u l a t i o n ) on the c o n d u c t a n c e are negligible (De Bruin, 1982). Even if K a c c u m u l a t i o n does n o t by itself induce c h a n g e s in c o n d u c t a n c e , the fact r e m a i n s t h a t it seriously c o m p l i c a t e s the analysis of the channels properties. W i t h the squid axon, the n o d e of R a n v i e r has been used for a b o u t thirty years as a m o d e l for excitable m e m b r a n e s . T h e recent findings on the frog node of R a n v i e r show that three classes of K channels can be kinetically a n d p h a r m a c o l o g i c a l l y s e p a r a t e d . F r o m this p o i n t of view, the frog n o d e of R a n v i e r is c o m p a r a b l e to the o t h e r excitable cells. In the near future, one can expect t h a t studies of K channels u n d e r v o l t a g e c l a m p c o n d i t i o n s on frog Ranvier n o d e a s s o c i a t e d with studies of K channels on o t h e r p r e p a r a t i o n s using new techniques such as single c u r r e n t r e c o r d i n g s (Conti a n d Neher, 1980) or i n c o r p o r a t i o n of channels in lipid bilayer ( C o r o n a d o a n d L a t o r r e , 1982) w o u l d p r o v i d e useful i n f o r m a t i o n a b o u t the m o l e c u l a r p r o p e r t i e s a n d p h y s i o l o g i c a l roles of K channels. ACKNOWLEDGEMENTS T h e a u t h o r w o u l d like to t h a n k Dr. M a r t i n F. S c h n e i d e r for v a l u a b l e discussions a n d critical r e a d i n g of the m a n u s c r i p t a n d P r o f e s s o r Claes H. B e r t h o l d a n d Dr. M a r t i n R y d m a r k for a p r e p r i n t of their p a p e r s (1983).

REFERENCES ADAMS,D. J., SMITH,S. J. and THOMPSON,S. H. (1980) Ionic currents in molluscan soma. A. Rev. Neurosci. 3, 141 167. ADAMS,P. R. (1982) Voltage-dependent conductances of vertebrate neurones. Trends Neurosci. 5, 116 119. ADELMAN,W. J., JR. and FRENCH, R. J. (1978) Blocking of the squid axon potassium channel by external cesium ions. J. Physiol. (Lond) 276, 13 25. ADRIAN,R. H., CHANDLER,W. K. and HODGKIN,A. L. (1970) Slow changes in potassium permeability in skeletal muscle. J. Physiol. (Lond) 208, 645 668. ARHEM,P. (1980) Effects of rubidium, cesium, strontium, barium and lanthanum on ionic currents in myelinated nerve fibers from Xenopus laeris. Acta physiol. Stand. 108, 7 16. ARMSTRONG,C. M. and BINSTOCK,L. (1965) Anomalous rectifications in the squid giant axon injected with tetraethylammonium chloride. J. gen. Physiol. 48, 859 872. ARMSTRONG,C. M. and HILLE,B. (1972)The inner quaternary ammonium ion receptor in potassium channels of the node of Ranvier. J. glen. Physiol. 59, 388~400. ARMSTRONG,C. M. and TAYLOR,S. R. (1980) Interaction of barium ions with potassium channels in squid giant axons. Biophys. J. 30, 473~488. ATTWELL,D., DUBOtS,J. M. and OJEDA,C. (1980) Fully activated potassium current-voltage relationship in the Ranvier node. p/tii.qers Arch. 384, 49 56. AULT, B. and EVANS,R. H. (1980) Depolarizating action of capsaicin on isolated dorsal root fibres of the rat. J. Physiol (Lond.) 306, 22P. BEGENISICH,T. (1979) Conditioning hyperpolarization-induced delays in the potassium channels of myelinated nerve. Biophys. J. 27, 257 266. BERGMAN,C. (1970) Increase of sodium concentration near the inner surface of the nodal membrane, p/[iiq~,rs Arch. 317, 287 302. BERGMAN,C. and ST~,MPFLLR. (1966) Difference de permeabilit6 des fibres nerveuses my61inis6essensorielles et motrices fi l'ion potassium. Heh'. Physiol. Acta. 24, 247 258. BERGMAN,J., DUBOlS,J. M. and BER~;MA~C. (1980) Post-tetanic membrane potential in single axon and mvelinated nerve trunk. Ann. N. Y. Acad. Sci. 339, 21 38.

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J . M . DUBOIS

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