Ca2+ release in both the presence and absence of perchlorate. dependences equally without altering the maxima in the amount of charge and in the

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1 J. Phy8iol. (1987), 390, pp With 9 text-fiure Printed in Great Britain PERCHLORATE AND THE RELATIONSHIP BETWEEN CHARGE MOVEMENT AND CONTRACTILE ACTIVATION IN FROG SKELETAL MUSCLE FIBRES BY L. CSERNOCH, L. KOVACS AND G. SZUCS From the Department of Physiology, University Medical School, Debrecen, Hungary, H-4012 (Received 8 July 1986) SUMMARY 1. The effects of perchlorate ions (1-8 mm) on intramembrane charge movement, myoplasmic Antipyrylazo III Ca2+ transients and contractile activation were examined in voltage-clamped cut skeletal muscle fibres of the frog. 2. Perchlorate shifted both the voltage dependence of charge movement and the rheobase of the strength-duration relation for contraction threshold towards more negative membrane potentials. 3. Both charge movements and myoplasmic Ca2+ transients were much slower at the new rheobase in the presence of perchlorate than in the control solution but there was no change in the threshold amount of charge or in the calculated peak binding of Ca2+ to troponin C. 4. The peak release rate had a steeper voltage dependence than the non-linear charge, but a lower concentration (2 mm) of perchlorate shifted both voltage dependences equally without altering the maxima in the amount of charge and in the rate of Ca2+ release. 5. The voltage dependence of the difference between total charge and charge at the threshold of Ca2+ transients agreed well with the voltage dependence of the rate of Ca2+ release in both the presence and absence of perchlorate. 6. It is concluded that the effect of perchlorate on contractile activation can be accounted for by its action on the intramembrane charge movement responsible for contraction, without significant effects on subsequent Ca2+ release from the sarcoplasmic reticulum or on Ca2+ binding to regulatory sites of troponin C. INTRODUCTION Several recent experiments have examined the possible relation between charge movement (Schneider & Chandler, 1973) and signal transmission between the transverse-tubule and the sarcoplasmic reticulum (s.r.) membranes. In this connection perchlorate anions have been reported to affect both the development of tension (Foulks, Miller & Perry, 1973; Foulks & Perry, 1979) and the voltage dependence of charge movement (Liittgau, Gottschalk, Kovacs & Fuxreiter, 1983) without affecting other voltage-dependent processes (Gomolla, Gottschalk & Liittgau, 1983).

2 214 L. CSERNOCH, L. KOVACS AND G. SZUCS The present experiments examined whether perchlorate altered charge movement alone or also modified subsequent steps of excitation-contraction coupling. Alterations in contraction threshold, myoplasmic Ca2+ transients and troponin saturation were compared with the modified kinetic and steady-state properties of the intramembrane charge. METHODS Preparations and solutions Single fibres were dissected from the semitendinosus muscles of frog (Rana esculenta) in Ringer solution (115 mm-nacl, 2-5 mm-kcl, 1-8 mm-cacl2, 2 mm-tris sodium maleate buffer), cut in a relaxing solution (120 mm-potassium glutamate, 2 mm-mgcl2, 0-1 mm-egta, 5 mm-tris sodium maleate buffer) and mounted in the experimental chamber. Experiments recording displacement currents and Ca2+ transients near the contraction threshold were performed in a single Vaselinegap voltage-clamp system (sarcomere length ,um) described by Kovacs & Schneider (1978). Experiments over a wider potential range studied stretched fibres (sarcomere length longer than 3-4,um) in a double-vaseline-gap system (Kovacs, Rios & Schneider, 1983). After the fibre had been mounted, the relaxing solution was replaced by the external solution (75 mm-tetraethylammonium sulphate, 10 mm-cs2so4, 8 mm-caso4, 3-1 x 1IO- M-tetrodotoxin, 5 mm-tris sodium maleate buffer) in the closed end or in the middle pool and by the internal solution (108 mm-caesium glutamate, 5-5 mm-mgcl2, 0-1 mm-egta, mm-cacl2, 4-5 mm-tris sodium maleate buffer, 13-2 mm-tris caesium maleate buffer, 5 mm-atp, 5-6 mm-glucose) in the open end pools. Perchlorate was applied as its sodium salt, prepared from perchloric acid (Merck, suprapure quality). The experiments were carried out at 3-5 C, the chambers being cooled by Peltier devices. Experimental arrangement and digital recording of the data The membrane potential of the fibres was held at -90 or -100 mv, and both the current (VI) and the optical (AI) transients accompanying the voltage-clamped depolarizing and hyperpolarizing pulses were measured. The VI signals were filtered by 10 khz upper-frequency cut-off. The details of the optical arrangement were described earlier (Kovacs & Sziics, 1983). To follow the light intensity changes (AI) the a.c. coupled amplifier was replaced by a track-and-hold circuit and a d.c. amplifier with 1 khz upper-frequency cut-off. Both the VI and AI transients were recorded on-line using a microcomputer system (HT-680X, HTSZ, Hungary) with a 10-bit analog-to-digital converter. The sampling interval was set to 100 or 200,us. Five consecutive sample points were added and the sums obtained represented data points for 0 5 or 1 ms intervals. In the case of simultaneous recording of VI and AlI, 200,ss sampling intervals were applied. To improve the signal-to-noise ratio the transients were averaged, the data points being collected in a 16-bit memory space. To attain maximum gain without saturating the analog-to-digital converter, the 100 or 200,s sampling points were also monitored graphically. 255 data points were collected from each transient. They were then stored using a hard-disk system (SZM 5400, IZOT, Bulgaria). The timing of the pulses was also controlled by the microcomputer, so that analog-to-digital conversion and pulse generation were perfectly synchronized. Determination of the contraction threshold The contraction threshold was determined by depolarizing pulses of increasing amplitude at constant duration. The just detectable contraction at the terminated segment of the fibre was observed visually using a compound microscope (x 400) equipped with a water-immersion objective. All further details have been reported earlier (Kovaics & Sziics, 1983). Measurement of intramembrane charge movement Charge movements were determined as described by Horowicz & Schneider (1981 a, b). Depolarizing pulses of length 60 or 100 ms and differing amplitudes were applied. After removing linear capacitive and ionic components from the current traces, the currents (IQ) were normalized to the linear capacitance of the fibre. The amounts of charge moved during depolarization (Q..) and

3 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 215 repolarization (Q.,,) were calculated by integrating the 'on' and 'off' parts of the IQ traces, respectively. The current necessary to charge the linear capacity was obtained using 100 ms, 30 mv hyperpolarizing pulses. The ionic component was removed from the current traces by subtracting a straight line fitted to the current between 60 and 100 ms. On analysing the current traces belonging to depolarizing pulses, first the capacitive, then the ionic component, was removed. The latter subtraction from the 'on' part was performed either in the way described for hyperpolarizing pulses (in the case of 100 ms duration) or by subtracting the mean of the last five to ten points (60 ms duration). Perchlorate prolonged the recovery of charge movement. Therefore, the 'off' part was recorded for at least 150 ms and the fitting procedure for the subtraction of the ionic component took place between the 100th and 150th points of the 'off'. In this way the integration of the total 'on' and 'off' parts gave good agreement between the QOn and Q., both in the control solution and in the presence of perchlorate. Measurement of myoplasmic Ca2+ transients The changes in intracellular Ca2+ concentration caused by depolarizing pulses were monitored by Antipyrylazo III at 720 nm. The dye was applied at a concentration of mm at the open end pools and reached the intracellular space of the voltage-clamped segment of the fibre through free diffusion. The change in the intrinsic absorbance of the fibre was corrected for by a procedure similar to the one described by Melzer, Rios & Schneider (1986a) using the signals measured at 850 nm. The calculation of changes in intracellular Ca2+ concentration was done as reported by Kovacs et al. (1983). Analysis of the data The first derivative of the Ca2+ transients was determined by calculation. The derivative of the nth point of the signal was obtained as the slope of the straight line fitted to data points (n-2), (n-1), n, (n+ 1), (n+2). The Ca2+ binding to the regulatory sites of troponin C was determined from the Ca2+ transient by the numerical integration of the equation: d [Ca-Trop]/dt = [Ca2+] [Trop] k.n- [Ca-Trop] k.,f, (1) where [Ca-Trop] is the concentration of the calcium-troponin complex, while the concentration of the free binding sites [Trop] is: [Trop] = [TroptOt,] - [Ca-Trop], (2) where [Trop0t.,,] is the total concentration of binding sites in the myoplasm. The parameters used in the calculations, [Tropt,t1] = 240,UM; kof, = 57.5 s-1; kl. = 2-9 x 107 M-1 S-1, were found to be acceptable for intact fibres in our circumstances. Further details are given elsewhere (Kovacs, Sziics & Csernoch, 1987). The steady-state voltage dependence of charge movement was fitted by the two-state Boltzmann model. Supposing that the charge (Q) moves between two positions in response to a voltage step, its distribution is described by: Q = Qmax/(1+exp{-(V-V)/k}), (3) where Qm.x is the maximal available charge, V is the voltage during the pulse, which 50% of the charge has moved and k is the steepness factor. The significance of differences was determined by Student's paired t test. V is the voltage at RESULTS Intramembrane charge movement and contraction threshold To obtain data about the perchlorate effect on the contraction threshold, the strength-duration relation was determined using pulse durations from 5 to 100 ms before and after the perchlorate treatment (Fig. 1). Perchlorate shifted the rheobasic potential in the hyperpolarizing direction from mv to mv

4 216 L. CSERNOCH, L. KOVACS AND G. SZUCS (mean + S.E. of the mean, n = 5). However, at a pulse duration of 5 ms the curve was not significantly altered ( mv to mv, n = 5, continuous lines in Fig. 1 were fitted with eqn. (4): P > 0 1). The (V-C)t= BT (4) E Ringer solution 0 Perchlorate 0. CL 0 E Pulse duration (ms) Fig. 1. Effect of perchlorate anions on the strength-duration curve of the contraction threshold. The data points represent the averages (± S.E. of the mean) of values measured on five fibres under control circumstances (0) and in the presence of 8 mm-perchlorate (El). In the course of the analysis eqn. (4) was fitted to the values obtained in the individual experiments. The parameter values calculated in this way were averaged and used to construct the continuous lines. The averaged parameters: BT = mv ms; C = mv (control) and BT = mv ms; C = mv (perchlorate). Five fibres; s = /sm; d = 56-98,um (s = sarcomere length; d = horizontal diameter in this and subsequent Figures). where V is the membrane potential during depolarization, t is the pulse duration, C and BT are the parameters of fit (Costantin, 1974; Adrian, Chandler & Hodgkin, 1969). One can see that the function gave a good fit for values between 5 and 20 ms both in the control and in perchlorate. To explain the shift in the strength-duration relation we examined the corresponding alterations in the charge movement induced by perchlorate (Liittgau et al. 1983) and also determined whether the amount of charge related to threshold movement was modified. The latter was explored by a comparison of charge movement in the presence and the absence of perchlorate at rheobasic potentials using 100 ms voltage steps. The investigations were restricted to this potential range to avoid the uncertainties in determining threshold charge at shorter pulse durations. In the experiment shown in Fig. 2, the rheobase potential and the threshold charge were determined. The contraction threshold was in absence of perchlorate and and mv in 1 and 8 mm-perchlorate, respectively. The amounts of charge moved at these potentials were 8-5, 8-5 and 7.4 nc 1zF-1 (filled symbols),

5 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 217 respectively. Hence, despite the shift in contraction threshold the amount ofrheobase charge was not measurably altered, though the time course of the charge movement was slower at more negative membrane potentials, in agreement with earlier reports (Liittgau et al. 1983). I/ 15 1;AAF' MoM-C ms mv 10 0 (nc AF' -632 mv mm-c mv 8 mm-c04-a A O- I Membrane potential (mv) Fig. 2. Effect of perchlorate on charge movement at the contraction threshold. On the right-hand side of the Figure the charge displacement currents at rheobase voltage are shown under control circumstances and in the presence of perchlorate anions (C104-). The records are averages of eight sweeps. The amounts of charg Tmoved were determined from the time integral of the corresponding current transients and plotted as a function of membrane potential during depolarization. The filled symbols on the left-hand side represent the charge-movement values at the rheobase, the open ones illustrate measurements below and above the contraction threshold at the given membrane potential. One fibre; no data recorded. Similar results were found in eleven fibres where the amount of charge belonging to rheobase potential was similar before and after treatment with 2 mm-perchlorate ( and nc uf-' mean+ S.E. of the mean, respectively). This observation is consistent with an unaltered relationship between charge movement and s.r. function. Fig. 2 also confirms the shift in the steady-state voltage dependence of charge movement in the hyperpolarizing direction previously reported (Liittgau et al. 1983). The charge movement observed in the presence of 1 mm-perchlorate at mv is about twice as much as one would expect from the control voltage dependence, and in 8 mm-perchlorate the charge moved at mv (A) was approximately three times that measured in the control. Because perchlorate did not alter the threshold amount of charge, the shift in the strength-duration relation can be explained in terms of an alteration of the voltage

6 218 L. CSERNOCH, L. KOVACS AND G. SZUCS dependence of charge movement. In the range of rheobase potentials the shift in the steady-state distribution is large, and therefore the rheobase is shifted to more negative potentials by perchlorate. At short pulse durations the threshold movement is attained at more positive potentials where the effect of perchlorate on the voltage dependence or the kinetics of charge movement is small. The shift in the contraction threshold then is minimal. Ringer solution Perchlorate 2 3ApF 50 ms Fig. 3. Effect of perchlorate on the secondary rising phase ('hump') appearing on the charge-displacement current. The records are averages of eight sweeps under control conditions (Ringer solution) and in the presence of 8 mm-sodium perchlorate. The membrane potentials during depolarizations are indicated between the corresponding traces. One fibre; s = 2-2,sm; d = 98 /Sm. Where a 'hump', called the Q, component (Huang, 1982; Hui, 1982), appeared in the transients near rheobase, the shift in the rheobase was accompanied by a similar shift in the appearance of the hump. Thus, in Fig. 3 the rheobase potentials were at mv in the control solution and mv in the presence of perchlorate, but the hump appeared around the rheobase in both cases. Fig. 3 also shows that perchlorate prolongs 'off' transients, in agreement with an earlier report (Liittgau et al. 1983). Intracellular application It is widely accepted that the chaotropic ions exert a significant lyotropic effect on membrane processes. Gomolla et al. (1983) found no effect on voltage-dependent processes other than contractile activation in the concentration range applied in our experiments (1-8 mm). Measurements were also carried out in the course of our work to rule out the possibility of a lyotropic mechanism. The effect on the contraction threshold and charge movement when replacing 8 mm-caesium glutamate with 8 mm-sodium perchlorate in the intracellular relaxing solution at the open end pool was studied. The perchlorate ions reached the voltageclamped terminated segment of the fibres through free diffusion. After changing the solution the membrane potential was checked by micro-electrodes but the bath

7 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 219 Ringer solution Perchlorate mv _ I 7 \ mv I I I /0 1 MA AF 50 ms Fig. 4. Effect of intracellularly applied perchlorate anions on the charge displacement currents. The records in the Figure are the averages of eight sweeps. The membrane potentials during depolarization are displayed between the traces. Data collection in the presence of perchlorate anions started 40 min after the addition of 8 mm-sodium perchlorate into the open end pool. One fibre; s = 2-8,um; d = 114,um. A mv-'- *. N 0m -. Sm [Ca2] I;M B 88 mv- 11 : Xk c D ;' -\ mv mv.. I 11 Fig. 5. Effect of 2 mm-sodium perchlorate on the Ca2+ transients measured at the contraction threshold. The membrane potentials displayed at the individual traces mean the ones sufficient to evoke just visible movement under control circumstances (A and B) and in the presence of perchlorate anions (C and D). The duration of depolarization was 100 ms (A and C) and 5 ms (B and D). The transients were obtained by averaging four sweeps in each case. One fibre; s = 2-2 /sm; p (vertical diameter) = 88,um; DT (total intracellular dye concentration) was in the range of /UM.

8 220 L. CSERNOCH, L. KOVACS AND G. SZUCS electrodes did not need rebalancing. The rheobase, determined by using 100 ms pulses of mv, started to decrease 5-6 min after the substitution min later it reached a new steady level at - 68' mv (mean + S.E. of the mean, n = 4). This shift in rheobase was accompanied by an increase in the charge movement and changes in its kinetics (Fig. 4), similar to that shown in Fig. 1. Perchlorate therefore has similar effects whether applied from inside the cell or extracellularly. This agrees with earlier results (Gomolla et al. 1983) in which a shift in contractile activation was observed after electrophoretic injection of perchlorate. Ca2+ transients and troponin saturation at the contraction threshold To assess the effect of perchlorate on intracellular Ca2+ redistribution and troponin saturation, Ca2+ transients were recorded with short (5 ms) and rheobase (100 ms) depolarization (Fig. 5) in fibres with a normal sarcomere length ( ium). In the control solution the rheobase signal (Fig. 5 A), had a smaller peak amplitude, a slower rising phase and a longer latency than that resulting from a 5 ms pulse (Fig. 5B), in agreement with earlier reports (Kovacs & Sziics, 1983; Kovatcs et al. 1987). Addition of 2 mm-perchlorate shifted the rheobase by 23-1 mv and reduced the rheobase signal (Fig. 5C) by about 20% compared to the control (Fig. 5A). Additionally, the rising phase was slower, the signal reaching its maximum after about 80 ms. The similar shapes of the transients obtained with a 5 ms pulse (Fig. 5B and D) demonstrate that removal of Ca2+ was not significantly influenced by perchlorate. Fig. 6 compares Ca2+ transients, charge displacement and Ca2+ binding to the regulatory sites of troponin C. In this experiment perchlorate shifted the rheobase from to mv, but in both cases the charge transfer was about 12 nc uf-1. In perchlorate the running integral of the charge-movement current rose and also recovered more slowly (Fig. 6A). A similar alteration was also observed in the rising phase of the Ca2+ transient (Fig. 6B). Nevertheless, the peak values of troponin binding of Ca2+ required to reach threshold (Fig. 6C) were almost identical. Table 1 shows that even though perchlorate reduced the peak amplitudes of the Ca2+ transients at the rheobase by about 20%, the calcium concentrations on the regulatory sites of troponin C are about the same. Finally, it was shown that the slower binding of Ca2+ to troponin in perchlorate is reflected in the contractile response by examining the minimum pulse duration (critical duration) required just to elicit movement at rheobase potential. Thus perchlorate increased the critical duration from about 55 ms to at least 80 ms, a lengthening comparable to the change in the time to peak troponin saturation. The finding that the same level of contractile activation, i.e. a just perceptible movement, corresponds to the same amount of charge and with the same maximal troponin saturation, demonstrates that the effect of perchlorate, at least at the low concentrations applied, is specifically attributable to an effect on charge movement in the absence of any influence on later steps in excitation-contraction coupling. Voltage-dependence of charge movement and Ca2+ release on stretched fibres Measurements over a wide membrane potential range were carried out on stretched fibres (sarcomere length longer than 3-4 /im) to avoid movement artifacts beyond the

9 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 221 Q (nc pf-1) ms 2. A[Ca2+] (#M) A[Ca-Trop] (#M) Fig. 6. Effect of 2 mm-sodium perchlorate on different steps in the excitation-contraction coupling at rheobase. 100 ms depolarization to -37X5 mv in the control solution (dotted lines) and to mv in the presence of perchlorate anions (continuous lines) was necessary to reach the contraction threshold. At these membrane potentials charge displacement currents and dye absorbance signals were recorded. The upper part of the Figure presents the charge movement as the time integral of the corresponding current transients (Q). The middle part shows myoplasmic Ca2+ transients (A [Ca2+], average of six sweeps each). The lower part illustrates the calculated time course of change in the concentration of the calcium-troponin complex. To calculate the Ca2+ binding to troponin eqn. (1) was used. One fibre; 8 = 2-4,um; p = 76 #m; DT was in the range of #m. contraction threshold. Fig. 7 compares Ca2+ transients recorded in control solutions (Fig. 7 A) and those observed in the presence of 2 mm-perchlorate (Fig. 7 B). To avoid run-down of fibres, pulse duration was decreased at large depolarizations. By applying appropriate pulse sequences efforts were made to minimize the distortion of perchlorate effect caused by increased dye concentration, which itself can modify the shape of the Ca2+ transients (Kova*cs et al. 1983).

10 222 L. CSERNOCH, L. KO VACS AND G. SZUCS Fig. 7 shows that the Ca2+ transients appeared at more negative membrane potentials in perchlorate (Fig. 7B) than in the control solution. The shape of Ca2+ transients evoked by a moderate depolarizing pulse (-61-6 mv in column B) resembled the one measured at the rheobase (Fig. 5C), proving that stretching did not alter the perchlorate effect significantly. Using large depolarizing pulses TABLE 1. Effect of perchlorate anions on the myoplasmic Ca2 + transients and on the Ca2+ binding of troponin at rheobase A [Ca2]max (um) [Ca-Trop]max (/tm) Fibres Control Perchlorate (3)/(2) Control Perchlorate (6)/(5) (1) (2) (3) (4) (5) (6) (7) * [ P Mean+s.E. 1P P <0-05 n.s. Columns (2) and (3) give the peak amplitudes of Ca2+ transients ([Ca2+]max) evoked by 100 ms depolarizing pulses under control circumstances and in the presence of 2 mm-perchlorate. The peak concentrations of the calcium-troponin complex ([Ca-Trop]max) shown in columns (5) and (6) were calculated from the corresponding Ca2+ signals using eqn. (1). (membrane potental -4.7 mv), it was found that perchlorate ions did not affect the rising phase and the amplitude of the signals. The effect of perchlorate on the steady-state voltage dependence of charge movement and of Ca2 + release was then compared in order to assess whether the effect of perchlorate was explicable in terms of its effect on intramembrane charge movement over the whole potential range. Current and optical transients were recorded simultaneously. Integrals of 'on' and 'off' charging currents were compared with the maximum rate of Ca2+ release, estimated from the maximum of the first derivative of Ca2+ transients assuming negligible Ca2+ uptake during the first few milliseconds (Baylor, Chandler & Marshall, 1983; Melzer, Rios & Schneider, 1984). In the course of the experiment, the charge-displacement current traces under depolarization could be evoked repeatedly with the same magnitude and kinetic features. On the other hand, the maximum of the first derivative of Ca2+ transients evoked by large depolarizing pulses generally decreased owing to the fibre run-down. Therefore, the sequence of pulses was selected in such a way that the possible changes both in the peak value and in voltage dependence could be described properly. Fig. 8 compares charge-voltage curves with Ca2+ release in the control solution (Fig. 8A) and in 2 mm-perchlorate (Fig. 8B) normalized to their maximum values. The continuous lines represent fitted two-state Boltzmann functions. Both the maximum charge and the maximumrv.0te of Ca2+ release at 0 mv were unchanged by perchlorate. The equality of Qmax values was obtained directly from the fits (Qmax = O-Of and ncuf-1, mean +s.e. of the mean, n = 2 and 4, in the control solution and in the presence of 2 mm-perchlorate, respectively).

11 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 223 A B N N.. N. *N mv N mv N N *42 6 mv N A[Ca2l 1 IM -61'6 mv 50 ms mv i I I - I I --'' I Fig. 7. Effect of 2 mm-sodium perchlorate on the myoplasmic Ca2+ transients. Membrane potential was decreased from -90 mv to the values indicated between the traces. At larger depolarizations the duration of pulse was shortened to avoid deterioration of the fibre. Traces are averages of two or four sweeps. One fibre; s = 3-4,um; p = 76,m; DT was in the range of ,M. The averages of the relative values at each membrane potential from different fibres are given in Fig. 8. The curves were constructed from the best fits of the model using every individual value (for the charge movement V = -35'1 and -50O1 mv, k = 12-7 and 12-2 mv; for (d [Ca2+]/dt)max V = and mv, k = 7-2 and 9-4 mv in the control solution and in perchlorate, respectively). The voltage dependence of charge movement and maximal release rate measured in control solution is shown in Fig. 8A. Ca2+ release appeared at more positive membrane potentials than the foot of the charge movement and showed a steeper voltage dependence, in agreement with the data of Rakowski, Best & James-Kracke (1985) and Melzer, Schneider, Simon & Sziics (1986b). In the presence of 2 mmperchlorate (Fig. 8B) the voltage dependence of charge movement and that of Ca2+

12 224 A L. CSERNOCH, L. KOVACS AND G. SZUCS 1-0*5-6' B I1 i *5-0 O Membrane potential (mv) Fig. 8. Membrane potential dependence of charge movement (0) and the maximum rate of rise of myoplasmic Ca2+ transients (El) in control solution (A) and in the presence of 2 mm-sodium perchlorate (B). The experimental data were obtained from two (control) and four (perchlorate) fibres, respectively. The symbols give the means (±S.E. of the mean) of the values at identical membrane potentials. The dotted (charge movement) and continuous (maximum rate of rise of Ca2+ signals) lines, representing the fit of the twostate Boltzmann model to the data, were constructed from the parameter values presented in the text. Five fibres; 8 = ,um; p = 82-98,um. release shifted towards the resting potential by about the same extent (the change in V is 15-0 and 11-9 mv, respectively) with no significant change in the steepness factors. Melzer et al. (1986b) have shown that the steady-state distribution of suprathreshold charge movement obtained by subtracting the threshold amount of charge (Qth) at the appearance of Ca2+ transients from the total charge (Q) was similar to the voltage dependence of the maximal rate of release. Thus Fig. 9 plots Q Qth against - membrane potential. The curves represent the voltage dependence of Ca2+ release

13 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 225 (taken from Fig. 8). These voltage dependences were similar in the control solution and shifted in a similar way in the presence of perchlorate. Thus the action of perchlorate is explicable entirely in terms of an action on the charge movement and not an influence on its relation to Ca2+ release. 0*5~~~~~~~ o Membrane potential (mv) Fig. 9. Suprathreshold charge movement and the maximum rate of rise of myoplasmic Ca2+ transients. In the experiments presented in Fig. 8 the amount of charge moved at the appearance of the measurable Ca2+ signal was taken as threshold charge and subtracted from the charge-movement values obtained at more positive membrane potentials. The differences, defined as suprathreshold charge, were averaged and plotted here for control solution (0) and for perchlorate (El). The maximum rate of rise of Ca2 + signals is represented by the lines describing the fit of Boltzmann distribution to the experimental points (same as the continuous lines in Fig. 8; here the continuous line gives the control values, the dotted one the data in perchlorate). DISCUSSION Gomolla et al. (1983) showed a selective effect of perchlorate on contractile activation with no influence upon either the inactivation of force or the threshold of the action potential and delayed rectifier. Liittgau et al. (1983) demonstrated a shift in the voltage dependence of intramembrane charge movement. These results indicate that perchlorate selectively modifies charge movements and has no direct effect on later steps in excitation-contraction coupling. For example, the effect of perchlorate on the rheobase potential was accompanied by a similar shift in the charge-voltage relation in such a way that the amount of charge associated with threshold movement remained constant and the saturation of troponin C reached the same maximum at rheobase. Additionally, the steady-state distribution for suprathreshold charge movement agreed with the voltage dependence of Ca2+ release, and both were altered in parallel in the presence of perchlorate. The parallel shift observed in the voltage dependence of both charge movement and Ca2+ release can be taken as evidence to prove that perchlorate shifts the voltage dependence of both sub- and suprathreshold charge. We did not test whether perchlorate facilitates all charged particles or whether its effect is restricted to a PHY 390

14 226 L. CSERNOCH, L. KOVACS AND G. SZUCS certain group of charges, for example to the Q,8or Qr component (Adrian & Peres, 1979; Huang, 1982; Hui, 1982). C. L.-H. Huang (personal communication) has found that the voltage dependence of the tetracaine-sensitive charge, which can be identified roughly with the Q, component and thought to regulate contractile activity, is altered by perchlorate treatment, whereas the tetracaine-resistant charge is not affected. Further experiments are needed to resolve the virtual or real contradiction: why does perchlorate alter the tetracaine-sensitive charge responsible for contraction selectively when it has no selective effect on suprathreshold charge movement responsible for Ca2+ release? Using perchlorate at a concentration of 1 or 2 mm a parallel shift was found in the voltage dependence of charge movement in stretched fibres. In earlier experiments a 3-fold increase in the steepness factor was observed in fibres with normal sarcomere length after using 8 mm-perchlorate. The differences obtained in the perchlorateinduced modification of steady-state distribution can be explained by the differences in the concentrations and the sarcomere lengths or simply by seasonal fluctuations in the sensitivity of the fibres. Our results show that the kinetics and voltage dependence of charge movement play an essential role in the regulation of the corresponding properties of Ca2+ release and contractile activation. The unique facilitating effect of the chaotropic anion perchlorate on charge movement can be utilized in further studies on the details of this role. We are indebted to Professor E. Varga for continuous support, to Margit Fuxreiter for participation during the initial period of the experiments and to Miss R. Ori for skilled technical assistance. The work was sponsored by the Hungarian Academy of Sciences (Grant No. 17/2-06/072 and OTKA 119). REFERENCES ADRIAN, R. H., CHANDLER, W. K. & HODGKIN, A. L. (1969). The kinetics of mechanical activation in frog muscle. Journal of Physiology 204, ADRIAN, R. H. & PERES, A. (1979). Charge movement and membrane capacity in frog muscle. Journal of Physiology 289, BAYLOR, S. M., CHANDLER, W. K. & MARSHALL, M. W. (1983). Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients. Journal of Physiology 344, COSTANTIN, L. L. (1974). Contractile activation in frog skeletal muscle. Journal of General Physiology 63, FOULKS, J. G., MILLER, J. A. D. & PERRY, F. A. (1973). Repolarization-induced reactivation of contracture tension in frog skeletal muscle. Canadian Journal of Physiology and Pharmacology 51, FoULKS, J. G. & PERRY, F. A. (1979). The effects of temperature, local anaesthetics, ph, divalent cations, and group-specific reagents on repriming and repolarization-induced contractures in frog skeletal muscle. Canadian Journal of Physiology and Pharmacology 57, GOMOLLA, M., GOTTSCHALK, G. & LUTTGAU, H. CH. (1983). Perchlorate-induced alterations in electrical and mechanical parameters of frog skeletal muscle fibres. Journal of Physiology 343, HOROWICZ, P. & SCHNEIDER, M. F. (1981 a). Membrane charge movement in contracting and noncontracting skeletal muscle fibres. Journal of Physiology 314, HoRowIcz, P. & SCHNEIDER, M. F. (1981 b). Membrane charge moved at contraction thresholds in skeletal muscle fibres. Journal of Physiology 314, HUANG, C. L.-H. (1982). Pharmacological separation of charge movement components in frog skeletal muscle. Journal of Physiology 324,

15 PERCHLORATE ANIONS AND CONTRACTILE ACTIVATION 227 Hui, C. S. (1982). Pharmacological studies of charge movement in frog skeletal muscle. Journal of Physiology 337, KovAcs, L., Rios, E. & SCHNEIDER, M. F. (1983). Measurement and modification of free calcium transients in frog skeletal muscle fibres by a metallochromic indicator dye. Journal of Physiology 343, KovAcs, L. & SCHNEIDER, M. F. (1978). Contractile activation by voltage clamp depolarization of cut skeletal muscle fibres. Journal of Physiology 277, KoVAcs, L. & SztUcs, G. (1983). Effect of caffeine on intramembrane charge movement and calcium transients in cut skeletal muscle fibres of the frog. Journal of Physiology 341, KoVAcs, L., SzUcs, G. & CSERNOCH, L. (1987). Calcium transients and calcium binding to troponin at the contraction threshold in skeletal muscle. Biophysical Journal (in the Press). LPTTGAU, H. CH., GOTTSCHALK, G., KovAcs, L. & FUXREITER, M. (1983). How perchlorate improves excitation-contraction coupling in skeletal muscle fibers. Biophysical Journal 43, MELZER, W., Rios, E. & SCHNEIDER, M. F. (1984). Time course of calcium release and removal in skeletal muscle fibers. Biophysical Journal 45, MELZER, W., Rios, E. & SCHNEIDER, M. F. (1986a). The removal of myoplasmic free calcium following calcium release in frog skeletal muscle. Journal of Physiology 372, MELZER, W., SCHNEIDER, M. F., SIMON, B. J. & SzUcs, G. (1986b). Intramembrane charge movement and calcium release in frog skeletal muscle. Journal of Physiology 373, RAKOWSKI, R. F., BEST, P. M. & JAMES-KRACKE, M. R. (1985). Voltage dependence of membrane charge movement and calcium release in frog skeletal muscle fibres. Journal of Muscle Research and Cell Motility 6, SCHNEIDER, M. F. & CHANDLER, W. K. (1973). Voltage dependent charge movement in skeletal muscle: a possible step in excitation-contraction coupling. Nature 242,