Outward currents in isolated snail neurones—II. Effect of TEA

Comp. Biochem. Physiol., 1975, Vot. 51C, pp. 265 to 268. Pergamon Press. Printed in Great Britain

O U T W A R D C U R R E N T S IN ISOLATED SNAIL N E U R O N E S - - I I . EFFECT OF TEA P. G. KOSTYUK,O. A. KRISHTAL AND P. A. DOROSHENIZO A. A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev, U.S.S.R. (Received 4 November 1974) Abstraet--1. The effect of TEA on fast and delayed outward currents was investigated in the isolated soma of snail (Helix pomatia) neurones under voltage clamp conditions. 2. Both outward currents were inhibited by TEA applied either to the outer or to the inner side of tbe membrane. 3. Much lower external concentrations of TEA were effective in the isolated soma as compared to the intact neurones; a 50 per cent block of delayed outward current was reached by the action of less than 1 mM of TEA. 4. External application of TEA depressed the delayed outward current more effectively. Internal application of TEA (by iontophoresis) caused a more effective depression of the fast outward current. Sufficiently large injections of TEA caused its almost complete disappearance. 5. The bell-shaped steady-state inactivation curve for the inward current could be turned into the usual S-shaped curve by inhibition of the fast outward current due to the injections of TEA.

INTRODUCTION

The designations are the same as in the previous communication (Kostyuk et al., 1975).

IN THE investigation on intact snail neurones it was shown that T E A affects the two o u t w a r d currents in a different way (Neher & Lux, 1972). W e have continued the study o f the effects o f T E A on the neuronal somata completely isolated f r o m the ganglia. T w o reasons can be suggested for this w o r k : (I) The isolation of the cell m a y m a k e its m e m b r a n e m u c h m o r e accessible to external T E A and thus create better possibilities to study its effect on the m e m b r a n e currents. (2) A n intracellular injection o f T E A m a y cause a depression o f the fast outward current. The existence o f this current creates some difficulties in the measurements o f C a - N a inward current in the s o m a o f mollusc neurones (Neher, 1971 ; K o s t y u k etaL, 1974b). In fact, a true inactivation curve and a reliable value for the reversal potential for the inward current could not be obtained. After depression o f the fast outward current these characteristics m a y be determined m o r e precisely.

RESULTS Application o f T E A to the external surface o f the membrane

MATERIALS AND METHODS The main details of the experimental procedure have been reported in previous communications (Kostyuk et al., 1974a, 1975). Three intracellular microelectrodes were used in the experiments with TEA injections into the cell. One of them was filled with 1 M TEA-chloride. The iontophoretic current passed through the second microelectrode which was used simultaneously in the feedback circuit. Thus the injections did not cause any polarization of the membrane. TEA-chloride was added to the external saline without any correction of osmolality.

The dependence o f m a x i m u m slope conductance A l I A V for the peaks o f the delayed outward current on the external concentration o f T E A is demonstrated in Fig. 1. A 50 per cent inhibition was obtained with a T E A concentration lower than 1 m M and 0.25 m M T E A was already effective. (According to Neher & Lux, 1972, a 50 per cent inhibition o f delayed outward current in the intact neurones was obtained with T E A concentrations between 6 and 8 m M . ) With 1-2 m M o f T E A the delayed outward current was inhibited in our experiments to 10-30 per cent o f its initial value. Also the fast outward current was depressed by external T E A , but somewhat less effectively. Therefore, only a partial separation of b o t h outward currents could be achieved by external application o f T E A .

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The kinetics of both outward currents were slightly affected by T E A , ~-f and ~'d being insignificantly increased. The inward current, when measured at Vh = - 40 mV, i.e. in the conditions of its m i n i m u m superposition with the fast outward current (Kostyuk et al., 1975), was not altered with a T E A concentration of up to 1 m M but decreased with any further increase.

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Fig. I. Dose-response curve for extracellular TEA action on the delayed outward current. Abscissa: TEA concentration in the normal Ringer (logarithmic scale); ordinate: normalized maximum slope conductance (A1]AV) for the peaks of delayed outward current.

tion is very close to that made by Neher & Lux (1972). Such injections depressed both the fast and delayed outward currents. Contrary to the external T E A application its action on the fast outward current was more effective. A n example of the internal T E A effect is shown in Fig. 2; while the delayed current was depressed for about 50 per cent (Fig. 2b), the fast outward current disappeared almost completely (Fig. 2a). The current which remained after T E A injection in Fig. 2(a) exceeded the leakage current because of the activation of a partially maintained delayed outward current (for an explanation see Kostyuk et al., 1975). A difference was observed in T E A action on the inactivating and steady-state components of the delayed current. The steady-state c o m p o n e n t either was less subjected to depression or was not affected at all (in four neurones out of eleven). The peak time and the inactivation time constant of the delayed outward current were not significantly altered (Fig. 2c), contrary to the data by N a k a j i m a (1966).

Application o f TEA to the internal surface o f the membrane

Separation o f the inward current by the internal action o f TEA

A n iontophoretic current of 50 n A was used to inject T E A into the isolated neurones. The injections lasted f r o m 1 to 5 rain. Only an approximate estimation o f the final concentration of T E A in the neurone is possible. Suppose that all the iontophoretic current is carried by T E A ions (which may bring about overestimation of their injected quantity) and suppose that the injected ions are uniformly distributed over the cell volume (which m a y bring a b o u t underestimation o f their acting concentration), we should obtain the value of 15-20 m M . This estima-

The steady-state inactivation curve h a (V) for the inward current has a m a x i m u m when measured in mollusc neurones (Geduldig & Gruener, 1970; Neher, 1971 ; Kostyuk et al., 1974b). A decrease in the peak inward current observed with the shift of the holding potential to hyperpolarization was assumed to be due to the superposition of the inward current with the fast outward current (Neher, 1971). A real degree of inward current inactivation at the holding potential between - 4 0 and - 5 0 mV (i.e. near the m a x i m u m of the measured inactivation curve)

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Fig. 2. Effect of intracellular TEA. (a) Effect of TEA injection on the fast outward current. The current traces (superimposed) were obtained before injection, after 25, 50 and 100 sec of injection, (50nA). F t = - 3 4 m V , V h = - 1 0 0 m V . Calibrations: 5 × 1 0 - 8 A , 10msec. (b) Effect of TEA injection on the delayed outward current. The current traces obtained before and after injection (50 nA, 100 sec) are superimposed. F t = 40 mV, F h = - 4 0 inV. Calibrations : 2 × 10-7 A, 100 msec. (c) The time course of delayed outward current inactivation (obtained from the curves presented in b). Abscissa: time from the beginning of the pulse, ordinate: In ( I - ls~) where Iss is the steady-state value of delayed current. ~'d = 225 msec. (a, b and c)--The same neurone.

Effect of TEA on outward currents in snail neurones remained unknown. As mentioned above, the fast outward current could be completely blocked by intracellular injections of TEA. This effect was used to obtain the steady-state inactivation curve for the inward current. The results of these experiments are summarized in Fig. 3. The injections of T E A clearly altered the shape of the hoo (V) curve; its fall with the negative shift of Vb either diminished or disappeared ----4O

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current. The cause of this phenomenon (also observed by Neher & Lux, 1972) is not clear. In any case, from the data presented in Fig. 3 it is obvious that (1) the inactivation curve for the pure inward current in the somatic membrane has its usual S-shaped form and (2) hoo is already close to I at Vh = - 4 0 inV. In other words, activation of the fast outward current does not introduce any substantial errors into the peak values of the inward current which is measured at Vh = - 40 mV (Fig. 4a). In two cases, however, the internal TEA caused an increase in the inward current elicited by depolarization from Vh = - 4 0 mV. The reversal potential for the inward current was shifted to a more positive value after TEA injection in these cases (Fig. 4b).

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Fig. 3. Effect of intracellular TEA on the steady-state inactivation curve hoo (V) for the inward current measured in normal Ringer. The dashed line is drawn for the points obtained before the injection of TEA. The solid line describes the points obtained after the injection of TEA (50 nA, 150 sec). With the holding potentials more positive than - 4 0 mV both both curves coincide. Test pulses, 10 inV. Different signs correspond to different neurones. completely. Some scattering in the results may be easily explained if one takes into account the difficulties in controlling the acting intracellular concentration of TEA. Also, it should be noted that large T E A injections caused a progressive (as the injection continued) depression of the inward

The shift of the dose-response curve for external T E A application to the lower concentrations of the drug demonstrates the high accessibility of the membrane of isolated neurones to external pharmacological influences as compared to that of intact cells. As previously shown (Kostyuk et al., 1974a), the surface of isolated neurones being treated with trypsin is free of any connective tissue or glial covering. At the same time, all the components of the membrane current found in the intact neurones are present in the isolated neurones, and all their features already known are also preserved. This confirms that the procedure of isolation does not modify the ion-carrying mechanisms of the somatic membrane. Both the fast and delayed outward currents in the somatic membrane are subjected to the blocking action of TEA. As in the Ranvier node (Koppenh6fer & Vogel, 1969), T E A affects them from both sides of the membrane. A preliminary conclusion may be that the T E A receptors of both

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Fig. 4. Effect of intracellular TEA on the current-voltage relations for the peaks of inward current. (a) O, Initial inward current and /x, inward current measured after the injection of TEA (50 hA, 150 sec). Vh = - 4 5 mV. (b) •, Initial inward current and O, after the injection of TEA (50 nA, 150 sec). Vh = - 45 mV.

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P.G. KOSTYUK,O. A. KRISHTALAND P. A. DOROSHENKO

outward currents have an opposite location; equal but unknown concentrations of internal T E A affect the fast outward current more, while the delayed current is more sensitive to external TEA. This conclusion corresponds to the suggestion made by Neher & Lux (1972). The anomalous increase in the inward current after TE A injection observed in the two experiments has to be especially discussed. This effect was revealed by depolarization from Vh = - 40 mV, i.e. when the fast outward current was expected to be completely inactivated. It may have the following explanation: the inactivation curve for the fast outward current cannot be exactly measured at sufficiently positive Vh levels because of a decreased difference between Vh and the most positive V~ at which the fast outward current is not yet contaminated by other currents. In these cells the initial relation between the fast outward current and the inward current was probably especially unfavourable for the measurement of the latter. These exceptions show that reliable values of the reversal potential for the inward current may be obtained in the mollusc neurones after the injection of T E A blocking the fast outward current. The true value of the reversal potential for the inward current in isolated neurones could be close to 65 mV, as this value was not altered by the injection of TEA. The reversal potential between 60 and 80 mV is also characteristic for the slow inward current in the heart muscle fibres, which is in many respects similar to the inward current in the somatic membrane (Trautwein, 1973). This value is obviously much more negative than that calculated from the data about the free Ca concentration in cytoplasm (Ashley & Ridgway, 1969) and from the experimentally found data for the calcium inward current in barnacle muscle fibres (Keynes et al., 1973). The observed low value of the reversal potential for the inward current in the somatic membrane may be due to the properties of the channels being permeable to both Ca and Na ions.

REFERENCES ASHLEYC. C. d~. RIDGWAYE. B. (1969) On the relationships between membrane potential, calcium transient and tension in single barnacle muscle fibres. J. Physiol., Lond. 209, 105-130. GEDULDIGD. ~. GRUENERR. (1970) Voltage clamp of the Aplysia giant neurone: early sodium and calcium currents. J. Physiol., Lond. 211, 217-244. KEYNES R. D., ROJAS E., TAYLORR. E., & VERGARAJ. (1973) Calcium and potassium systems of a giant barnacle muscle fibre under membrane potential control. J. Physiol., Lond. 229, 409-455. KOPPENHOFER E. & VOGEL W. (1968) Wirkung von Tetrodotoxin und Tetra~ithylammoniumchlorid an der Innenseite der SchniJrringsmembran von Xenopus laevis. Pfliigers Arch. ges. Physiol. 331,361-380. KOSTYUKP. G., KRISHTALO. A. & DOROSHENKOP. A. (1974a) Calcium currents in snail neurones--I. Identification of calcium current. Pfliiger's Arch. ges. PhysioL 348, 83-94. KOSTYUK P. G., KRISHTALO. A. tg. DOROSHENKOP. A. (1974b) Calcium currents in snail neurones--II. The effect of external calcium concentration on the calcium inward current. Pfliiger's Arch. ges. Physiol. 348, 95105. KOSTYUK P. G., KRISHTALO. A. & DOROSHENKOP. m. (1975) Outward currents in isolated snail neurones--I. Inactivation kinetics. Comp. Biochem. Physiol. 51C, 259-263. NAKAJIMAS. (1966) Analysis of K inactivation and TEA action in the supramedullary cells of puffer. J. gen. Physiol. 49, 629-640. NEHER E. (1971) Two fast transient current components during voltage clamp on snail neurons. J. gen. Physiol. 58, 36-53. NEHER E. & Lux H. D. (1972) Differential action of TEA + on two K+-current components of a molluscan neurone. Pfliiger's Arch. ges Physiol. 336, 87-100. TRAUTWEIN W. (1973) Membrane currents in cardiac muscle fibers. Physiol. Rev. 53, 793-835.

Key Word Index--Helix pomatia; nerves; outward currents; TEA.