Behaviour of delayed current under long-duration voltage clamp in snail neurones

Camp.Biochem.P&viol., 1971, Vol. 4OA, pp. 715 to 722. Pergamon Press. printed in Great Britain

BEHAVIOUR OF DELAYED CURRENT UNDER LONGDURATION VOLTAGE CLAMP IN SNAIL NEURONES I. S. MAGURA,

0. A. KRISHTAL

and A. G. VALEYEV

Department of General Physiology of Nervous System, Bogomoletz Institute of Physiology, Academy of Sciences, Kiev, U.S.S.R. (lie&wed 9 March 1971) Abstract-l. When the depolarization of the somatic membrane of Helix pomatia giant neurones under a long-duration voltage clamp exceeds - 25 mV, the outward current after reaching the maximum value declines. The time course of this decline is approximately exponential with a time constant of 500 msec. 2. Double-step voltage clamp experiments show that the outward current decline is caused both by a decrease in the membrane conductance and by a shift in the equilibrium potential in the positive direction. 3. The maximum value of the outward current for a given level of depolarization can be considerably altered by preceding shifts in the membrane potential. The preceding depolarization decreases and the hyperpolarization increases the maximum value of the outward current, probably causing changes in the degree of the potassium inactivation. 4. The time course of the potassium inactivation removal by hyperpolarization is approximately exponential. The time constant of this process varies from 0.5 set to several seconds. 5. In some cases, beginning with a certain level of the preceding hyperpolarization so large an increase in the dutward current during depolarization is observed that a transient breakdown of the membrane can be supposed. 6. The mechanisms of the effects produced by preceding shifts in the membrane potential upon the action potential generation are discussed. INTRODUCTION IN PREVIOUS papers

it has been shown that the preliminary change of the somatic membrane potential (conditioning depolarization or hyperpolar~ation) of mollusc neurones causes complex changes in the action potential (Krishtal & Magura, 1970; Magura & Krishtal, 1970). Thus conditioning depolarization considerably decreases the maximum rate of rise of the action potential. Simultaneously, the appearance of a plateau on the falling phase of the spike may be observed. In this case the spike overshoot may stay unaffected. Conditioning hype~ol~~ation often does not change the maximum rate of rise, but the spike overshoot may be decreased. This finding differs from that obtained by Narahashi (1964) on lobster giant axons in similar experiments. One may suppose that such an action of the conditioning polarization on the spike generation in some of the mollusc neurones may be due to the effect upon the mechanisms which are responsible for a rise of potassium conductance. 715

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I. S. MAGURA,0. A. KRISHTALANDA. G. VALEYEV

The aim of the present paper is to describe on the basis of voltage clamp experiments. MATERIALS

AND

the properties

of these mechanisms

METHODS

Experiments were performed on neurones of the visceral complex of the ganglia of Helix pomatiu. Composition of the “normal” saline solution was similar to that described by Krishtal & Magura (1970). The clamping device consisted of two operational.amplifiers. One of them controlled the membrane potential whilst the other was used as a “virtual ground” for the current measurement. The system’s drift was less than 1 mV/hr. Two microelectrodes filled with 3 M KC1 were inserted into the soma of a giant neurone. Their resistance was usually from 4 to 7 MQR. All experiments were done at room temperature (20-22°C). RESULTS

The time course of the delayed outward current A long-lasting, sufficiently large depolarization of the somatic membrane in a voltage clamp condition produced a delayed outward current which declined with time. Figure l(a) presents a family of the delayed outward current traces corresponding to the different levels of the depolarization. After reaching a maximum value the

(a)

lb)

.

I h I 50 z

O -50 E

_ 0-+

2

J_ -40

-20

0

20

FIG. l(a). Currents under long-duration voltage clamp, above, and membrane potential, below, in soma of H. pomatia neurone. Resting potential, -50 mV, holding potential, -50 mV. (b). Current-voltage relationship in the same cell. Filled circles measured at the peak of the currents. Triangles -2.5 set after the beginning of depolarization. A broken line represents the current-voltage relationship for leakage conductance.

DELAYED

CURRENT

IN

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outward current starts decreasing. The time course of this decline is approximately exponential with a time constant of about 500 msec. A similar observation was made on the supramedullar neurones of the puffer fish (Nakajima & Kusano, 1966), pacemaker neurones of ~~~~~u (Alving, 1969) and fibres of frog sartorius muscle (Adrian et d., 1970). Figure l(b) illustrates the current-voltage relations for the maximum values of the delayed outward currents and for its values after 2.5 set from the beginning of depolarization. The broken line shows the value of the leak current. The leak

current was extrapolated from the records with hyperpolarizing shifts of membrane potential. The extrapolation was based on the assumption that the non-specific leak current through the membrane is linearly proportional to the size of the potential step. litactivation of the potassium conductance In order to analyse the events resulting in a decline of the outward current twostep voltage clamp experiments were made and instantaneous current-voltage relations were obtained. They allowed one to measure conductances and determine equilibrium potentials for the delayed outward current at various moments. The current corresponding to the second (lower) step of the depolarization declines exponentially. The rate of decline increases when the potential difference between the first and the second steps becomes larger (Fig. Z(a)), Precise measurement of the current after the instantaneous shift of the membrane potential was difficult because of the presence of a capacitive surge associated with a sudden change in the voltage. (al

(b)

_L_ f

mV

FIG. 2(a). Tracings of membrane current, above, and membrane potential, below, during a double-step voltage clamp. (b). Instantaneous curren+voltage relations at different phases of the delayed outward current. These phases are indicated above. Resting potential, - 42 mV, holding potential, -SO mV. 24

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I. S. MAGURA, 0. A. KRISHTAL AND A. G. VALEYEV

Figure Z(b) demonstrates the instantaneous current-voltage relations for the delayed outward current at various moments after the beginning of depolarization. Judging from the slope of the lines obtained in this experiment the chord conductance of the membrane was about 1. 1O-5 Q-1, where the outward current reached its maximum value. Then the conductance of the membrane declined to O-3. 1O-5 fi-l being still about five times higher than the conductance of the resting membrane. Extrapolation of the current-voltage line to the abscissa gives the equilibrium potential for the outward current. In the experiment represented in Fig. 2, at the moment of maximum outward current the equilibrium potential was about - 70 mV (the resting potential being 45 mV). After 2.5 set from the beginning of a depolarization the equilibrium potential was about - 60 mV. Thus the outward current decline during the long-lasting, voltage-clamp depolarization is due to a decrease in the membrane conductance and to a decrease in its e.m.f. A similar phenomenon was observed by Alving (1969) on the Aplysia pacemaker neurones and by Adrian et al. (1970) on fibres of the frog sartorius muscle. Considering the outward current to be carried by potassium ions (Hodgkin & Huxley, 1952), it is possible to assume the presence of potassium inactivation which develops during the long-lasting depolarization. If the potassium ions leaving the cell accumulate near the outer surface of the membrane the potassium equilibrium potential becomes less negative (Frankenhaeuser & Hodgkin, 1956). The e&feet of the preceding polarization

upon the delayed

outward

current

The value of the delayed outward current maximum for a given level of depolarization may be considerably altered by a preceding shift in the membrane potential. Hagiwara et al. (1961) were the first to note that the preliminary hyperpolarization causes a significant transient rise of the delayed outward current in Onchidium neurones. Figure 3(a) demonstrates a family of the outward current traces obtained by a depolarizing shift up to - 5 mV which was performed from different preceding levels of the membrane potential (the preceding shift in the membrane potential lasted for 2 set before each depolarizing step). It can be seen that relatively small changes in the preceding level of the membrane potential are able to cause a considerable shift in the value of the delayed outward current. The relations between the maximum outward current and the preceding level of the membrane potential are shown in Fig. 3(b). Qualitatively, similar results were obtained in the experiments on supramedullary cells of puffer fish (Nakajima, 1966) and fibres of frog sartorius muscle (Adrian et al., 1970).

DELAYED CURRENT \a1

fN

SNAIL Ik(V)

719

NEURON730

(b)

6 7 0 Q

4 2 0

FIG. 3(a)_ Dependence of the outwardcurrents (I) upon the preiimimry Ieve1of the membrane potential. Bmation of the p&imimuy shifts of membrane potential was 2 sec. The inset diagram illustrates a method of measuring I. (b). The peak of the outward currant as a function of the preliminary level of the membrane potential for the same cell. (c). Traces of the membrsne currents under the voltage clamp from different levels of holding potential,

set I -00

-60

t -40

I -20

1

0

mV

FIG. %a>. Demndence of the outwaxdcurrent (I) upan the preliminary level of the membrane potential. Inset, meth0.d of measuring I. Note the transient “breakdown” of the membrane after a strong preceding hyperpolarization. Duration of the preliminary shifts of the membrane potential was 2 sec. (b). The peak of the outward current as a function of the preliminary level of the membrane potential.

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I. S. MAGURA,0. A. KRISHTALANDA. G. VALEYEV

In the experiment represented in Fig. 3 practically a complete removal of the steady-state potassium inactivation was obtained when the preceding membrane potential reached - 110 mV. Figure 3(c) represents ionic currents at fast sweep records when the somatic membrane was depolarized up to - 18 mV from different holding levels. The shift of the holding level from - 60 mV to - 50 mV and - 40 mV slightly increased the transient inward current, simultaneously decreasing the rate of rise of the delayed outward current. One may suppose that this change in the transient inward current is due to the slower rise of the delayed outward current. It was difficult to determine the value of the hyperpolarization needed for a complete removal of the steady-state potassium inactivation in some experiments. Beginning with certain levels of the preceding hyperpolarization further small increases in its value could produce large increases in the outward current developing under the following depolarization so that a transient breakdown of the membrane could be supposed in such cases (Fig. 4). Figure 5(a) presents two families of the delayed outward current traces obtained under depolarization up to - 25 mV which was performed after preceding hyperpolarization of various durations.

6

0L q

2v

set

4

FIG. 5. Dependence of the outward currents upon the duration of preliminary hyperpolarization. Resting potential, - 45 mV, holding level, - 50 mV. Depolarization for both families of current traces, - 25 mV. Preliminary hyperpolarization for upper family - 70 mV, for lower, - 78 mV. Duration of the preliminary hyperpolarization for upper traces: 0, 0.5, 1, 2.5, 3.5 sec. For lower traces: 0, 0.4, 1, 2.5, 3.5 sec. Right peak of the outward currents as a function of duration if preliminary hyperpolarization. Filled circles, hyperpolarization to -70 mV, triangles, hyperpolarization to -78 mV.

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Such experiments indicated that potassium inactivation is removed approximately exponentially (Fig. S(b)). Th e t ime constant of its removal varies in different experiments from O-5 set to several seconds. DISCUSSION

The voltage clamp experiments described here provide some basis for the conclusion that the properties of the delayed rectifier in the somatic membrane of snail neurones are somewhat different from those described for the squid giant axon (Ehrenstein & Gilbert, 1966; Armstrong, 1969): (1) The delayed outward current is subjected to a relatively quick inactivation when the somatic membrane is depolarized. (2) The value of the delayed outward current is highly sensitive to the changes of the preceding level of the membrane potential. (3) The preceding hyperpolarization increases the rate of rise of the delayed outward current in the somatic membrane. (According to Frankenhauser & Hodgkin (1957) in squid giant axons the preceding hyperpolarization produces an opposite effect.) The properties of the delayed rectifier in the soma of Helix neurones are similar to those observed by Nakajima & Kusano (1966) and Nakajima (1966) on the soma of the supramedullar neurones of the puffer fish. The preceding hyperpolarization of the soma up to -65 to -75 mV (the resting potential is - 40 to - SO mV) does not usually affect the maximum value of the transient inward current whilst it increases the delayed outward current. It may be supposed that the steady-state inactivation of the sodium carrying system is practically absent when the membrane potential equals - 40 to - 50 mV (Geduldig & Gruener, 1970; Magura & Krishtal, 1970). These observations correlate with the effect of the preceding polarization upon the action potential. The spike overshoot may stay unaffected under a moderate preceding depolarization because both sodium and potassium systems of the membrane become inactivated. In this condition the soma generates the action potential with a low maximum rate of rise and with a plateau on its falling phase. The preceding hyperpolarization may decrease the spike overshoot because the removal of the potassium inactivation induces a more rapid increase in the potassium conductance whilst the sodium system stays unaffected. Our findings about the influence of the preliminary hyperpolarization upon the delayed outward current in the soma of Helix pomatia neurones are somewhat inconsistent with those obtained by Geduldig & Gruener (1970) on ApZysia giant neurone. In that study the preliminary shifts in the membrane potential did not change the value of the delayed outward current. Such inconsistency may be considered as an example of the wide variations in the properties of the giant neurones belonging to different species of molluscs. Acknowledgement-The authors wish to thank Dr. P. G. Kostyuk for his guidance and encouragement in this work.

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I. S. MAGURA,0. A. KRISHTALANDA. G. VALEYEV REFERENCES

ADRIANR. H., CHANDLERW. K. & HODGKINA. L. (1970) Voltage clamp experiments in striated muscle fibres. J. Physiol., Lond. 208, 607-644. ALVINGB. 0. (1969) Differences between pacemaker and nonpacemaker neurons of Aplisiu on voltage clamping. J. gen. Physiol. 54, 512-531. ARMSTRONG C. M. (1969) Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injected in squid axons. J. gen. Physiol. 54, 553575. EHRJZNSTEIN G. & GILBERT D. L. (1966) Slow changes in potassium permiability in squid giant axon. Bi0phys.J. 6, 553-564. FRANKENHAEUSER B. & HODGKINA. L. (1956) The after effects of impulses in the giant nerve fibres of Loligo. J. Physiol., Lond. 131, 341-376. FRANKENHAEUSW B. & HODGKINA. L. (1957) The action of calcium on the electrical properties of squid axons. J. Physiol., Lond. 137, 218-244. GEDULDIC D. & GRUENERR. (1970) Voltage clamp of the Aplisiu giant neurone: early sodium and calcium currents. J. Physiol., Lond. 211, 217-244. HAGIWARAS., KUSANOK. & SAITON. (1961) Membrane changes of Onchidium nerve cell in potassium-rich media. J. Physiol., Lond. 155, 470-489. HODGKINA. L. & HUXLEY A. F. (1952) Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol., Lond. 116, 449-472. KRISHTAL0. A. & MAGURA1. S. (1970) Calcium ions as inward current carriers in mollusc neurones. Comp. Biochem. Physiol. 35, 857-866. MAGURAI. S. & KRISHTAL0. A. (1970) The effect of conditioning polarization on the action potential of mollusc giant neurones. Neurophysiology 2, 91-99. (In Russian.) NAKAJIMAS. (1966) Analysis of K inactivation and TEA action in the supremedullary cells of puffer. J. gen. Physiol. 49, 629-640. NAKAJIMAS. & KUSANOK. (1966) Behavior of delayed current under voltage clamp in the supramedullary neurons of puffer. J. gen. Physiol. 49, 613-628. NARAHASHI T. (1964) Restoration of action potential by anodal polarization in lobster giant axons. J. cell. camp. Physiol. 64, 73-96. Key current;

Word Index-Nerve physiology; potassium inactivation; membrane; conductance; voltage clamp; snail neurones; Helix pomatia.

delayed