A slowly inactivating calcium current in molluscan neurons—II. Kinetics and steady-state properties

0300-9629179/1001-0225102.oo/o

Camp. Bwhem. Phpd.. Vol. MA, pp. 225 to 234 gi Pergamon Press Ltd 1979. Pnnted m Great Britam

A SLOWLY INACTIVATING CALCIUM CURRENT IN ~OLLUSCAN NEURONS-~~. KINETICS AND STEADY-STATE PROPERTIES Institute of Neurophysiology

E. &ilLE and M. &LA and Psychophysiology, C.N.R.S., 31, Chemin Joseph~Aiguier, 13274 Marseilles, France

(Receitu?d 23 January 1979) Abstract-l. The process causing the slow calcium current decline during a long lasting depolarization was investigated in EDTA/EGTA injected cells. 2. Intracellular EDTA or EGTA induced prolonged action potentials. The EDTA/EGTA plateaus were abolished by Co or La, they persisted in presence of lo-’ M TTX. 3. Intra~llul~ EDTA/EGTA increased the amplitude of the slow Ca current and shifted its reversal potential towards positive values. 4. Substitution of calcium by barium in the external solution led to the same results as the EDTA/ EGTA injection. 5. The experimental results were analyzed according to two hypotheses which could account for the slow current decline; shift of the Ca reversal potential or inactivating process. Tests performed in various conditions are in favour of a slow inactivating process. 6. It is concluded that the Ca channel of Heiix neurons has fast and slow inactivating processes. The slow Ca current is involved in the lengthening of the action potential during repetitive firing.

INTRODUCTION

In the first paper of this series (Ehile & Gala, 1979), we have described several experimental faots which strongly suggest that the plateaus induced by TEA in some molluscan neurons are due to a long lasting calcium current. In voltage clamp conditions, long depolarizing voltage pulses led to a slowly developing

outward current which was interpreted as the slow decline of a calcium current, fastly activated by the pulse. However one important point still remains obscur concerning the membrane mechanism which slowly reduces the Ca current. Two different mechanisms have been anticipated: a slowly inactivating process or a change in the electromotive force due to Ca accumulation inside the cell. In order to test the hypothesis of a slowly inactivating calcium current it was necessary to find experimental conditions in which the hypo~etical slow Ca current could be easily identified among the various other ionic currents. A first attempt to increase the relative weight of the Ca current consisted in the inhibition of part of the K current with TEA. Another way was to increase the calcium current by lowering the intracellular Ca concentration with Ca chelating agents (Meech, 1974a; Kostyuk & Krishtal, 1977; Krjevic, et al., 1978). This method was introduced by Meech (1974a) who injected EDTA (ethylene diamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) in molluscan neurons by pressure applied to a microelectrode filled with the chelating agents. METHODS

The preparation and the electric arrangement have been described in the preceding paper. EDTA or EGTA microelectrodes were obtained by pulling glass pipettes with 1-3 225

glass fibers. The microelectrodes were filled with 0.5 M EDTA or 0.7 M EGTA neutralized to DH 7 bv NaOH. Two microelectrodes were inserted in’ the &ma; the EDTA/EGTA electrode and a classic KC&electrode used for potential recording. Intra~ll~~ iontophoretic injections of EDTA/EGTA were operated by passing current pulses (i_lOnA) between the two electrodes. In voltage clamp conditions the EDTA/EGTA electrode was used to inject the feedback current. The effects of intracellular EDTAFGTA were observed after 2-4 hr iontophoresis. The transport of one chelating molecule needs 4 x 96.500 24 10’ cb. The current pulses were about 8 nA, equivalent to a constant current of 4 nA which transports 10- ‘*M EDTA/EGTA per sec. For a cell having a diameter of 2OOpm, the intracellular volume is about 4 10m91. Thus, the concentration of EDTA after 1 set of current injection amounts to 2.5 PM and after 2 hr: 18 mM. This value is a theoretical maximum since we suppose that all the current is transported by the EDTA/ EGTA molecules and that the injected molecules do not diffuse out of the cell soma. The corresponding intracellular calcium cannot be calculated directly from such theoretical concentrations of chelating agents since an unknown part of them is continuously blocked by Cal+ ions entering the cell (Stinnakre & Taut, 1973; Eckert et at., 1977; Gorman & Thomas, 1978). However, it appeared that after 224 hr injections, the effects of EGTA/EDTA reach a steady-state which is then kept constant for several hours even if the iontophoresis is stopped. This steady state must correspond to a constant level of the EDTA/EDTA-Ca equilibrium. RESULTS

After 2-4 hr injection, the first effect of intracellular EDTA/EGTA was an increase of the excitability of the cell (Fig. la). Then the cells displayed slight depolarizations which could further trigger action poten-

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Fig. 1. Long lasting action potentials in EDTA-injected cells. Series a: Excitability increase induced by intracellular EDTA. a, : cell in normal saline before EDTA injection a2 : after 2 hr iontophoretic injection of EDTA; the same current pulse (5 nA) induces one spike in normal cell and two spikes in the injected cell. Series b: Same cell as illustrated in a. The spikes in b2 were observed after 3 hr injection of EDTA. Series c: Intracellular EDTA prolongs the plateau induced by TEA. c, : spikes triggered by a 6 nA constant current in the cell bathed in normal saline; c2: plateau after 20min in presence of 20 mM TEA: c3: after 4 hr iontophoretic injection of EDTA. the TEA plateau is prolonged.

tials characterized by slow repolarizing phases (Fig. lb). This effect is very similar to that described by Meech 1974ab in R,, Aplysia neurons injected with EGTA. The spontaneous firing was irregular and the spikes were either singly or in doublets or followed by a long plateau (Fig. lc). The plateau level was close to the spike overshoot and it lasted several seconds. The plateau ended suddenly so that the cell generated long square shaped potential waves. These square potentials bear a close resemblance with those induced by substituting Ba for Ca in molluscan neurons (Ducreux & Gala, 1977). Since in Ba-treated cells it is assumed that Ba2+ ions flow in the Ca channel more easily than the calcium itself (Magura, 1977) it is very likely that intracellular EDTA/EGTA has a similar effect, i.e. it increases the Ca current. When the cell was first treated with TEA, the delay for the effects of the iontophoresis was not significantly modified but the plateau duration was twice that induced by TEA alone (up to 16 set), (Fig. lc). The production of spikes followed by long plateaus was facilitated by repetitive stimulation: the higher the frequency, the sooner the plateau appeared (Fig. 2). Conversely, after a plateau spike, l-2 min of recovery were necessary to trigger another plateau spike. The plateaus were reversibly suppressed by a number of Ca blockers such as cobalt (Fig. 2) and lanthanum which reinforces the idea that during the plateau, the cell is mainly permeable to Ca ions. low5 M TTX did not prevent the plateau formation. II Slow current in EDTAiECITA injected cells The cell was activated by long pulses (duration lO20sec) applied in voltage clamp conditions; the current was recorded at a low speed in order to observe the slow components of the current following the initial fast events (inward Na + Ca current, outward K current). In normal saline, after the fast events, the current slowly reached a steady state with a time constant of several set (Fig. 3a). In EDTA/ EGTA injected cells the initial and final levels of the slow current during a long lasting pulse were shifted towards negative values (Fig. 3b). This effect was simi-

lar but more pronounced than that produced by extracellular TEA. In particular. EDTA/EGTA induced an inward current which slowly disappeared (Fig. 4). If we denote AT the amplitude of the slow current changes during long lasting pulses, this quantity was increased 2~-3 times in injected ceils. However, the threshold (between -30 and -4OmV) and the shape of the slow current were not affected (Fig. 3c and 3d). By increasing the pulse amplitude, AI increases in both control and injected cells, up to a maximum and further declines. The potential at which the slow current vanished was located at positive values. This apparent reversal potential was shifted to positive values in the injected cells. In control cells treated with TEA (in order to reduce the potassium current which obscured the initial part of the slow currents) the reversal potential was at EDTA -TEA

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Fig. 2. Repetitive stimulation of an EDTA-injected cell bathed in 20 mM TEA-saline. The membrane was activated by repetitive current pulses at the frequencies indicated on the recordings. With frequencies higher than 0.75 Hz, a plateau is produced. Adding 1OmM cobalt to the TEA-saline blocks the plateau production which is restored after 6 min washing with the TEA-saline.

Slow calcium current in molluscan neurons-II

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Fig. 3. Slow current in EDTA-injected cells. (a) In both series, the membrane was depolarized by long voltage pulses (2Osec duration) at the levels indicated on the traces. The long pulses induced fast current transients (not perceptible in these slow recordings) followed by a slowly developing current (holding potential -48 mV). Upper series: cell in normal saline. Lower series: after 5 hr EDTA injection; the slow current amplitude is enhanced and it becomes partly inward. (b) Amplitude of the slow current (AI) versus potential of the pulse, in normal saline and after EDTA injection. (c) Same representation as in b for a cell bathed in normal saline, then in 20 mM TEA-saline and after 3 hr EDTA injection. The reversal potential of the slow current passes from + 16 mV in normal saline to + 50 mV in the injected cell bathed in TEA-saline (holding potential: -53 mV).

-I-33mV (mean of 15 cells). In injected cells (without TEA) the reversal potential varied greatly from cell to cell: from + 18 to + 56 mV. If we assume that the reversal potential is the true equilibrium potential of the slow current, we calculate that the maximum conductance of the slow channel is 1.44 nA~mV in TEAtreated celis and 3.24nA/mV in injected cells: Fig. 4 illustrates the currents recorded during long voltage

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Fig. 4. Cadmium ions block the EDTA/EGTA-induced slow current (a) Currents produced by 20sec voltage pulses. The potential during the pulse is indicated on the current traces (holding potential: -42 mV). (b) Slowly developing current after 4.5 hr iontophoresis of EGTA. Note that most of the slow current is inward. (c) The slow current is blocked by 2mM cadmium chloride. The current is outward in the three recordings. The slow tail current following the pulses in series b is also inhibited by Cd’+. (d) Cadmium-inhibited current obtained by substracting the corresponding current traces in b and c.

pulses in a cell bathed in normal saline before and after 4 hr injection of EGTA. As already described, EGTA enhanced considerably the slow component of the currents the amplitude of which increased with the pulse amplitude, up to a maximum (for voltage pulses near 0 mV). This slow current had an inward phase which was fully suppressed by adding inhibitors of the calcium current: Co’+, Cd”, La’+ and Mn’+. The steady-state current during the pulse was only slightly affected by the Ca blockers, so that the current suppressed bi the Ca blockers appeared as a slowly declining inward current (Fig. 4d). The slow current persisted in normal saline containing 5 x lo-’ M TTX. Therefore it is very likely that the slow current induced by EDTA/‘EGTA is similar to the slow Ca current described in TEA-treated neurons (Ehile & Gola, 1979).

The effects of substituting Ba*+ to Ca” in excitable membranes are well documented. In particular, in molluscan neurons Ba*” ions induce long lasting potentials produced by a persistent or slowly inactivating inward current (Magura, 1977; Gola et aI., 1977). The effects of the Ba-Ca substitution on the slow current are shown in Fig. 5, where it appears that the time course of the slow current is not modified except during the first second of the pulse where its initial phase become inward. This can be ascribed to the potassium blockade produced by Ba’+ ions (Magura, 1977). The second effect of Ba” was to reduce the leakage current. The siow current in Batreated cells is inhibited by the same calcium blockers than those acting on the slow current in normal ceils.

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‘OnAt-_ 10 set 25nA L 1Osec Fig. 5. Slow current in barium-treated cells Series a: slow currents produced by long voltage pulses (11 set duration). The potential during the pulse is indicated on the traces. Holding potential = -54mV. Series a,: cell in normal saline (Ca ‘+ = 8 mM) Series a, : after replacing Ca” by the same amount of Ba ‘+. In Ba saline, the slow current amplitude is increased but its time course is not altered. The slow current becomes partly inward. Series b and c: blockade of the slow current in Ba-treated cells by 5 mM mephenesine (series b) and by 2 mM Cd’+ (series c). (a, b and c are from three different neurons). In current clamp conditions, the Ba-Ca substitution led to the production of spike plateau patterns closely resembling those produced by the intracellular injection of EDTA/EGTA in TEA treated cells. However, the plateaus lasted up to 50sec in Ba-treated cells instead of 16 see in injected cells. They were abolished by the Ca blockers and they displayed the same frequency dependence than the EGTA/TEA plateau: the duration of the plateau was reduced by repetitive stimulation. These analogies are not surprising since both experimental Methods result in a common modification, that is to say a reduction of the intracellular calcium concentration. Barium is known to flow in normal calcium channels more easily than calcium itself (Heyer & Lux, 1976; Magura, 1977; Akaike et (II., 1978). Moreover, barium is more electronegative than calcium and it can displace most of the intracellular calcium. Since intracellular calcium is necessary for the activation of part of the potassium conductance (Meech, 1974; Meech & Standen, t975), barium causes a strong reduction of that potassium current, unmasking the initial part of the slow current carried by barium. IV Properties of the slowly declining CQ current All the experimental evidences presented above and in the preceding paper are in favour of a slowly decreasing Ca current, activated by depolarization.

M. WXA However, the lack of information concerning the membrane conductance changes during the slow current decline (Ehile & Gala, 1979) does not permit to ascribe this decline to an inactivating process or to a shift in the equilibrium potential of Ca ions. In order to give a clear response to this question, the experimental data have been analyzed according to these two hypotheses and the reconstructed currents have been compared to the experimental currents. I .iZ~tit.clrion-inicc’tir:uticlri .s~~quen~‘e. in this hypothesis. the slow decline would be due to a slow inactivating process which develops upon depolarization. The steady-state level of inactivation was determined by measuring the slow current at a given test potential after a long conditioning at various potential levels. The slow current on the test potential was plotted versus the conditioning potential and the current was normalized to the maximum slow current obtained with a conditioning hyperpolarization. The results obtained in 7 cells bathed in TEA saline are shown in Fig. 6a. The experimental points can be approximated by a Z-shaped curve. from -5OmV (inactivation fully removed) to about 0 mV tslou current fuliy inactivated). The time constant of inactivation (ri) was obtained by semi-logarithmic plots of the slow current and of the slow tail current in TEA-treated cells. The values obtained in 15 cells are indicated in Fig. 6b which shows that the inactivation is very slow at -15 mV (ri up to 15 set) and that it becomes faster at positive potentials and near the resting potential. The rate constant of inactivation (r, and J1) were obtained from the steady-state inactivation curve (i) and the time constant of inactivation from:

rxi increases at negative potentials (Fig. 6c and 6d). The experimental by the following relations:

while pi decreases points were fitted

and

Zi and /Ii are expressed in set _‘. The curves relating the steady-state level and time constant of inactivation to the membrane potential were derived from the analytical expressions of ai and fli. They are represented as continuous lines in Fig. &a and 6b. The steady-state activation-voltage curve-~(V)was deduced from the slow current amplitude (AI) during long voltage steps. Changes in the slow conductance during the step are given by g = AI/ (I’ - E,,,) where E,,, is the apparent reversal potential of the slow current corresponding to the intersection of the AI = f(v) curves with the zero current baseline. The points were normalized according to the maximum conductance obtained at positive potentials. The results from 10 cells bathed in TEA-saline are shown in Fig. 7a. The continuous line passing through the experimental points is a sigmdid curve:

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Fig. 6. Kinetics and steady-state properties of the slowly inactivating calcium current. In order to reduce the delayed potassium current, all the experiments were performed on TEA-treated cells. a: Steady-state inactivation curve. b: Time constant of inactivation (see text for details) The continuous curves in a and b were traced according to the equations of the rate constants illustrated in c and d. c and d: rate constants a, and /I$versus membrane potential. The experimental points were approximated with a sigmdid curve for ai and with an exponential curve for pi.

In these cells, the maximum conductance was gca = 1, 44nAJmV. The tacit assumption in calculating the steady-state activation curve was that the slow decrease of the current was equal to its fast increase. The shape of the inactivation-voltage curve shows that this assumption is valid at positive potentials only. At negative potentials, the current is never fully inactivated which minimizes the actual level of activation. Thus, the experimental steady-state activation curve must be corrected for incomplete inactivation. This is achieved by correcting the experimental points of Fig. 7a according to:

potential corresponding to half activation passes from -18mV to -30mV. 2 Activatio~accumulation sequence. In this section we tried to evaluate the influence of an intracellular accumulation of Ca2+ ions on the kinetic of a calcium current deprived of inactivating process. We assume that the calcium current is activated at the levels corresponding to the experimental steady-state activation curve of Fig. 7a. The calcium current at potential V is given by: with gtd. = 1,2 nA/mV

Cc(V) = ii(V)/(l - i) the corrected levels of activation are shown in Fig. 7b where the points are reasonably well fitted by the following expression. &(v)=[l+exp(-y)]-l The correcting factor has the effect to shift the steadystate activation curve towards negative values: the

and E,,, = 29 log e

a, [Cal, and [Cali are the external and internal concentrations of calcium. The electric flux or (in nCb) corresponds to a calcium inflow of &, = dr/k (in nM) with k = 2 105. This inflow increases the intracellular Ca

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Fig. 7. Steady-state activation curve of the slowly inactivating calcium current Z(V): activation curve deduced from the AI (V) plots of the slow current amplitude in TEA-treated cells. The experimental points are approximated with a sigmoid curve. The half activation occurs at - 18 mV. &(v: corrected steady-state activation curve. The experimental points were corrected for incomplete inactivation according to ZC= Z (1 - 7) where 7 is the steady state level of inactivation illustrated in Fig. 6a. The half activation is shifted to - 30 mV.

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clearly indicates that the ;&t>\c mrtdrl cann~~i ~zccoun~ for the slow current changes ohser\cd 11)long ~oltagc pulse experiments: the speed ()I’ decline i\ much slower than in experimental situ:\tions and the le\el of the current after 20 see dcpolarlration it X0-90”,. of its initial values. More th;m .SOOse’i‘ of depolarilation are needed for the \tcad>-\t:tt< to be reached instead of 10 20 see in
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Fig. 8. Slow current changes associated with the activation accumulation model. a: Slow decline of the inward calcium current produced by intracellular accumulation of calcium entering the cell. (See text for details of the model). The hypothetical cell is depolarized for 20 set at the levels indicated on the traces, from a holding potential of -5OmV. The current decline is obviously slower than in experimental recordings. b: Same conditions as in a. with longer voltage steps. by :

con~n~ation

[Ca]i = - #M/C where t’ is the cell volume. The sign minus comes from the convention that inward currents are negntive. The instantaneous change in [CaJ will be:

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(a) Efl&rs rlf’u conditiorung plrlst,. These experiments were performed in TEA-treated cells. The membrane was first depolarized by a long conditioning pulse (5 set duration near 0 mVI and then by a test pulse similar to the conditioning one. The two pulses were separated by a variable delay. Thr initial level of the slow current (I, clI during the test pulse was measured. With a small delay. I, 0 was shifted towards positive values: it exponentially recovered its control level with a time constant of several seconds (8.3 set in the experiment illustrated in Fig. 9). similar to that of the slow tail current following the conditioning pulse. (b) Kinetics of’jivrt,ard mtl htrd ~trrtf sh c’frrrenfa. In these experiments he compared the slow current kinetics at the same potential level following either a conditioning hyperpolarization or a conditioning depolarizations. The experiments were performed in TEA-treated cells. In the experiment iliustrated m Fig. IO, the cell was depolarized at -70 mV b! :I 15 set pulse and the slow current was recorded. Then. the cell was first depolarized at a more positive potential (- 10 mV in Fig. IO) and then partly rcpolarizcd 31 -20 mV: the current was recorded during the sccanrl voltage step at --?OmV. The first recording appeared as a 4~~wly increasing current whereas the second was a slowly decreasing current: both currents had similar time constant (4.4 and 4.2 hec 111Fig. 101. The two tests described aho\c can be Interpreted according to the two hypothesc\. inactivation or accumuIation. In the inactn ation hypothesis. the first test (two successive pulses) is the measure of the time constant of removal of inacti! atinn. whereas according to the second hypothchlh it must rotlcct the SIUW ing down of [CaJ by ;Icti\v proccsscs. Similarly. in the second test. the foraard backyard
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The inactivation hypothss1\ vrr> easily accounts for the Fact that the time cfmstants in the two tests are similar to that of the \lo\\ current. (‘onversely. according to the second hypothesis IL, account for these facts we must a\\urnt that the passive accumulation of calcium h:i> the same kinetic properties as its active extrusion or uptake, cium.

This non linear differential equation was integrated by numerical methods with a PDP 11 computer. The cell volume was of 4. 10e9 1 (correspon~ng to a 2OO/lrn diameter). From the [CaJ (f) curves, the changes in I,, were easily calcuiated. The resulting slow current changes are shown in Fig. 8. The upper series (Fig. 8a) shows the slow current decline during 2Osec pulses at different potential levels. This figure

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The following experiment5 \\crc performed in EDTA-injected cells bathed in TF+I zal~ne. The mem-

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Fig. 9. Time course of recovery from inactivation. a: Cell bathed in 20mM TEA-saline for 1 hr. The cell was activated by a conditioning pulse (5 set at - 10 mV; holding potential: - 50 mV) followed by a test pulse (13 set at - 10 mV). The delay between the two pulses is indicated (in set) on the voltage traces. With a short delay, the slow current amplitude is reduced owing to the shift of its initial level towards positive currents. b: Recovery of the initial current level (measured as indicated in inset): the exponential curve has a time constant of 6.1 sec. Note that the curve passes by the

black dot corresponding to the current at the end of the test pulse.

brane was depolarized (near 0 mV) by repetitive short current pulses (50-100 msec). The fast inward current was simultaneously recorded from an oscilloscope and with a pen recorder. Following a long depolarizing pulse (lG15 set near 0 mV) which produced the slow current, the fast inward current was considerably depressed; it slowly recovered its test amplitude with

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a time constant of several set similar to that of the slow tail current (T = 10 set in Fig. 11). Within the framework of the activation-inactivation hypothesis, the above result shows that the slow inactivating process is also acting on the fastly inactivated inward current. Therefore, during a long potential step, the calcium channels must display the following events: a fast initial activation, a fast inactivation which reduces the calcium current almost to zero followed by a slow inactivation acting on the remaining calcium current. This conclusion is reinforced by noting that the potential dependence of the slow Ca inactivation is similar to that described by Kostyuk et al. (1977) for the fast Ca inactivation.

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Fig. 10. Time course of forward and backward slow currents at the same potential level. Cell bathed in 20 mM TEA-saline; holding potential: - 50 mV. The cell was first activated at - 20 mV during 16.5 sec. In the right hand part of the recording, the cell was first activated at -1OmV for lOsec, then the potential was reset to - 20 mV. In both recordings at -20 mV, the slow current has the same time constant (4.2 and 4.4sec in the righthand and left-hand recordings, respectively).

DISCUSSION

The experimental facts presented in the preceding paper have shown that the long plateaus of TEAtreated cells were due to a sustained calcium current. This conclusion is in accordance with the results of Bryant (1978), Horn & Miller (1977) and Klein & Kandel (1978). Tillotson & Horn (1978) reported that in caesium loaded neurons of Aplysia the time constant of inactivation of the Ca current is of 4.44.7 sec. The intracellular injection of calcium chelating agents amplifies the effects of extracellular TEA: ad-

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Fig. II. Long term inactivation of the fast transient current. Ceil injected with EDTA and bathed in TEA-saline. The membrane is activated near OmV by repetitive short pulses t,F = 0.2 Hr. duration l~msec). The fast inward current during the pulse is shown in the recording T of series a and as vertical deflections in raw h,. Following a 15 set depolarization at -5 mV which produces the slow current, the fast inward current is strongly depressed: (successive recordtngs in series a). The number attached to the recordings of a referred to those of b,. The fast transient current recovers its control amplitude with a time constant of IOsec.

ditional lengthening of the TEA-plateau and increase in the amplitude of the slow current produced by Iong voltage pulses. The same effects were obtained by replacing Ca’+ by Ba” + in the normal saline. All these effects are accounted for if we assume the existence of a slowly decreasing Ca current. This current must be increased by reducing the intracellular Ca which is the effect common to the intracellular EDTAi EGTA and to the Ba-Ca substitution. Besides, TEA, barium and intracellular EDTA,‘EGTA reduce part of the potassium current, which reveals the slowly decreasing calcium current. Several pieces of indirect evidence have been presented that support the existence of a slow inactivating process. These pieces of evidence are based on the kinetic properties of the slow current and of the slow tail current. To sum up it has been shown that the slow decrease (upon depolarization), the slow increase (upon repolarization) and the recovery have similar kinetic properties which strongly suggest that they are due to the same mechanism. This would not be the case if the slow decline was caused by Ca accumulation since other processes would be needed to restore the normal Ca concentration. In addition, the time constant of the slow current during a long voltage step is not related to the current amplitude even when this current is increased with barium or with intracellular EDTA. We can conclude that the decrease of the Ca current is mainly caused by a slow inactivating process. The existence of inactivation is supported by the sensitivity of the slow current to conditioning depolarization which allowed the steady-state inactivation curve to be determined. Kostyuk et al. (1977) and Tillotson & Horn (1978) reported that the Ca inactivation may include a very stow process in addition to the fast inactivation. However, the level of the reversal potential may indicate that shifts of the Ca reversal potential do occur during the fast initial inward current; even in presence of TEA, EDTA-injected cells have reversal potentials far from the accepted value of. the Ca equilibrium potential The steady-state inactivation curve is located between -50 mV and about 0 mV; it follows that in

this potential range the slow current is not fully inactivated and that it must persist in steady state conditions in accordance with the observations of Kostyuk ut uI. (1977). This conclusion is corroborated by the following observations: in Fig. IO the passage from -- 10 mV to -20 mV leads to an inwardly directed slow tail current having the same time constant than the inactivating process at this potential level. This slow tail current does represent the removal of part of the inactivation produced by the - 10 mV pulse. The second fact is illustrated in Fig. 4 in which the slow current of an EGTA-injected cell is blocked by cadmium ions. The cadmium-blocked current obtained by substracting the current traces before and after adding Cd ions is an inward current which does not fall to zero. This method has been used by Kass ct al.. (1976) to show that part of the slow calcium current in Purkinje fibres fail to inactivate. From these observations we can conclude that a persistent inward calcium current exists between -50 and 0 mV. Moreover, this could be true also at more negative potentials: the TEA-resistant slow tail current does not vanish for potentials more negative than -SO mV. Since the slow tail current represents the removal of the inactivation induced by a long pulse, it follows that the activation must not fall to zero upon repolarization, otherwise the change in the inactivation could not be observed. This permanent activation is probably very small since the slow tail current amplitude is of a few nA only. In order to evaluate the role played by the slowly inactivating calcium current in the electrical behaviour of the cell, the currents were reconstructed from the data relative to the activation and inactivation processes. Since we were mainly concerned with the slow current changes, the fast ionic events (fast transient inward currents) were not considered. Conversely, the leakage current and the delayed outward current, which both flow in opposite direction must be taken into account. The kinetics and steady-state parameters of the Ca current are indicated in Section IV. The leakage current was determined by the potential changes induced by steps of inward currents, In the model, the leakage

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&=L!( x2;

Fig. 12. Reconstructed slow current changes. The model includes the slowly inactivating Ca current, the delayed K current and the leakage current (see text for the parameters). a: The three upper diagrams show the current during 20sec depolarizing pulses in three situations corresponding to: a cell in normaf saline (gr = 14 nA/mV), a cell bathed in TEA or in Ba-saline or injected with EDTA/EGTA (gK = 5 nA/mV), a cell deprived of outward X current (gK = 0). b: The “apparent” activation curve deduced from the current traces with gK = 5 nA/mV. The curve is constructed according to the same criteria as used in the experimental part. The steady-state activation curve in S-shaped from -40 mV to 0 mV with a half activation at - 17 mV. The actual activation curve introduced in the program was that illustrated in Fig. 7b with a half activation at -3OmV. c: Current-voltage curves obtained with slow ramp pulses (slope lOmV/sec). The 1-V curve becomes N-shaped when the delayed K current is reduced. is given by I, = 0.7 (V + 5.5). The kinetics and steady-state properties of the delayed outward current are derived from the analytical expressions given by Cola et al., (1977) except that the inactivating process acts on 80% of the potassium current. This modification, suggested by the results of Leicht et al. (1971), was found necessary to account for the marked rectification displayed by the neurons we used. Three experimental situations were simulated:

current

1. the maximum conductance (gK) of the delayed K current was of 14nA/mV. This would correspond to a cell in normal saline; 2. gK reduced to 5 nA/mV, which simulates the effects of TEA, of intracellular EDTA/EGTA and of barium; 3. gK = 0 The reconstructed currents induced by 20 set depolarizing pulses (from holding potential = -40mV) are shown in Fig. 12 for the three situations. With gx = 14nA/mV (Fig. 12 al) the steady state current is always outward, and part of the calcium current is masked by the K current. Reducing g, to 5 nA/mV reveals the inward phases of the calcium current (Fig. 12 az). The steady-state current becomes inward for potentials more negative than -2OmV; the cell would tend to have this potential level which may lead to firing. The calcium current with gK = 0 is

shown in Fig. 12 as. The current voltage relationships of the hypothetical cells were obtained by using slow potential ramps (lOmV/sec) instead of long pulses. In the control condition (gk = 14nA/mV) the I-V curve is monotonous; its null current point is at -5OmV (Fig. ltb): such a cell must be silent with a resting potential of - 50 mV. When g, is reduced to S nA/mV the I-V curve becomes N-shaped and the null current point is now at - 20 mV, that is to say at a potential level more positive than the spike threshold. The membrane potential tends towards - 20 mV inducing a spiking. Then the slow inactivation reduces the N-shape of the I-k’curve and shifts the null current point towards negative values. Such changes would lower the frequency of spiking. Therefore, the slow calcium inactivation would participate to the frequency adaptation together with the slowly deveIoping potassium current described by Partridge & Stevens (1976). The plateaus produced by the slowly inactivating Ca current were observed in situations in which the delayed potassium current was reduced. It is known that in normal conditions the repetitive stimulation leads to the gradual reduction of the available delayed potassium current and consequently to the lengthening of the spikes (Gala, 1974; Thompson & Getting, 1977). This situation partly imitates the effects of the I< current blockade. The shape of the action potential

E.

234

EHILE

and M.

has a particular functional significance in the synaptic transmission; the release of the neural transmitter increases with the duration of the depolarization of the presynaptic membrane. The increase in spike duration during repetitive firing may increase the Ca influx per action potential (Gorman & Thomas, 1978) and may have a role in synaptic facilitation.

GOLA

KASS R. S.. SIEC;ELHACM S. & TSIIX K. W. (19761 Inconi-

plete inactivation of the slow inward current m cardiac Purkinje fibres. J. Physiol. (Lontl.) 2%. 127 118P KLEIN M. & KANDELE. R. (1978) Presynaptic modulatmn of voltage-dependent CaL + current. mechanism for hchavioral sensitization in .4pl~.sir1( ~rli/ornk~1.Proc tu~ru Acud. Sci. C’.S.il. 75, 3512 35th KOS~YUKP. G. & KRISHTAI.0. A. 11977) Eficcts of calcium and calcium-chelating agents on the inward and outward current in the membrane of mollusc neurones. J Phv.sio/.

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