Membrane currents related to configuration changes in the action potential of frog atrial muscle in Na- and Ca-free conditions

Journal

of Molecular

and Cellular Cardiology

(1982) 14, 371-379

Membrane Currents Related to Configuration Changes in the Action Potential of Frog Atria1 Muscle in Na- and Ca-free Conditions M.

Urata

and

M.

Goto

Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka 8 12, Japan (Received 4 August 1981, accepted in revised form 1 March 1982) M. URATA AND M. GOTO. Membrane Currents Related to Configuration Changes in the Action Potential of Frog Atria1 Muscle in Na- and Ca-free conditions. Journal of Molecular and Cellular Cardiology (1982) 14, 371-379. Changes in membrane currents and action potential in frog atria1 muscle under Ca-free or Na-deficient conditions were investigated using double sucrose-gap techniques. In Ca-free Ringer, the instantaneous current (Zkr and others) decreased and the delayed outward current (I,) was markedly retarded. The slow inward current (Z,,,,) d’rminished in the absence of the calcium inward current (Zcs). The remaining slow sodium inward current (I xa stow) was less than one fourth in amplitude of the control Zslow, and showed a faster activation and a slower inactivation. In Na-deficient Ringer when LiCl or sucrose replaced NaCl, the Isrow increased in amplitude, despite the elimination of Zxa slow. The Zkt also increased, but the amplitude of fully activated ZX diminished although the activation of ZX appeared much faster than in the control. Thus, it became clear that in a Ca-free condition, the overshoot of the action potential (AP) decreased due to the absence of Zca, and the AP-duration was prolonged due to the pronounced delay of activation of IX and the depression of Zk,. The Zxa stow was too small to contribute to the delayed repolarization of AP. In Na-deficient conditions, the AP-amplitude increased due to enhancement of Zca, and the AP-duration was shortened mainly as a result of the fast activation of Z, and due to the increase of Zkr. KEY WORDS: Frog atria1 muscle; Voltage Action potential.

clamp; Slow channel;

Membrane

current;

Na- or Ca-free;

Introduction Na and Ca ions play an important role in excitable membrane, particularly in cardiac cells, and it is well established that Na ions participate in the fast inward current while both Na and Ca ions contribute to the slow inward current (Zsrow). Na and Ca ions also regulate Na-K pump and Na-Ca exchanges [.5, 6, 131, and inducing manifold effects, finally affect excitation-contraction coupling mechanisms [ZZ, 24, 281. Although numerous investigations of ZsrOw have been done since the early research by Rougier et al. [31], differences in the characteristics of the slow Na and Ca currents (Zxa slow, Zca) have not been fully elucidated. Chesnais et al. [4] investigated the sensitivity to H, Li and Mg ions of the Zxa slow and ZcIL, and suggested that Na and Ca ions did not necessarily penetrate through the same channel. Reuter and Scholz the kinetic properties of the [301 analyzed IslOw and estimated the selectivity of the slow 0022-2828/82/070371+09 M.C.C.

$03.00/O

channel for Ca, Na and K ions. Recently, however, Ca ions were found to ‘relate to Islow not only from the outside but also from the inside of the cell membrane [Z6, 201, and also relate to tlhe instantaneous and delayed outward currents such as Zki and Z, [7, 17, 181. Configuration of tlhe action potential, therefore, can be regulated by Ca and Na ions [I, 19, 22, 31, 321 in very compllex ways, and changes may be induced either in the inward Islo,.,, outward Zki and Zx or in their combinations. As to the configuration changes seen in Ca-free media, Garnier et al. [9] attributed the long duration of action potentials mainly to the presence of slow inward currents, whi,le Miller and Morchen [ZZ] ruled out that possibility and suggested Ca-dependent changes in Zt,,. We attempted to clarify changes in these membrane currents in the absence of Ca or Na ions and to determine the current components responsible for configuration changes of the action 0 1982 Academic Press Inc. (London)

Limitled u

372

M. Urata

and M. Goto

potential, under these conditions. The current components were clarified and evidence was obtained that changes in the outward current

Materials

components play an important role in determining the duration of action potential in Ca-free and Na-deficient conditions.

and Methods

Experimental procedures The experiments were muscle bundles from the and procedures were foregoing studies [IO, apparatus and electrical

carried out using atria1 bullfrog (Rana catesbeiana), similar in approach to 1.21. The experimental circuits were the same as

previously described. The membrane potential, current and contractile tension were measured mainly by means of the double sucrose-gap technique with diaphragms.

Preparations and chamber “Winter” bullfrog (November to March) of both sexes were used. The excised hearts were opened and rinsed in frog-Ringer solution at low temperature (5 to 10%). Thin muscle bundles of 0.4 to 0.5 mm width and 5 to 6 mm length which run in parallel were isolated from the left atrium and the epicardium was removed under a binocular was microscope (N 120 x ). The low temperature preferable for the isolation because it reduced the beat frequency and movement of preparation, and seemed to improve the eventual viability. The

muscle bundles were mounted in a five compartment chamber, for the double sucrose-gap experiments [I,?, 331. The central compartment (0.3 to 0.5 mm width) was perfused with normal or test solutions, the left and right intermediate compartments (1.0 mm width) with isotonic sucrose solution (250.5 mM contamination of residual Ca ions of about 0.1 mM), and the terminal compartments (10 x 5 mm pools) with isotonic KC1 solution ( 127 mM).

Solutions The normal Ringer solution contained, NaCl 110.0, KC1 2.5, CaCl, 1.0, Na,HPO, 2.15, NaHsPO, 0.85 and glucose 20.0 in mM, and pH was 7.4 f 0.1. The Ca-free solutions were simply obtained by omitting CaCI,. In Na-deficient solutions when sucrose was substituted for NaCl, the content

was 10 mM NaCl to maintain steady the current and voltage conditions. In complete Na-free solutions, NaCl was substituted by equivalent Tris-Cl or LiCl. Tetrodotoxin (TTX) was used at a concentrathe fast Na tion of 5 x lo-’ g/ml to block current.

Experiments and recordings All experiments were performed at a constant temperature (17 & 0.5%). The muscle in the test compartment was usually driven at a frequency of 0.1 Hz with square wave pulses (5 to 10 ms, about twice the threshold voltage), and was equilibrated in normal Ringer solution for about 1 h before changing the solution.

The membrane potential, current and contractile tension were monitored on a triple beam oscilloscope (Nihon Kohden, VC-9), photographed with a long recording camera (Nihon Kohden, PC9B), and simultaneously recorded on a four-channel rectified pen recorder (Nihon Kohden, PMP 3004).

Voltage clamp experiment The membrane potential was clamped by using a voltage clamp feedback amplifier (Nihon Kohden, CEZ 1100). The potential was usually held at the resting gap potential or at a slightly hyperpolarized state up to 10 mV (-80 to -90 mV) except where noted in the text, and small de-

polarizing pulses (60 mV, 200 ms) were applied at basic intervals of 20 s. In order to determine the voltage-current relationship, a long hyperpolarizing or depolarizing pulse of 2.0 s and different voltage were applied at the same or longer intervals (up to 60 s). Usually, three series

Membrane

Currents

and Action

of examinations were repeated in each: the control, a test solution, and after washing with control solution. The mean of the pre- and postcontrols was plotted in comparison with that of test solution. It was possible to obtain from one preparation a series of 15 to 20 voltage-current relationships with good reversibility. In analyses of the slow inward current (Isrow),

Potential

373

50 N 60 mV depolarizing step pulses of various durations (0.02 to 4.0 s) were applied at the intervals of 20 s. In analyses of the delayed outward current (Ix), the large depolarizing prepulse (100 mV) of various durations (0.2 to 6.0 s) and second step depolarizing pulses (40 to 50 mV, 10.0 s) were applied at intervals of 60 s (see Figure 6 legend).

Results General features of the efects of Ca- or Na-deficiency Miller and Morchen [ZZ] studied the effects of Ca-free on action potential (AP) of the frog atrium extensively. Reconfirming their results, we observed that the early phase of the overshoot fell rapidly by more than several millivolts [Figure l(a)], the plateau also reduced but assumed a more horizontal form as the onset of the fast repolarization was progressively delayed [Figure 1 (b)]. The prolonged tail potential was sometimes accompanied by a step. AP duration increased time-dependently and the resting potential was depolarized and depended on the driving frequency. Twitch tension fell to a very low level in these Ca-free solutions, with an approximately exponential time course. In contrast to the effects of Ca-free, Na-deficiency (NaCl 10 mM, other substituted by sucrose) or completely Na-free solutions (see Method) induced a shortening of AP and a contracture [3] which was followed by more or less complete spontaneous relaxation of the muscle. Relaxation phase of the twitch contraction was however markedly delayed and the effect was sustained [Figure l(c)]. The resting potential was slightly hyperpolarized (- 10 mV), and in some preparations an increase of the overshoot was noted. These Na-free contractures and hyperpolarizations were not only due to changes in passive conductance but were related to the functioning of an electrogenic Na-Ca exchange mechanism [IO, II] which will carry inwardly one Ca and outwardly three or more Na [5, S, 131.

These effects are well known [I, 3, 101, but the changes in current components which underlie these effects are poorly understood. With regard to the effect of Ca-free, the prolongation of AP may be produced by a long duration of the response of Ina sr0w [9, 311, while an inhibition of outward currents may also contribute to some extent [ZZ].

FIGURE 1. Effects of &-free and Na-deficient conditions on AP and tension. (a) AP and tension immediately before, and 5 min (n), and (b) 3 min (0) and 15 min (a) after changing to Ca-free solution. (c) AP and tension immediately before, and 5 min (0) after changing to Na-deficient solution. Vertical calibrations are 50 mV and 50 mg, and the horizontal one is 1 s.

Membrane currents in Ca- or Na -free conditions under voltage clamp Figure 2(a) shows the effects of Ca-free Ringer solution on the membrane currents. The basal current level slightly shifted inward in the Ca-free condition, though such is hardly visible in this figure. A small depolarizing step pulse of 35 mV from the holding potential (- 85 mV) induced a time-independent outward current, 1kr, following the fast inward current [Figure 2(a), a]. The depolarizing step was subthreshold for generation of the slow inward current, IslOw, and tension did

not develop. It was also subthreshold for generation of the time-dependent outward current, 1%. The thus identified I& was clearly reduced in the Ca-free conditions. On the other hand, for large depolarizing pulses of about 130 mV which were close to the reversal potential of Isrow [Figure 2(a), b], a distinct time-dependent IX was elicited. In Ca-free solution, the 1, was strongly inhibited and the contractile tension disappeared. u2

374

M. Urata

and M. Goto

The current-voltage relationships in the control solution and in Ca-free conditions are illustrated in Figure 3.(a). The inward background current for hyperpolarizations (Zb), instantaneous outward current for depolarizations of low voltages

current for stronger (Id 2 and net outward depolarizations (Zki + Ix), were all inhibited in the Ca-free condition. A decrease in slow inward current may be due to a diminution of the calcium inward current (Zoa).

Cb)

FIGURE 2. Effects of Ca- and Na-free conditions on the membrane current under voltage clamp. (a) Membrane currents in Ca-free condition. Holding potential was - 85 mV. a, Clamp step was 35 mV where Zki was reduced in Ca-free (white dot). b, Clamp step was 130 mV where net outward current was inhibited in &-free conditions. (b) Membrane currents in Na-free conditions. a, Clamp step of 40 mV; b, 90 mV and c, 130 mV from holding potential of -85 mV. White dots mark the records in Na-free conditions. Note an increase of Zsi in “a” and difference in time-course of Ix in “c”. In both figures, vertical calibrations are 2 PA, 100 mV and 50 mg. The horizontal one is 2 s.

9-

(a)

(b)

6-

I -50

0

50

100

150

-50

0

50

100

15G

Voltage CmV) Figure 3. Effects of Ca-free and Na-free on current-voltage relationship in the constant presence of TTX (5 x lo-’ g/ml). Depolarizing and hyperpolarizing step pulses of 2.0 s duration were applied from a holding potential of -80 mV. In each figure, squares denote terminal current level for 2.0 s pulse, and circles denote peak value of inward currents. (a) Relationship in control (0, 0) and in Ca-free (m, 0) condition. Note an inhibition of Zb, Zsl, I, and ZSiO,w.(b) Relationship in control (0, 0) and in Na-free (m, 0) in which NaCl was replaced by Tris-Cl. Note an augmentation of Zkt. (c) Relationship in control (0, 0) and in Na-free (m, 0) condition in which NaCl was replaced by LiCl. Note similar changes except for Zs,,.

Membrane

Currents

Figure 2 (b) shows the effects of Na-free in which external NaCl was completely replaced by equimolar Tris-Cl. The basal current level shifted outward and such may contribute to the hyperpolarization of the membrane in the Na-free condition. In contrast to the effect of Ca-free, the steady outward current (mostly Zkr) for 40 mV depolarization was enhanced [Figure 2 (b), a]. hlthough in this case the slow inward current was activated, the enhancement of the outward current was confirmed by step pulses of lower voltages. Whereas, for larger depolarizing pulses, ZX was increased in the early phase [Figure Z(b), b and c], but when depolarizing pulses over 100 mV were used, the current trace crossed down to the control. Thus, the Z, appeared finally inhibited at the end of 2 s pulse [Figure 2(b), c], the tail current of Z, also being inhibited. The current-voltage relationships under conditions of replacement NaCl with Tris-Cl are illustrated in Figure 3(b). Here also the Zkr

and Action

375

increased, Zb decreased and the basal current level was shifted upward. The net outward current for depolarizing pulses over 100 mV was inhibited. The slow inward current in this Na-free Tris Ringer diminished, and after washing with control solution, the inward current showed a poor recovery, thereby suggesting that Tris-Cl suppressed generation of the slow inward current

[a. The current-voltage relationships in control and in Na-free condition in which NaCl was replaced by LiCl are illustrated in Figure 3(c). In this Na-free Ringer, It, was somewhat inhibited but later tended to recover to the controi level. As previously noted, however, Zkr increased in Na-free, and Z, was inhibited for larger depolarizing pulses, so that the curves showed a ‘6cross-over” at about 100 mV depolarization. It must also be noted the Zca was augmented in this Na-free condition. The details are described in the next section.

Effects of Ca- or Na-removal As the peak amplitude and the time course of . . Isrow were modrfied m Ca- or Na-free conditions, such may indicate that Zna srow and Zcs have different kinetics. Figure 4(a) shows the time course of control Islow for 60 mV depolarization of different durations (80 to 800 ms) . The holding potential was - 100 mV in this case and the terminal current level indicated roughly Zkr as the depolarization was still subthreshold for IX, the threshold potential of which is between -40 and -30 mV (see current level of 40 to 50 mV depolarization in Figure 3 where the holding potential was -80 mV). The effect of Na-free on Isrow is shown in Figure 4(b), in which NaCl was replaced with LiCl. The basal current level shifted to outward, and Isrow, which means Its, was activated at a slower rate than the control. Thus, the opening rate of the slow channel was apparently slower in the Na-free condition. The peak current of Zca measured from the terminal current level at 800 ms after depolarization was initially reduced, but was then gradually augmented until it became larger than control Isrow, as was also noted by Noble and Shimoni [27]. There was a tendency toward reduction with time. The time to peak for control Isrow was 70 ms, and 90 ms for Zca. The inactivation time course was slightly faster for Zca [Figure 4(c)], the time constant (~1) being 175 ms for control Islow and 135 ms for Zca. When NaCl was replaced with Tris-Cl or sucrose

Potential

on Islow

the time constant tended to be much faster for Zca. The time course of the tail current was somewhat slow in the Na-free solution [Figure 4(d)] and the time constant jr&) was about 30 ms for Isrow and 35 ms for Zca, although in case of the sucrose substitution the dacay tail appeared much slower for Zc,. These results suggest that there was no remarkable change for the time course of Zca when LiCl replaced NaCl. The mechanism related to the augmentation in the amplitude of Zca remains to be determined. The effect of Ca-free on the Isrow is shown in Figure 5. The basal current level shifted slightly inward, and Zstow that is Zna slow, was activated faster than control Zsrow, the interval between the end of the current trace at the holding potential and the point where Islow first crossed the basal current level during depolarization being shorter in Ca-free. This suggests that the opening rate of the slow channel was faster in Ca-free conditions. The peak current of Zxa slow measured from the terminal current level of 800 ms depolarization was about 0.5 PA, that is about one-quarter of time course was the control Islo,. The inactivation slower for ZN~ srow, the time constant (rr) being 150 ms for control Isrow and 350 ms for ZNa sr0w. The time course of the tail current was distinctly faster in Ca-free solution, the time constant (74) being 32 ms in control and 12 ms in Ca-free. Therefore, activation was faster and inactivation was slower for Zna ~r,,w.

M. Urata

376

and M. Goto

Time (ms) Time (ms)

FIGURE 4. Effect of Na-free on Islo,., in the presence of TTX. (a) Control response to various short (80 to 800 ms) step pulses of 60 mV depolarization from holding potential of - 100 mV. (b) Response in Nafree solution in which NaCl was replaced by LiCl. The peak inward current meant Zca. Vertical calibrations are 2 kA, 100 mV and 50 mg. The horizontal one is 300 ms. (c) Semi-logarithmic plots of inactivation of control IsloW and IQ+ (d) Semi-logarithmic plots of current decay tails. In each of (c) and (d), where 0 = control and 0 = Na-free, the mean for three different preparations was plotted.

FIGURE 5. Effect of Ca-free on Z,,, in the presence of TTX. (a) Control response to various short (80 to 350 ms) step pulses of 70 mV depolarization from holding potential of -90 mV. (b) Response in Ca-free solution. Step pulses were 60 mV depolarization, where Vertical calibrations are ZIYaslow was maximum. 2 PA, 100 mV and 50 mg. The horizontal one is 200 ms. (c) Semi-logarithmic plots of inactivation of control Islo, and ZN~ s~0W.(d) Semi-logarithmic plots of current decay tails. In each of (c) and (d), where 0 = control, l = Ca-free, the mean for three different preparations was plotted.

Analysis of delayed outward current in Ca- or Na-deJicient conditions As the delayed outward current (Ix) differed in and in Na-deficient appearance in Ca-free conditions, the properties of Z, under both conditions were analyzed. The time course of activation of Ix in Ca-free or in Na-deficient conditions differed distinctly as shown in Figure 6. There was a latency of Z, a&ivation in the Ca-free condition, but later Zx d&eloped rapidly to the extent of the control or over. In Na-deficient conditions, the activation of Ix was accelerated initially but the maximal amplitude attained was about half that of the control. A biphasic activation of I, was occasionevident in the Na-deficient condition, ally however, this point was not further investigated. Actual records of these experiments are shown in Figure 7. In Ca-free conditions, the activation of Z, during the first step pulse was slower, but

FIGURE 6. Activation curve of the delayed outward current, I,. The experimental protocol is shown in the inset. After a large initial test pulse (+ 100 mV) of different durations (0.1 to 6.0 s), the preparation was

Durafion of test pulse (s I

depolarized to a second step (140 mV, 10 s). The holding potential was -80 mV. The activation curve was obtained by plotting the amplitude of the tail current as a function of duration of the test pulse. (0) Control, (0) Ca-free and (,J) Na-deficient conditions. In Na-deficient condition, NaCl was replaced by sucrose with NaCl 10 rn~ remaining. Similar results were also obtained when NaCl was replaced with Tris-Cl.

Membrane

Currents

the amplitude of Ix in the tail was larger [Figure 7(c)] and the time independent outward current, Iki, was small [Figure 7(d)]. In Na-deficient conditions,. the activation of ZX was faster but the amplitude of 1x in the tail was smaller [Figure 7(b)], the time independent outward current

and Action

Potential

377

Iki being large [Figure 7(e)]. From these results, it is apparent that the development of outward current differs in Ca-free and in Na-deficient conditions, thus, the process of repolarization in the action potential may be directly influenced.

(d)

FIGURE 7. Actual records of tail current of delayed outward current, I,. (a) control, (b) in Na-deficient and (c) in Ca-free conditions. In each panel, the durations of the first step pulse were 2 s, 3 s and 4 s from the left to the right. (d), Current tail in Ca-free solution was compared with that in (e) Na-deficient conditions. The first step pulse was 100 mV, 3 s, and the second was 50 mV, 10 s.

Discussion In the present studies, we found that the instantaneous outward current, Zki, and the delayed outward current, IX, mainly contributed to the configuration changes in AI’. The prolongation of AP in Ca-free conditions appears to be basically due to the inhibition of background current (mainly Zk,), but maintenance of the plateau principally depended on the delay of the activation of IX (Figure 6). This conclusion is derived from evidence that the changes of 1x were larger in comparison with the change of 1~~ sr0W toward the end of a sustained depolarization to the plateau potential. For example, the maximum amplitude of INa slow was only one-quarter that of the control IsioW and such may rather contribute to the shortening of AP. The inactivation time constant (or) of I Na slow at 70 mV depolarization was not so prolonged [less than 0.4 s, see Figure 5(c)]. The extremely delayed [more than 2.0 s, Figure l(b)] but not diminished repolarization can hardly be explained by these characteristics of INa stow,

yet such would seem feasible if the delayed activation of Ix was indeed a contributing factor. The long tail potential of AP commonly observed in Ca-free conditions was not given particular attention, but it is highly likely that the membrane potential was sustained at the equilibrium potential of Ix (&) because a distinct decrease of K-permeability (Zk,) occurred aftler Ca-removal [see Figure 7 (d)] . In contrast to the form of AP in Ca-free condlitions, the shortened AP in Na-deficient conditions is attributed to the augmentation of Iki, and such may be induced by an increase in the intracellular Ca concentration [1, 17, 181. As the delayed outward current, Ix, was activated earlier, such may also contribute to the fast repolarization. The amplitude of 1, at the end of the long depolarizing pulse was inhibited (Figure 6), but did not seem to participate as the AP-duration never exceeded one second under these Na-deficient conditions. The slow inward Ca current, Zca, tended to increase when Na was replaced by Li, as was also

378

M. Urata

and M. Goto

found by Noble and Shimoni [27]. This may result from the finding that removal of extracellular Na results in a rise in intracellular Ca due to Na-Ca exchange mechanisms [25, 291 and the rise in turn causes an increase in conductance of the slow inward current system [%I. The time course of Zslow was modified in Cafree conditions (Figure 5). Here the remaining Isrow meant Zxa slow and its inactivation time constant (q) increased and that of tail current decreased. This means that the opening rate of the slow channel was fast while the closing rate was slow in the absence of Ca ion. The observations are well in accord with those of Rougier et al. [31], although they did not report the time constants. In Na-free Li Ringer, the inactivation time constant (q) of Zca and that of Zca tail current were not appreciably modified in our experiments. In the Na-deficient sucrose Ringer, the inactivation was accelerated and the deactivation was retarded. These changes, however, cannot be ascribed to the effect of Na ions until the role of Cl ions is clarified. With regard to the activation of the delayed outward current, IX, the activation curve (Figure 6) was similar to that reported by Brown, Clark and Noble [.?I. In comparison with their results,

the activation curve in Ca-free condition seemed to correspond to that of “ix fast”, and the activation curve in Na-deficient conditions, to that of “’ it was reported zx slow + k33Un “. Additionally, that in the case of rat uterine smooth muscle, the fast component of Zx remained unchanged in Ca-free conditions [23]. These results suggest that the Ca ions are related to the slow component of Zx or “i Bccum”, while the Na ions are related to the fast component of Zx, since the fast component diminished markedly in Na-free conditions [Figure 7(b), (ej]. These Z, components are now being studied in detail. The effect of Na-deficiency on Zki was similar to the effect of CaCl, injection reported by Isenberg [17, ZB]. As noted, Na-deficient conditions induced an increase in intracellular Ca concentration and this increase may be related to the conductance. Extrainfluence on potassium current related to the Na-Ca exchange may also contribute to alterations in the outward K current [II]. The details will be reported elsewhere, but many more studies have to be done to settle the problem. In conclusion, the effects of Na or Ca deficiencies on the membrane currents were much injection the same as those of CaCl, or K-EGTA [14-181.

Acknowledgements This research was supported by a Grant-in-Aid of Education, Science and Culture of Japan. reading of the manuscript.

for Scientific Research (00548090) from University, We thank M. Ohara, Kyushu

the Ministry for critical

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