Decline of myoplasmic Ca2+, recovery of calcium release and sarcoplasmic Ca2+ pump properties in frog skeletal muscle.

639

Journal of Physiology (1991), 414, pp. 63-671 With 10 figures Printed in Great Britain

DECLINE OF MYOPLASMIC Ca2l, RECOVERY OF CALCIUM RELEASE AND SARCOPLASMIC Ca2l PUMP PROPERTIES IN FROG SKELETAL MUSCLE

BY MICHAEL G. KLEIN, LASZLO KOVACS*, BRUCE J. SIMONt AND MARTIN F. SCHNEIDERt From the Department of Biological Chemistry, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201, USA (Received 7 January 1991) SUMMARY

1. The two calcium indicators Antipyrylazo III (AP III) and Fura-2 were used simultaneously to monitor free myoplasmic [Ca21] in voltage-clamped cut segments of frog skeletal muscle fibres (8-10 °C). Antipyrylazo III was used for the relatively large [Ca21] transients during 100-200 ms depolarizing pulses to -20 to 0 mV and for the rapid decline of [Ca2+] during the 200 ms after the pulses. Fura-2 was used to follow the slow decline of the small remaining elevation of [Ca2+] during the following 16 s (slow recovery period) and to monitor resting [Ca2+]. 2. From 1 to 16 s of the slow recovery period [Ca2+] declined with two exponential components, having time constants of 19+ 0-3 and 13-5 + 1-5 s (these and all other values are means+ S.E.M. of eleven runs from seven fibres). At 1-2 s after the end of the pulses the amplitudes of the fast and slow exponential components of decline of [Ca21] were 34+7 and 31+4 nm, respectively. The resting [Ca2+] in these runs was 40+4 nM. 3. The time course of calcium bound to parvalbumin ([Ca-Parv]) was calculated from the [Ca2+] records using literature values for the parvalbumin kinetic constants. From 1 to 16 s of the slow recovery period the total calcium [Ca]T outside the sarcoplasmic reticulum (SR) was assumed to equal [Ca-Parv] + [Ca-Fura]. During this period [Ca]T declined with two exponential components having time constants of 1-7 + 0-2 and 14-2 + 1-4 s, the same as those for [Ca2+]. Assuming the total concentration of parvalbumin cation binding sites to be 1000 ,SM, the fast and slow components of [Ca]T had amplitudes of 117+21 and 147+16 4UM, respectively, at 1-2 s after the pulses. 4. The rate of decline of [Ca]T, -d[Ca]T/dt, was used as a measure of the net rate of removal of calcium from the myoplasm by the SR. From 3 to 16 s of the slow recovery period and in the resting fibre -d[Ca]T/dt varied with [Ca2+] according to Present address: Department of Physiology, University Medical School, H-4012 Debrecen, Hungary. t Present address: Department of Physiology and Biophysics, F41, University of Texas Medical Branch, Galveston, TX 77550, USA. t Author for correspondence and reprint requests. *

MS 9060

640

M. G. KLEIN AND OTHERS

A[Ca2"]'-L. The term A[Ca2+] represents the pump rate and L represents a constant rate of calcium leak from the SR. 5. For 40 nm < [Ca21] < 80 nm, the power n for the [Ca2+] dependence of pump rate was 3-9 + 06. This indicates that at least four highly co-operative calcium binding sites are present in the functional unit of the SR calcium pump. Since the SR calcium ATPase binds two calcium ions per monomer, a simple interpretation is that ATPase dimers constitute the functional pump unit. 6. The rate of release (Rrei) of calcium from the SR was calculated from the [Ca2+] records. The recovery of releasable calcium following a conditioning pulse was determined by applying test pulses at various intervals from 1 to 16 s after the conditioning pulse and determining the relative suppression of the test release compared to the conditioning release. 7. The recovery of releasable calcium occurred with two exponential components, having time constants of 1-5 + 02 and 31-6 + 5-6 s. The slower phase of recovery of releasable calcium was significantly slower than the slower phase of decline of [Ca]T, perhaps indicative of a delay between removal of calcium from the myoplasm and its availability for release from the SR. 8. The rate of recovery of releasable calcium varied with [Ca2+] to a power not significantly different from that obtained from the decline of -d[Ca]T/dt. Also, the magnitude of the calcium pump flux estimated from the decline of - d[Ca]T/dt was similar to the flux estimated from the recovery of releasable calcium after determining values for the SR calcium content and fibre parvalbumin concentration. 9. Thus using either the rate of recovery of releasable calcium or the rate of decline of [Ca]T as a measure of the rate of SR calcium uptake, the results were consistent with a fourth power dependence on [Ca2+] and with pump dimers being the functional unit of the SR calcium pump in voltage-clamped skeletal muscle fibres. INTRODUCTION

Activation of a skeletal muscle fibre is produced by the release of calcium ions from the sarcoplasmic reticulum (SR) in response to fibre depolarization. Cytosolic [Ca2+] becomes elevated and calcium ions bind to regulatory binding sites on thin filament troponin C molecules, removing the inhibition of mechanochemical interaction between the thick and thin filaments and permitting force production and fibre shortening. Fibre relaxation occurs when repolarization terminates calcium release, [Ca2+] falls, calcium ions dissociate from troponin C and inhibition of filament interaction is restored. In frog skeletal fibres relaxation can occur within a few hundred milliseconds of the cessation of stimulation, yet the return of calcium to the SR requires tens of seconds for completion (Miledi, Parker & Zhu, 1983; Cannell, 1986; Schneider, Simon & Szuics, 1987). Lowering of [Ca2+] and fibre relaxation without full return of calcium to the SR is accomplished by calcium binding to the soluble protein parvalbumin, which is present in relatively high concentration in frog skeletal fibres and serves as an intermediate sink for released calcium (Gillis, Piront & Gosselin-Rey, 1979; Gillis, Thomason, LeFevre & Kretsinger, 1982). During relaxation much of the calcium bound to troponin C during activation becomes bound to parvalbumin. During the subsequent slow recovery period the calcium

SR CALCIUM PUMP IN FROG MUSCLE 641 bound to parvalbumin is slowly transported back into the SR, eventually leading to full return of the released calcium. During the slow recovery period, when calcium ions are moving from parvalbumin back to the SR, [Ca2"] is only slightly elevated above the resting level. The degree of elevation of [Ca2l] is determined by a pseudo steady state in which the rate of calcium dissociation from parvalbumin approximately equals the rate of calcium removal from the cytosol by the SR (Cannell, 1986; Melzer, Rios & Schneider, 1986). Since [Ca21] is changing relatively slowly during the slow recovery, calcium removal by the SR should be predominately via steady-state turnover of the SR calcium pump. Thus, measurements of both the rate of calcium removal by the SR and of [Ca2+] during slow recovery should provide sufficient information for determining the steady-state calcium dependence of the SR calcium pump in functioning muscle fibres. This was a major goal of the present paper. In order to characterize the calcium movements occurring both during the relatively large [Ca2+] transients accompanying calcium release and during the slow recovery period after release when [Ca2"] is low, we have used a dual-indicator [Ca2+] recording system (Klein, Simon, Sziics & Schneider, 1988) that employs simultaneous measurements of Fura-2 fluorescence and Antipyrylazo III (AP III) absorbance signals. The Fura-2 signal, which provided a reliable measure of the [Ca2+] time course during slow recovery, showed that [Ca2+] declined with two exponential components having time constants of about 2 and 14 s during slow recovery. Using the [Ca2+] measured during the slow recovery by Fura-2 and the time course of calcium occupancy of parvalbumin calculated from the AP III and Fura-2 signals, our results indicate that the steady-state rate of calcium transport by the SR pump has a steep calcium dependence, varying with the fourth power of [Ca2+]. Since the SR calcium ATPase has two calcium binding sites per monomer (Martonosi & Beeler, 1983; Inesi, 1985), this result is consistent with the SR calcium pump molecules operating as functional dimers having four interacting calcium binding sites, two contributed by each pump monomer. Parallel measurements of the rate of recovery of calcium release indicate that recovery of release may exhibit a somewhat slower time course than the removal of calcium from parvalbumin. The rate of recovery of calcium release also exhibits a high power relationship to [Ca2+]. Some aspects of this work have been presented in abstract form (Kovacs, Klein, Simon & Schneider, 1989),. METHODS

Frogs (Rana pipiens, northern variety) were killed by decapitation and the spinal cord destroyed. Single muscle fibres from the ileofibularis or semitendinous muscles were dissected, cut at both ends and mounted in a double Vaseline gap chamber, all in a relaxing solution (Kovacs, Rios & Schneider, 1983). Fibres were stretched to 3-8-4 2 ,um per sarcomere and were notched just beyond the walls in both end-pools. After forming Vaseline seals and positioning covering pieces (Kovacs et al. 1983) the solutions in the middle and end-pools were changed to those used for the experiments. The 'internal' solution applied to the cut ends of the fibres contained (mM): 102-5 caesium glutamate, 5.5 MgCl2, 5 ATP (disodium salt), 4-5 sodium tris-maleate buffer, 13-2 caesium tris-maleate buffer, 0.1 EGTA, 5 creatine phosphate (disodium salt), 1 AP III, 0 05 Fura-2 and 6 glucose. The 'external' solution applied to the intact portion of the fibre in the middle pool contained (mM): 75 (TEA)2S04, 5 Cs2SO4, 7-5 total CaSO4, 5 sodium tris-maleate buffer and 10- g ml-' tetrodotoxin. Both solutions were adjusted to pH 7 0 at room temperature. Fibres were 21

PHY 441

642

M. G. KLEIN ANVD OTHERS

voltage clamped and current and voltage were monitored using standard circuits (Kovacs et al. 1983). Experiments were carried out at a holding potential of - 100 mV and at 8-10 'C. Optical absorbance and fluorescence measurements were carried out as described by Klein et al. (1988). Calcium transients (A[Ca2"]) were calculated from the AP III absorbance signals at 700 nm (Klein et al. 1988) using the calibration of Kovacs et al. (1983). Fura-2 fluorescence emission signals for excitation at 380 nm (F380) were recorded simultaneously with the absorbance signals (Klein et al. 1988). Fura-2 fluorescence for excitation at the isosbestic wavelength of '358' nm (F3; Klein et al. 1988) was determined in the resting fibre at various times during the course of each experiment. The value of F358 at the time of each pulse was calculated by interpolation between the measured values of F358. The F380 signal recorded during the pulse was divided by the interpolated value of F358 to give the Fura-2 fluorescence ratio signal for that pulse. Simultaneous Fura-2 fluorescence ratio signals and AP III calcium transients were used to adjust the on and off rate constants for Fura-2 calcium binding in each fibre so that the Fura-2 signal predicted from the AP III calcium transients gave a best fit to the measured Fura-2 signal (Klein et al. 1988). Fura-2 fluorescence ratios in the resting fibre were then used with this calibration to calculate resting [Ca21]. Each AP III [Ca2+] record was constructed as the sum of the AP III A[Ca 2] record plus the resting calcium determined from the Fura-2 fluorescence just prior to the pulse. Monitoring, acquisition, processing and storage of electrical and optical signals were all carried out as described by Klein, Simon & Schneider (1990). Two different sampling rates were used for many of the stored data records. For both sampling rates two electrical signals, fibre current and voltage, and three optical signals, fibre light transmission at 700 and 850 nm and fibre fluorescence at 510 nm (excitation at 380 nm), were monitored sequentially at 40 ,ts intervals during a 200 ,ts period. For time intervals over which the faster sampling rate was used, each point in the stored record for a given signal consisted of the average of five or ten such determinations of that signal over a 1 or 2 ms interval for sampling at 1 or 2 ms per point, respectively. For time intervals over which the slower sampling rate was used, each point in the stored record consisted of the average of ten determinations of the signal spaced equally over a 100 ms sampling interval. In one fibre in which a 200 ms slow sampling interval was used the ten determinations were equally spaced over the 200 ms interval. For most pulses fast and slow sampling rates were employed sequentially in order to monitor both fast and slow events during and after the pulse. In these cases composite [Ca2+] records were constructed from the AP III and Fura-2 [Ca2+] records. Throughout the fast sampling interval, which comprised the baseline period prior to a pulse, the depolarizing pulse, and 100-200 ms after the pulse, the AP III [Ca2+] signal was used in the composite [Ca2+] record. During the slow sampling interval, which began immediately after the fast sampling interval and lasted for up to 16 s, the Fura-2 [Ca2"] signal was used in the composite [Ca2+] record. If a second pulse was applied after the slow sampling interval, fast sampling was restarted immediately after the end of the slow sampling interval and the AP III [Ca2"] signal was again used in the composite [Ca 2] record. In all cases in which Fura-2 fluorescence was monitored for periods of more than 1 s the recorded F380 signal was corrected for bleaching of Fura-2 during the recording period. The bleaching correction was calculated from the rate of decline of F380 during 16 s slow sampling periods which were not preceded by a depolarizing pulse so that [Ca2+] was stable. Such measurements made on several fibres showed that over periods of 16 s bleaching caused a relatively small linear decline of F380 with time and that the rate of decline of F380, dF380/dt, was proportional to its magnitude. The mean value of (dF380/dt)/F380 found during 16 s recording periods without preceding pulses was - 1-76 x 10-4 + 015 x 10-4 s-'. Based on these observations the component due to bleaching in F380 records after pulses was calculated as t times the product of 1-76 x 10-4 s-I and the value of F380 just prior to the pulse in the fibre under study, where t is time during the record. The measured F380 signal was corrected by subtracting the calculated component due to bleaching and the corrected p80 was then used to calculate the Fura-2 [Ca2+] record. In a typical fibre the bleaching correction at 16 s after a typical pulse was about 4% of the change in F380 produced by the pulse. The bleaching correction assumes that resting [Ca2+] did not change during the 16 s records without pulses used to characterize bleaching. The same relative rate of bleaching was observed in recordings of fluorescence at 358 nm for 16 s without stimulation in several fibres, indicating that changes in[Ca2+] did not interfere with the estimation of bleaching at 380 nm. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum (SR) was calculated from each [Ca2+] record using the general approach developed by Melzer, Rios & Schneider (1984, 1987).

SR CALCIUTM PlUMP IN FROG MLrSCLE

643

Details regarding the specific myoplasmic calcium binding sites, both rapidly and slowly equilibrating, used in the present calculations of calcium removal and Rrel were as described by Klein et al. (1990). The calcium-specific binding sites on thin filament troponin C were assumed to be present at 250 /tM (referred to myoplasmic water) and to have on and off rate constants of 1 3 x 108 M-1 s-I and 103 s-'. The calcium binding sites on the SR calcium pump were assumed to be present at 200 /M. The various values of the calcium removal system parameters were set or adjusted as described by Klein et al. (1990). The on and off rate constants for calcium binding to the troponin C used in our standard calculations give a dissociation constant of 7-7 /tM, which is relatively large compared to that reported from other studies (cf. Baylor, Chandler & Marshall, 1983, for tabulation). We used the higher value so that our removal model can reproduce the close to exponential decline of A[Ca2+] during the first few hundred milliseconds after most calcium transients (Melzer, Rios & Schneider, 1986, 1987). Use of considerably lower values results in appreciable deviations from simple exponential decline in the A[Ca2+] predicted by the removal model due to saturation of troponin C. Throughout the text average values are given as the mean+ S.E.M. Statistical significance was determined using a two-tailed t test assuming significance for P < 0-05. Fitting of various models to the calcium dependence of calcium removal or return to the SR was carried out using the generalized data-fitting program NFIT (Island Products, Galveston, TX, USA) developed by Dr Simon. RESULTS

Decline of myoplasmic [Ca21] following SR calcium release Fast and slow decline of [Ca2+] following calcium release Figure 1 presents AP III and Fura-2 calcium signals recorded during a 200 ms depolarizing pulse to 0 mV (bottom) and for 16 2 s after the pulse. The early part of each record in Fig. 1 was obtained using relatively fast sampling (Methods), in this case 2 ms per point. Fast sampling was carried out continuously during the 20 ms baseline interval just before the pulse, during the pulse and for 200 ms after the pulse. Immediately after the last point of fast sampling, relatively slow sampling (100 ms per point) was begun and was continued for 16 s. The change from fast to slow sampling is apparent in Fig. 1 A as the first gap in the falling phase of the record. Figure 1 A gives the [Ca2"] record calculated from the AP III absorbance change at 700 nm, with the resting [Ca2+] before the pulse set equal to the value (33 nM) determined from the Fura-2 fluorescence ratio prior to the pulse (Methods). The resting [Ca2+] determined from Fura-2 was used for this and all other AP III [Ca2+] records because resting [Ca2+] cannot be determined from AP III due to its relatively low calcium affinity (Klein et al. 1988). Figure IA shows that [Ca2+] increased continuously during the depolarizing pulse, reaching a maximum level of about 4 9 /m at the end of the pulse. Immediately after the pulse the AP III [Ca21] record (Fig. 1A) declined relatively rapidly and had returned to a level close to the resting [Ca2"] within a few points of the start of the slow sampling interval. This rapid component of decay of [Ca2+] within a few hundred milliseconds of the end of a pulse has been characterized previously (Melzer et al. 1986). The dip in the AP III [Ca2+] record in Fig. 1A near the start of the slow sampling interval was probably due to a small movement artifact, to which the AP III absorbance signal was very sensitive (Klein et al. 1988). Movement artifacts were not present in most fibres studied. However, even in the absence of movement artifacts the actual recovery time course of [Ca2+] during most of the slow sampling period in Fig. 1 and in other fibres and runs may not be reliably represented by the AP III 21-2


644

[Ca2+] signal. The AP III absorbance signal produced by the slight elevation of [Ca21] during this period was relatively small. Thus, other small non-[Ca21] components in the AP III absorbance signal, arising either from imperfect correction for intrinsic signals or from a magnesium component in the 700 nm AP III signal (Baylor, A

[Ca2+l

(from AP 1II) (pM)

_100

B

Fura-2 saturation

_ ...._.____...__._ ,___ __.....,_ 20o......s

[Ca2+] (from Fura-2)

C

200

(nM)

0 mV

JI

3s

-100 mV

Fig. 1. Time course of the decline of [Ca2+J after a depolarization. A, [Ca2+] during and after a 200 ms depolarization to 0 mV. The record was calculated from the absorbance signal measured by AP III, with the resting level of [Ca2+] before the pulse set at the value determined from the Fura-2 fluorescence ratio in the resting fibre. Fast sampling rate 2 ms/point and slow sampling rate 100 ms/point. B. the Fura-2 saturation (%) recorded during and after the pulse. C, [Ca2+] calculated from the Fura-2 percentage saturation record in B, displayed on a 20-fold expanded ordinate compared to A. The part of the signal above 200 nM is clipped. [Ca2+] was calculated from the Fura-2 signal assuming on and off rate constants of the Ca-Fura-2 reaction to be 2-70 x 108 M-1 s-1, and 26-0 s-', respectively. The line superimposed on the record is a least-squares two-exponential fit beginning 1 s after the start of the slow sampling interval. The parameters of the fit are: AIC = 19 nm, rTc = 2-0 s, A2C = 40 nM, r2 = 127- s. Fibre 434, resting [Ca2+] 33 nM. [AP III] 595 /UM, sarcomere length 3-9 /tM, temperature 9 'C.

Chandler & Marshall, 1982; Baylor, Quinta-Ferreira & Hui, 1985), could constitute a significant fraction of the absorbance signal during most of the slow sampling interval and make the AP III [Ca2+] record unreliable during this period. In contrast to AP III, Fura-2 has a relatively high calcium affinity (Grynkiewicz, Poenie & Tsien, 1985) and its fluorescence signal is not significantly influenced by movement artifacts (Klein et al. 1988) or by intrinsic (Klein et al. 1988) or magnesium

SR CALCIlTM PUMP IN FROG MUSCLE

645

(Grynkiewicz et al. 1985; Klein et al. 1988) components. Thus, Fura-2 should provide a good measure of [Ca2+] during the period when [Ca21] was low and not reliably followed by AP III (Klein et al. 1988; Baylor & Hollingworth, 1988). Figure lB presents the Fura-2 percentage saturation calculated from the Fura-2 fluorescence signal recorded simultaneously with the AP III [Ca2"] record in Fig. IA. During most of the depolarizing pulse, when [Ca21] was relatively high and reliably followed by AP III, the Fura-2 signal was close to saturation as previously reported for relatively large and long pulses (Klein et al. 1988). Fura-2 was thus relatively unreliable for monitoring [Ca2+] during the pulse in Fig. 1. However, during most of the slow sampling period after the pulse, when [Ca21] was low and could not be reliably followed by AP III, the Fura-2 saturation record (Fig. iB) was near the middle of its range so that Fura-2 could provide a reliable measure of [Ca2+] during this time. Figure 1 C presents the [Ca2+] record calculated from the Fura-2 signal in Fig. lB. The [Ca21] scale in Fig. 1 C is expanded twentyfold compared to the scale for the AP III [Ca2f] record in Fig. IA. Although the calibration of the AP III and Fura-2 [Ca2+] records in Fig. 1A and C may underestimate the actual [Ca2+] due to binding of both AP III (Baylor, Hollingworth, Hui & Quinta-Ferreira, 1986; Maylie, Irving, Sizto & Chandler, 1987) and Fura-2 (Konishi, Olson, Hollingworth & Baylor, 1988) to myoplasmic constituents, the Fura-2 [Ca2+] record in Fig. 1C provides a reliable measure of the relative change of [Ca2+] with time during the slow sampling interval. During this time [Ca2+] was changing sufficiently slowly that kinetic delays in the equilibration of Fura-2 with [Ca21] (Klein et al. 1988; Baylor & Hollingworth, 1988) would be negligible compared to the time course of [Ca21]. Figure 1 C shows that [Ca2+] declined continuously during the slow sampling interval and provides data for analysis of the time course of decay of [Ca2+] during this period. In order to quantify the time course of the slow decline of [Ca2+] during the slow sampling the record was fitted with sums of exponentials plus a fixed constant. A single-exponential function of time plus a constant did not give a satisfactory fit to the observed decline of [Ca2+] in Fig. 1 C or in any other [Ca2+] record from 1 to 16 s after a similar pulse in any fibre studied. The continuous line superimposed on the Fura-2 [Ca2+] record in Fig. 1 C is the fit of the sum of two exponential functions of time plus a constant to the [Ca2+] record over a 15 s interval beginning 1 s after the start of the slow sampling. The fit is barely distinguishable from the [Ca2+] record (dotted) over most of the fitted interval. Similarly good fits were obtained in all fibres, indicating that two exponential components were sufficient to describe the slow decline of [Ca2+] from 1 to 16 s after a pulse. The value of the constant for the fit in Fig. 1 C was the resting [Ca2+] (39 nM) measured just prior to the next pulse, which was applied 1 min after the records in Fig. 1. The time constants and amplitudes of the two exponential components were determined by a least-squares fitting procedure. For the record in Fig. 1 C the values of the two time constants obtained from the fit were 2-0 and 12-7 s and the amplitudes of the fast and slow components were 19 and 40 nm, respectively, at 1 s after the start of the slow

sampling. The mean (± S.E.M.) values of the two time constants Tic and T2C for the slow decline of [Ca2+] obtained from fits similar to those in Fig. 1 C to a total of eleven Fura-2 [Ca2+] records from seven fibres during the slow recovery period were 1-9 + 0 3

646


and 13 5 + 1P5 s, respectively. The mean values of the respective amplitudes Alc and A2C were 34+7 and 31+4 nm. The mean value of the relative fraction f2c =

A2C/(Alc +A2C) of A2C compared to the total amplitude of [Ca2+] elevation at 1 s after the start of slow sampling was 51 + 5 %, indicating that the two components of the slow decay of [Ca2+] each contributed about half of the total elevation of [Ca2+] at 1 s after the start of the slow sampling period. The conclusion from this analysis is that after a depolarization, [Ca2+] declined with a time course that can be described by three exponential components: a single large amplitude fast component with a time constant of about 01 s that determined the initial fast decay of [Ca2+] after a pulse and two small amplitude components with time constants of about 2 and 14 s that determined the time course of [Ca2+] during the slow recovery period. The mean resting [Ca2+] for the eleven records was 40 +4 nm and the mean [Ca2+] before the next pulse applied to each fibre (equal to the mean value of the constant for the two exponential plus constant fit) was 42 + 3 nm. The mean value of the Fura2 dissociation constant (KD), which was set in each fibre so as to make the Fura-2 [Ca2+] signal agree with the AP III [Ca2+] signal (Klein et al. 1988), was 57+5 nM. Thus, if the AP III [Ca2+] calibration were too low due to myoplasmic binding of AP III (Baylor et al. 1986; Maylie et al. 1987), the Fura-2 KD would be too low by the same factor and the values of A c, A2C and resting [Ca2+] calculated using that value of KD would also be too low by the same factor, as would the value of [Ca2+] at any time during any record.

Calcium outside the SR during the slow recovery following release The change in free myoplasmic [Ca2+] that is observed in response to a depolarizing pulse represents only a small fraction of the change in total myoplasmic calcium [Ca]T. The majority of the released calcium is bound to various calcium binding sites intrinsic to the fibre or to the binding sites on the calcium indicators introduced into the fibres. Thus, the time course of decay of the [Ca2+] signal after a pulse does not by itself provide a direct measure of the time course of decay of the total calcium outside the SR. However, it is possible to use the measured [Ca2+] to estimate the time course of calcium occupancy of all sites that contribute significantly to [Ca]T and thus calculate [Ca]T (Baylor et al. 1983). The decline of the calculated [Ca]T then provides a measure of the return of calcium to the SR. This approach is illustrated in Fig. 2. Figure 2A presents a [Ca2+] record from another fibre for a 200 ms depolarizing pulse to 0 mV (bottom). The [Ca2+] record in Fig. 2A is a composite formed from both the AP III and Fura-2 signals. The AP III signal was used to calculate [Ca2+] during the fast sampling interval, whereas the Fura-2 signal was used during the slow sampling. Figure 2B presents an identical [Ca2+] record to that in Fig. 2A, but now on a 20-fold expanded [Ca2+] scale. As in the fibre in Fig. 1, [Ca2+] rose continuously during the pulse, in this case reaching a peak of 3-6 /aM (Fig. 2A). Following the pulse [Ca2+] declined rapidly to close to its resting level (Fig. 2A) and then declined more slowly during the 16 s slow sampling period (Fig. 2B). The line superimposed on the [Ca2+] record starting 4 s after the start of slow sampling in Fig. 2B is a twoexponential fit to the [Ca2+] record. This provides a good fit to the data and has been substituted for the [Ca2+] record over this interval in subsequent calculations in this

SR CALCIUM PUrMP IN FROG MlUSCLE

647

and other fibres in order to eliminate noise in records calculated from the [Ca2"] record. A major component of [Ca]T during the slow decline of [Ca2"] should be the concentration [Ca-Parv] of calcium bound to the relatively high-affinity calciummagnesium sites on the myoplasmic protein parvalbumin. The [Ca-Parv] time course was calculated from the [Ca2"] record in Fig. 2A and B and from the fit to the [Ca2+] record by numerical solution of the simultaneous differential equations for calcium and magnesium binding to parvalbumin,

d[Ca-Parv]/dt = kn Ca-ParJCa21] [Parv] koff,caParv[Ca-Parv], -

and

d[Mg-Parv]/dt =

kon, Mg- Parv [Mg2+] [Parv] - koff,Mg-Parv[Mg-Parv],

(1)

(2)

where [Parv] is the concentration of metal-free sites on parvalbumin and the total concentration [Parv]T of parvalbumin calcium-magnesium binding sites is equal to [Parv] + [Ca-Parv] + [Mg-Parv]. Here, and in all other cases in which [Ca-Parv] was calculated to estimate [Ca]T, the [Ca-Parv] time course was calculated using the on and off rate constants tabulated by Baylor et al. (1983) for Ca2+ and Mg2+ binding to parvalbumin (Fig. 2 legend). [Parv]T was assumed to equal 1 mm. Resting [Mg2+] was assumed to be 1 mm and d[Mg2+]/dt was assumed to equal -d[Mg-Parv]/dt. Figure 2 C, which presents the calculated [Ca-Parv] time course, shows that [Ca-Parv] rose continuously throughout the entire fast sampling interval. Thus, [Ca-Parv] increased both during the phase of rising [Ca2+] during the pulse and during the phase of rapid decline of [Ca2+] after the pulse. This is consistent with calcium binding to parvalbumin contributing to the rapid decline of [Ca2+] after a pulse (Cannell, 1986; Melzer et al. 1986; Irving, Maylie, Sizto & Chandler, 1989). The calculated [Ca-Parv] (Fig. 2C) began to decline only after the second point of slow sampling, a time at which [Ca2+] had returned to close to its resting level (Fig. 2A). From this time on both [Ca2+] and [Ca-Parv] declined continuously over the rest of the 16 s slow sampling interval. During most of the slow sampling period in Fig. 2 [Ca2+] was below 100 nm and was only slightly elevated above the resting level of 28 nM in this fibre. During such small elevations of [Ca2+], calcium binding to the troponin C regulatory sites would be negligible and the only myoplasmic binding site other than parvalbumin expected to have a significant calcium occupancy would be Fura-2. Figure 2D presents the time course of [Ca-Fura] obtained directly from the Fura-2 fluorescence signal. The [Ca-Fura] record in Fig. 2D is presented using the same ordinate scale as used for the [Ca-Parv] record in Fig. 2 C. Comparison of the two records shows that during most of the slow sampling period the change of [Ca-Fura] was negligible compared to that of [Ca-Parv]. Figure 2E presents a record of [Ca-Parv] + [Ca-Fura], calculated as the sum of the records in Fig. 2C and D. The continuous line which is superimposed on the record in Fig. 2E is the least-squares fit of two exponentials plus a constant to the record from 1 to 16 s after the start of slow sampling. The constant was set to the sum of [Ca-Parv] + [Ca-Fura] calculated from the value of resting [Ca2+] measured just before the next depolarization. The fit was so good that it is indistinguishable from


648 A

[Ca2+] 0

[Ca2+]

B

(nM)

100

_ _~ ~ ~ ~ ~0 J

0

c~~~~~~~~~~~~~

,_

I 05) from the mean value of 3.9 obtained including and heavily weighting the resting value. The mean value of the rate of leak (L) of calcium from the SR obtained from the fits of eqn (4) to the data for ten of the eleven runs (excluding one run that had a near first power dependence of net uptake rate on [Ca2+] and value of L over eight times larger than the overall mean) was 1f9 + 0-8 tM s-'. The net rate of calcium uptake by the SR at 3 s after the pulse in each fibre had a mean value of 29-9 + 2 9 tM s-1. Adding the mean leak to the mean net rate of uptake, the rate of calcium transport by the pump would be 32 uM s-1 using the mean value of L obtained without the single unusually high value. Using 250 /M for the effective concentration of calcium binding sites contributed by the SR calcium pump in these fibres (cf. tabulation by Baylor et al. 1983), the calculated rate of calcium transport by the pump would correspond to a turnover number of 32 tM s-1/250 JM or 013 s-'. Since there are two calcium binding sites on each calcium ATPase molecule, pump monomers or dimers would be present at 2 or 4 the concentration of pump calcium binding sites but would transport two or four calcium ions per monomer or dimer turnover, respectively. Thus, the same turnover number of 0-13 s-1 would apply to pump monomers or to functionally coupled pump dimers as was calculated for individual pump sites. The mean [Ca2+] at 3 s after the pulse when this turnover number was determined was 79 + 6 nm (above). The values of n and L in the preceding paragraph were obtained from fits of eqn (4) to the - d[Ca]T/dt data from each run assuming the SR calcium content remains

M. G. KLEIIN A-ND OTHERS

654

constant during the slow recovery period. Alternatively, if the SR calcium content is assumed to vary during the recovery interval and - d[Ca]T/dt data from each run are fitted by eqn (5), mean values of 40 + 0O6 and 22 + 0O7 /LM s-I are obtained for n and Lr (the Lr value from one run was again unusually large and was not

40 -4

30

C,0,,2 20-

-10 -// 0

30

50

70

90

[Ca2+] (nM) Fig. 4. The calcium dependence of the rate of change of [Ca]T. The symbols represent the mean (±S.E.M.) values of [Ca21] and -d[Ca]T/dt from eleven runs in seven fibres. S.E.M. values are not shown when they are smaller than the symbol. Same format as Fig. 3 for both the data and the fit. The power, n, was 3-9.

included in the calculation of the mean value of Lr). These values are very close to the mean values of 3 9 + 0-6 and 1 9 + 0-8 /IM s-1 obtained for n and L assuming the leak to be constant during the recovery interval (above). The mean of the values of the fractional SR calcium content at 3 s after the pulse in each run obtained from the recovery experiments (below) and used in the calculation of R* by eqn (6) was 0-81 +0-2. Figure 4 presents the mean [Ca2+] dependence of the rate of calcium transport by the SR calculated for all the records used for the above means. The error bars denote+S.E.M. The point at the highest [Ca2+] corresponds to the mean values of - d[Ca]T/dt and [Ca2+] at 3 s (above). The other open circles give the mean values at 4, 6, 8, 12 and 16 s and the filled circle gives zero net uptake rate at the mean resting [Ca2+]. The fit of eqn (4) to the mean data gave a value of n of 3.9, again indicating that at least four calcium ions appear to be transported in a co-operative manner by the functional unit of the SR calcium pump. The value of L obtained from the fit to the mean data in Fig. 4 was 2-3 ,tM s-'. This value is in good agreement with the mean value of 1-9 + 08 for L (above) obtained from the L values for fits to ten of the eleven individual runs but excluding the unusually high value of L from one fibre. Mean values of - d[Ca]T/dt were also calculated for 1 and 2 s after the start of slow sampling. When the value at 2 s was plotted at the corresponding mean [Ca2+], the point was located on the continuation of theoretical line generated for the points at 3-16 s according to eqn (4) at higher [Ca2+] (not shown), again consistent with steady-state operation of the pump and [Ca2+] far from saturation at 2 s of the slow

SR CALCIUM PUMP IN FROG MUSCLE A

655

[Ca2+]

0

I

'

1

~~~~~(PM)

10 | >, ****.*,:>,et..*....*..*..*.*... ......,,,,,,,,,,,,,,,.,,,,.......t

Nomalized ;

;

;

~~~~~~~~~~~Rrel

-100 3s

Fig. 5. Decline of [Ca2+] and recovery of the rate of release. A, [Ca2+] recorded with AP III and Fura-2 for a 200 ms conditioning pre-pulse to -20 mV, followed by a recovery interval of 1, 2, 4, 8 or 16 s at the holding potential (-100 mV), and then a 60 ms test pulse to -20 mV (bottom). The individual records represent a composite of the [Ca2+] from the AP III signal for the first 420 ms (fast sampling), the [Ca2+] from Fura-2 for the recovery interval (1-16 s, slow sampling), and the [Ca2+] from AP III for the test pulse (fast sampling). The [Ca2+] at the start of the test pulse interval was set equal to the [Ca2+] value for the last point of the recovery interval. B, [Ca2+] in A shown on a 20-fold expanded vertical scale. Points above 100 nm are clipped. C, the rate of release calculated from the [Ca2+] in A, normalized to the maximum value reached during the conditioning pulse. The individual, non-normalized records are shown in Fig. 6. On and off rate constants to convert the Fura-2 saturation to [Ca2+] were: kon = 2-61 x 108 M-1 s-1, koff = 15-3 s-1. The parameters used in the characterization of the removal properties of the fibre to calculate the rate of release were: koffMgParv = 6-2 s-', pump Vmax = 2824 aUm s-', [Parv] set to 600 ^UM. Fibre 435, resting [Ca2+j 27-34 nm, [AP III] 457-485 /LM, sarcomere length 4 0 ,um, temperature 9 'C.

sampling period. At 1 s of the slow sampling period the mean - d[Ca]T/dt value fell considerably below the continuation of the theoretical line, indicating deviation from one or more of the assumptions underlying the applicability of eqn (4) at that time.


656

Recovery of SR calcium following release Return of calcium to the SR estimated from the recovery of calcium release In the preceding analyses the AP III and Fura-2 [Ca2l] signals were used to calculate the concentration of total calcium outside the SR ([Ca]T). The rate of Test pulse

Pre-pulse

Recovery time (s)

Test/pre (%)

1

75.2

2

77.2

4

78.5

8

81.0

10 PM 16

ms11 83.1

100 ms J-L-\ -100 mV

Fig. 6. The rate of release during the pre-pulse and test pulse of the recovery experiment. The release records are the same as in Fig. 5C, shown on an expanded time scale for the fast-sampling intervals containing the pre-pulse (left column) and test pulse (right column). The part of the record during the recovery interval is not shown, but the recovery time corresponding to each pair of records is given to the left. The percentage recovery of the test release relative to its corresponding pre-pulse release is given to the right. The pre- and test pulse depolarizations are given at the bottom. Same fibre and conditions as in Fig. 5.

decline of [Ca]T during the slow decline of [Ca2+] after a pulse was used as a measure of the net rate of transport of calcium from the myoplasm to the SR. An alternative means of monitoring the return of calcium to the SR following conditioning pulse is

SR CALCIUM PUZMP IN FROG MUTSCLE

657

to use a constant test pulse applied at variable times after the conditioning pulse to determine the SR calcium content as a function of time during the recovery interval (Schneider et al. 1987). Figure 5 illustrates an experiment using this approach. Figure 5A presents five superimposed composite AP III and Fura-2 [Ca21] records. In each case a 200 ms conditioning pulse to -20 mV was applied to the fibre 20 ms after the start of the record. Fast sampling was carried out before, during and for 200 ms after the conditioning pulse, at which time slow sampling was started. Fast sampling was restarted at either 1, 2, 4, 8 or 16 s after the start of a slow sampling period and a 60 ms test pulse to -20 mV was applied to the fibre after a 20 ms baseline interval at fast sampling. In each case Fura-2 was used to calculate the resting [Ca2+] and the [Ca2+] during slow sampling, whereas AP III was used to calculate the change in [Ca2+] during fast sampling. Figure 5B presents the same set of superimposed [Ca2+] records as in Fig. 5A, but now on a 20-fold expanded [Ca21] scale. Except for the 1 s recovery, the [Ca2+] time course during common intervals following successive applications of the same conditioning pulse was quite

reproducible. Figure 5C presents superimposed records of the rate of release of calcium (Rrel) from the SR calculated from each of the [Ca2+] records in Fig. 5A and B. The peak rate of release during the conditioning pulses declined by about 2 % between successive records, indicating a slow run-down of release over the course of the experiment (Klein et al. 1990). To correct for the effects of fibre run-down each of the records in Fig. 5C have been normalized so as to have the same peak Rrei during the conditioning pulses. Inspection of the releases for the test pulses in Fig. 5 C shows that the peak rate of release during the test pulse was smallest after the 1 s recovery period, and increased as the recovery interval was increased from 1 to 16 s. The peak Rrel during the test pulse had still not completely recovered to the amplitude of the peak during the conditioning pulse after the longest recovery period (16 s) used in Fig. 5, indicating a slow recovery of release following the conditioning pulse. Figure 6 presents the fast sampling segments of release records from Fig. 5 C on an expanded time scale. The two records on each line of Fig. 6 give RreI for a conditioning pulse (left) and for the test pulse (right) that followed it in the same record in Fig. 5. The duration of the slow recovery interval between each conditioning and test pulse is indicated at the left of each line. The release records for the conditioning pulses in Fig. 6 were quite reproducible, although close examination indicated a slight run-down of release for the conditioning pulses over the course of the experiment (above). The records shown in Fig. 6 have not been corrected for fibre run-down. As indicated in relation to Fig. 5C, the peak test pulse Rrei was smallest for the 1 s recovery interval (top), and increased with increasing recovery time (top to bottom release records). Previous results indicated that Rrel recovers in two phases following a conditioning pulse (Schneider et al. 1987). The faster phase was attributed to recovery from inactivation of release and was completed within about 1 s (Schneider et al. 1987; Schneider & Simon, 1988). The subsequent slower recovery was attributed to recovery from depletion of calcium from the SR (Schneider et al. 1987). In the present experiments we have further examined the details of the time course of recovery from depletion during the period from 1 to 16 s after a conditioning pulse. During the phase of recovery from depletion the release record had the same time

658

M. G. KLEIYN AND OTHERS

course as prior to depletion but was simply decreased in amplitude (Schneider et al. 1987). Assuming the amplitude of Rrei during recovery from depletion to be proportional to the SR calcium content, the relative amplitude of the test pulse Rrei record can be used to provide a measure of the relative calcium content of the SR (Schneider et al. 1987). In order to obtain a quantitative measure of the relative amplitude of the average release during test pulses after the various recovery intervals in Fig. 6, both the conditioning and the test pulse Rrej records were integrated for 60 ms starting at the onset of the pulse. The integration period corresponded to the entire duration of the test pulse but to only the initial 60 ms of the 200 ms conditioning pulse. Use of the integrals of Rre, over 60 ms for comparing test and conditioning releases provided a more reliable measure of relative release during recovery from depletion than simply comparing the peak values of the test and conditioning releases as was done in previous work (Schneider et al. 1987). The relative size of each test pulse release integral, expressed as a percentage of the release integral for the corresponding conditioning pulse, is given at the right of each pair of records in Fig. 6. Since each value constitutes a comparison of a test release with the immediately preceding conditioning release, the contributions of run-down of release over the course of the experiment should be minimized in the indicated percentage values. Assuming the relative amplitude of the release integrals to represent the relative amount of SR calcium available for release, the values at the right of each pair of records in Fig. 6 give the relative amounts (%) of releasable calcium in the SR at the indicated recovery times (left) compared to the SR content prior to each of the conditioning pulses. Although the differences between conditioning and test releases with recovery time were small, they were reproducible and were observed in all fibres studied. Figure 7A presents the time course of recovery of releasable calcium for the experiment in Figs 5 and 6. The circles in Fig. 7A are values of test release as a percentage of the corresponding conditioning release obtained from the records in Fig. 6. The line in Fig. 7A is the least-squares fit of the sum of two exponential functions of time plus a constant to the recovery time course of the relative releasable calcium. The value of the constant was set equal to 0-98, the average ratio of release integrals for successive conditioning pulses, to account for the slight run-down of release with time over the course of the experiment. The values of the two time constants TiR and T2R of recovery of Rrei obtained from the fit in Fig. 7A were 25 and 69-1 s, respectively. Comparison of these values with the time constants for the decline of [Ca2"] and [Ca]T for the same run indicates that the time constant TrR of the faster component of the recovery of Rrel was similar to the values of 3 8 and 1P4 s for rlc and T1T, but that the time constant T2T of the slower phase of recovery of Rrei, 69 1 s, was considerably larger than the values of 19 5 and 14-8 s for Trc and T2T for the same run. Thus, the slower final phase of recovery of releasable calcium in the SR appeared to be slower than the final phase of recovery of both [Ca2+] and [Ca]T. It should be noted that TlR and T2R are both assumed to represent time constants in the process of recoverv from depletion of calcium from the SR. In a few fibres in which it was examined in the present experiments the faster process of recovery from inactivation of release (Schneider et al. 1987; Schneider & Simon, 1988) was complete prior to the first recovery point (1 s) used for determining TlR and TiR.

SR CALCIUM PUMP IN FROG MUSCLE

659

Two exponentials plus a constant were fitted to the recovery of Rrel from I to 16 s after the start of slow sampling for the fibre and run in Figs 5-7 and for all other fibres and runs used for the preceding means. The data for these determinations were obtained from a series of conditioning and test pulses at various separations as 90

A

C

o 85 0 8

a) 80 75

D

20I

B

0

\

a) >

0

(D

0.5

0

8 12 16 Recovery time (s) Fig. 7. Time course of the recovery of calcium release following a conditioning pulse. A, the recovery of the rate of release versus time. The symbols are the integral of the test release as a percentage of the integral of the control (pre-pulse) release. The values are from Fig. 6. The continuous line is a least-squares fit of a two-exponential function to the data, the parameters of which are: A1R = 3-7 %, T1R = 2-5 s, A2R = 31-5 %, T2R = 69-1 s. B, time derivative of the fit to the recovery time course in A. Same fibre and conditions as in Fig. 5. 4

in the release recovery protocol illustrated in Fig. 6, which was carried out for each of the runs. In fact, the records in Figs 1 and 2 represent 16 s recovery intervals in the recovery protocol for two of those runs, but the records presented in Figs 1 and 2 were truncated just before the test pulses. Comparison of the mean results confirms the result of the fit to Fig. 7A in showing that the recovery of Rrel exhibited a slower final time course than the decline of either [Ca21] or LCa]T. The mean value of the time constant T2R for the final phase of recovery of Rrel was 31 6 + 5-6s, which is significantly larger (P < 0-01) than the mean values for the time constants T2C and T-T for the final decline of both [Ca2] and [Ca]T ( 13-5 + 1.5 and 14-2 + 1 4 s, respectively, above). The mean value of the relative amplitude f2R of the slower component of recovery of Rrel compared to the total amplitude at 1 s (70 + 6 %) was also larger than the relative amplitudes 2c andf2T for the slower components of [Ca2+] and [Ca]T (51 + 5 and 57 ± 6 %, respectively; above).

660


The difference between f2R and f2c was significant (P < 005), but the difference between f2R andf2T was not significant (P > 041). Thus, on average the slower phase of recovery of Rre1 was slower and may also have constituted a larger fraction of the total amount than the slower phases of decline of [Ca2+] and [Ca]T. In contrast, the faster of the two slow phases of recovery had about the same time constant as the faster of the two slow phases of decline of [Ca2+] and [Ca]T. The mean value of the time constant TlR for the faster of the two slow phases of recovery of Rr, was 1-5+0-2 s, which is not significantly different (P > 0-2) from the values of 1V9+0 3 and 1-7+002 s for Tic and T1T, respectively (above). Taken at face value, the slower final recovery Of Rrel compared to the final decline of [Ca]T could indicate the existence of a delay between the removal of Ca2+ from the myoplasm by the SR and the return of the removed Ca21 to the pool of releasable calcium within the SR. However, it is also possible that the discrepancy arose from an overestimate of the final rate of decline of [Ca]T or from an underestimate of the final rate of return of calcium to the SR release pool. The former could conceivably arise from an error in the parameters used for calculating [Ca-Parv], and the latter could arise if some process in addition to the return of calcium to the SR contributed to the final recovery of Rrei after a conditioning pulse. If the discrepancy is real, it would indicate the existence of a pool of calcium between the myoplasm and the releasable calcium within the SR, either on the SR calcium pump or within the SR lumen. These various alternatives will be considered in the Discussion.

Calcium dependence of the rate of recovery of releasable calcium Figure 7B presents the derivative of the line in Fig. 7A, which corresponds to the rate of recovery of releasable calcium as a function of time during the slow recovery period. The time course of the rate of recovery of releasable calcium in Fig. 7B, together with the time course of decline of [Ca2+] during slow recovery in the same fibre and run (Fig. 5B), provide data that establish the [Ca2+] dependence of the rate of recovery of releasable calcium. The resulting relationship obtained from the data in Figs 7B and 5B is presented in Fig. 8. The six open circles in Fig. 8 present values of the rate of recovery obtained from Fig. 7B at 3, 4, 6, 8, 12 and 16 s of the slow sampling period, plotted as a function of [Ca2+] at the same time as determined from the [Ca2+] record for the 16 s recovery in Fig. 5B. The filled circle in Fig. 8 corresponds to zero rate of recovery, which is plotted at the resting [Ca21] in the fibre during the run. The continuous line in Fig. 8 is the least-squares fit of the right side of eqn (4) to all the points in Fig. 8. As was done for the fits in Figs 3 and 4, the point at resting [Ca2+] (@) was weighted six times compared to each of the open circles in fitting the continuous line in Fig. 8. This weighting tended to force the fit to go through the resting value, resulting in a fit (continuous line) that shows a systematic deviation from the points. Since it is possible that the final phase of recovery of release was underestimated in the data in Fig. 8 (above), the fit of eqn (4) was repeated, but now excluding from the fit the value at resting [Ca2+] (which corresponds to 'infinite' time). The resulting fit, which is shown by the dashed line, appears to fit the data better than the fit obtained when resting [Ca2+] was included and heavily weighted (continuous line). The value of the power n of the [Ca2+] dependence of recovery of

SR

CALCIUM PUMP IN FROG MUSCLE

661

releasable calcium obtained from the fit not including resting [Ca2l] (dashed line) was 6-8. This value was considerably larger than the value of n of 3-7 obtained from the fit including resting [Ca2l] (continuous line). Values of n were obtained from fits similar to those in Fig. 8 to data from each of the fibres and runs used for the preceding means. The mean value of n obtained from 't, 1.2

050-8 0

D 0.4

(0

o cc

0.0 30

40

50

60

[Ca2+1 (nM) Fig. 8. The [Ca2+] dependence of the rate of recovery of releasable calcium. The format is similar to Fig. 3, with 0 representing the rate of recovery of the SR contents (taken as the fractional release recovery rate) plotted as a function of the corresponding [Ca2+] at 3, 4, 6, 8, 12 and 16 s. * plotted at the resting [Ca2+] represents the corresponding resting rate of change of SR contents, taken to be zero. The continuous line is a fit of eqn (4) to the data with the resting value weighted by 6. The value of n from the fit was 3-7. The dashed line is a fit of eqn (4) to the data excluding the resting value. The value of n in this case was 6-8. Same fibre and conditions as in Fig. 5.

fits that did not include the zero point at resting [Ca21] was 5-4 + 1-3. The mean value of n obtained from fits that included and heavily weighted the point at resting [Ca2+] was 3-0 + 0O8. Because of the scatter in the individual values these means are not significantly different (P > 0-1), nor are these values significantly different (P > 0 1) from the mean values of n of 3-9 + 06 or 4-6 + 1 0 obtained for the [Ca2+] dependence of the rate of decline of [Ca]T either with or without inclusion of the zero point, respectively. The mean rate of recovery of releasable calcium was 1-9 + 0-2 % of the resting SR calcium content per second at 3 s after the conditioning pulse. In order to convert this recovery rate to an equivalent rate in terms of [Ca2+] it is necessary to have an estimate of the SR calcium content in the resting fibre. During pulses longer than about 100 ms the rate of release of calcium from the SR declines with two phases, the faster being due to inactivation of release and the slower phase being due to depletion of calcium from the SR (Schneider et al. 1987; Schneider & Simon, 1988; Simon, Klein & Schneider, 1991). The rate of the slower phase of decline of Rrei during a pulse can be used to obtain an estimate of the calcium content of the SR (Schneider et al. 1987; Schneider, Simon & Klein, 1989). The mean resting calcium content of the SR ([Ca]SR, r) obtained in this way for each of the runs was 1-06 + 0-08 mm, given in terms of the equivalent concentration that would be produced if all the SR calcium content were present as free calcium in the myoplasmic water. Multiplying the percentage

662

M.G. KLEIN AND OTHERS

rate of recovery of releasable calcium for each run by the corresponding resting SR calcium content in the same run gives the recovery rates in units of gm s-1. At 3 s after the pulse the mean rate of recovery of releasable calcium was 20+2 s-1. The mean value of 20+2/zM s-' for the rate of recovery of releasable calcium is about 2 the mean value of 30+ 3 /SM s- for the rate of decline of [Ca]T at the same time after the pulse in the same runs (above). In view of the fact that these values were obtained from different sets of experimental data using different assumptions, the agreement can be considered to be quite good. However, the magnitude of the estimate obtained from -d[Ca]T/dt is directly proportional to the total concentration [Parv]T of parvalbumin calcium-magnesium binding sites assumed to be present in fibre. For the calculations of -d[Ca]T/dt, [Parv]T was assumed to be 1000 /M. An analysis of the time course of decline of after various pulses can be used to obtain estimates of some of the properties of the various slow calcium binding sites and transport systems in a fibre, including parvalbumin (Melzer et al. 1986; Brum, Rios & Schneider, 1988a; Klein et al. 1990). Such an analysis was routinely carried out in each fibre in order to calculate Rreli The mean value of after various pulses was [Parv]T obtained from the analysis of the decline of 637 + 55 tM. This value is about of the value of 1000 /M assumed in calculating the above rate of decline of [Ca]T. If [Parv]T were set at 637 /M rather than at 1000 for calculating the rate of decline of the resulting value for the net rate of decline of [Ca]T would be 19-1 tM s-1, very close to the mean value for calcium uptake by the SR of 20 /IM s- obtained from the recovery-of-release results. Thus the two different methods used to estimate the uptake rate gave similar values for the calcium pump flux at relatively low [Ca2+]. Figure 9 presents the average data for all runs as a function of time during slow recovery. The open circles in Fig. 9A give the mean values (+S.E.M.) of [Ca2+] at 3, 4, 6, 8, 12 and 16 s after the start of the slow sampling period. The filled circle in Fig. 9A gives the mean value of resting for these runs, plotted at 'infinite' time of The circles in 9B the recovery. Fig present mean (±S.E.M.) values of the rate of of releasable calcium in the SR for the same recovery times as used in Fig. recovery 9A. The individual values used to calculate the means in Fig. 9B were obtained using the procedure of Figs 5-7 for each run. For comparison, the mean values of -d[Ca]T/dt for the same runs are also presented in Fig. 9 (El and right-hand ordinate). The two ordinate scales in Fig. 9B have been adjusted so that the values of rate of recovery (0) and - d[Ca]T/dt (El) at 3 s in Fig. 9B correspond to the same vertical displacement. Comparison of the circles and squares in Fig. 9 confirms the finding that the rate of recovery of releasable calcium followed a slower final time course than the decline of -d[Ca]T/dt during the slow recovery period. The [Ca2+] dependence of the rate of decline of obtained from the mean and - d[Ca]T/dt data in Fig. 9 has already been presented (Fig. 4). The fit of eqn (4) to the mean data in Fig. 4 showed that the data closely followed the relationship given by eqn (4), whether or not the zero value at resting [Ca2+] was included and of the rate of heavily weighted. Figure 10 presents the mean of recovery releasable calcium. The open circles in Fig. 10 give the mean rates of recovery of releasable calcium (O in Fig. 9B) plotted as a function of the mean [Ca2+] at the same time (Fig. 9A). The filled circle in Fig. 10 gives zero rate of recovery

mM

[Ca2+]

[Ca2+]

3

/tM

[Ca]T,

[Ca2+]

[Ca]T

[Ca2+] dependence

[Ca2+]

SR CALCIUM PUJMP IN FROG MUSCLE

663

plotted at the mean resting [Ca2+]. The continuous line in Fig. 10 is a fit of the right side of eqn (4) to the points in Fig. 10, with zero rate of recovery at resting [Ca2+] weighted six times. The resulting fit deviates slightly but apparently systematically from the data. The dashed line in Fig. 10 presents the result of an alternative fit of

90r

A l

I

701

T

T

0

0

+

T

01

CD 50L (-

T

T

0i

30

nl B 2.0-

,o

cn

T

30

a) o

1.5

cD

en

20> U,

CEC

0)

I~~~~~ l D

ci)

0

P-

T

X

10

0

o

0 O

0

a)

n.n 0

o 4

8

12

16

Recovery time (s)

Fig. 9. Time course of [Ca2"], of rate of decline of total myoplasmic calcium and of rate of recovery of release. A, [Ca2"] during the 16 s recovery interval. 0 represents the mean level of [Ca2"] in the resting fibres. B, the rate of recovery of releasable calcium (0, left ordinate), and -d[Ca]T/dt (EZ, right ordinate) during the 16 s recovery interval. Symbols represent the mean (±S.E.M) values from eleven runs in seven fibres used in this study. In B, the ordinate scales have been set so that the vertical position of the two values at 3 s is the same and the points at 3 s have been offset slightly from 3 s for clarity.

(4) to the data without the value at resting [Ca2"]. As in the case of the fit to the data from an individual run in Fig. 8, the fit without resting [Ca2"] seems to follow better the mean data than the fit including and heavily weighting the point at resting [Ca2"]. If there were a very slow phase of recovery of releasable calcium, due to an as yet unidentified very slow process, a fit to the recovery of releasable calcium that did not include the final return to resting [Ca2"] might provide a better representation of the [Ca2"] dependence of recovery than a fit forced to include zero rate of recovery at resting [Ca2+]. This might be the case if the final very slow phase of recovery were not strongly represented in the data for the period of recovery from 3 to 16 s that was actually monitored. The results of the fits in Figs 8 and 10 may support this interpretation.

eqn


664

The value of the power n for the [Ca2+] dependence of recovery of releasable calcium obtained from the fit in Fig. 10 excluding resting [Ca21] was 4-3. This value is in good agreement with the value of 3-9 obtained from the fit to the mean [Ca2+] dependence of the rate of decline of [Ca]T (Fig. 4). Thus, if zero rate of recovery of

_20

U)

o >

T

i-

10

M

0.5

0

O 0.0

'-

0

-0.5

1}

0

''

30

50

70

90

[Ca2+J (nM) Fig. 10. The [Ca2+] dependence of the rate of recovery of releasable calcium. Same format as Fig. 8, with the points representing the mean (± S.E.M.) values from all fibres and runs in this study. The continuous line is a fit of eqn (4) to the data with the resting value weighted by 6. The power, n, was 2-5. The dashed line is a fit of eqn (4) to the data excluding the resting value. The value of n in this case was 4-3.

releasable calcium at resting [Ca2+] is disregarded, the rate of recovery of releasable calcium followed essentially the same [Ca2+] dependence as the rate of decline of [Ca]T, whether using a fit to the mean data (Figs 4 and 10) or considering the mean value of n obtained from individual fits (above). The [Ca2+] dependence of the rate of decline of [Ca]T and of the rate of recovery of releasable calcium both indicate the involvement of four or more calcium ions in each of the functional units responsible for the respective processes. DISCUSSION

Time course of decline of myoplasmic [Ca2+] and of calcium outside the SR One of the major new results presented in this paper is the determination of the time course of the slow decay of cytosolic [Ca2+] to close to its resting level after calcium release from the SR. Calcium release was initiated by and maintained during a 100-200 ms depolarization of a voltage-clamped fibre. When the depolarization was terminated calcium release halted and [Ca2+] declined rapidly to slightly above resting [Ca2+] with a time constant of the order of tens of milliseconds as previously characterized (Kovacs et al. 1983; Cannell, 1986; Melzer et al. 1986; Brum, Rios & Stefani, 1988b). We now report that the relatively small elevation of [Ca2+] that remained after the initial rapid decline decayed along a two-exponential time course with time constants of 2 and 14 s. The two exponential components of decay of [Ca2+] probably do not represent two distinct processes but rather provide an empirical


665

approximation of a more complicated function that results from the 4th power pump operating to remove calcium from a space in which free [Ca2"] is in equilibrium with calcium bound to parvalbumin. Determination of the relatively slow final time course of decline of [Ca2+] in the present study was made possible by the use of Fura-2, which has a relatively high affinity for Ca21 (Grynkiewicz et al. 1985) and thus provides an acceptable signal for monitoring the relatively small elevation of [Ca2+] during the slow recovery. The Fura-2 signal is not contaminated by interference from changes in [Mg2+] or pH (Grynkiewicz et al. 1985; Klein et al. 1988) or by intrinsic signals (Klein et al. 1988) and is relatively insensitive to movement artifacts (Klein et al. 1988) and instability in the light source, all of which may contaminate the final decay of the AP III signal after a pulse. The photoluminescent protein aequorin has previously been used to provide some information regarding the slow final decline of [Ca2+] after calcium release. A single-exponential fit to mean values of [Ca2+] determined by aequorin during 8 s after four averaged contractions in one fibre gave a time constant of 2 8 s (Cannell, 1986), similar to the faster of the two slow components determined here, but the [Ca21] values were too scattered to resolve two exponential components (cf. Cannell, 1986, Fig. 8B). In contrast, each of the 16 s Fura-2 [Ca2+] records (eleven individual unaveraged records from seven fibres) analysed in the present study clearly revealed two exponential components of decline of [Ca2+]. The higher order [Ca2+] dependence of aequorin light emission (Blinks, Wier, Hess & Prendergast, 1982), which results in relatively smaller light/[Ca21] signals for smaller elevations of [Ca2+], probably made aequorin relatively insensitive to calcium during the slight elevation of [Ca2+] during its final decay. Because of the limitations of both the absorbance and luminescent [Ca2+] indicators used previously, it appears that the time course of the final slow decline of [Ca2+] to its resting level after release may not have been previously fully characterized. Using composite AP III and Fura-2 [Ca2+] records together with literature values for the rate constants for Ca2+ and Mg2+ binding to parvalbumin, it was possible to calculate the total amount of calcium ([Ca]T) outside the SR during the slow decline of [Ca2+] after release. From 1 to 16 s after release the time courses of decline of [Ca2+] and [Ca]T were found to be very similar. Both [Ca2+] and [Ca]T followed two exponential time courses with similar time constants and relative amplitudes for the two exponential components. However, [Ca]T was several thousand times larger than

[Ca21].

The time course of the calculated [Ca]T during the slow recovery period was determined by the values assumed for the on and off rate constants for Ca2+ and Mg2+ binding to parvalbumin. The rate constants used in our calculations were those tabulated by Baylor et al. (1983), which were obtained from measurements of the dissociation constants (Potter, Johnson, Dedman, Schreiber, Mandel, Jackson & Means, 1977) and the off rate constants (Johnson, Robinson, Robertson, Schwartz & Potter, 1981) for carp parvalbumin. Frog muscle contains two major parvalbumins which differ slightly in isoelectric point and are present in approximately equal concentration (Pechere, Demaille & Capony, 1971; Ogawa & Tanokura, 1986a). Measurement of the dissociation constants of calcium binding at 10 °C gave values of 5 and 20 nm for the two types of parvalbumin (Ogawa & Tanokura, 1986 a). The

666


dissociation constants for magnesium at 10 °C were 100 and 200 /Im (Ogawa & Tanokura, 1986a). These values are close to the values (= koff/kon) of 4 and 91 nm for calcium and magnesium, respectively, used in our calculations. Measurements of the off rate constants for frog parvalbumin (type JVb) gave values of 0-46 s-1 for calcium and 1P76 s-5 for magnesium at 10 °C (Hou, Johnson & Rall, 1990), close to the values of 05 and 30 s-1 used for the off rate constants for calcium and magnesium, respectively, in our calculations. These values and the above values of dissociation constants would give on rate constants similar to those used in our calculations. Thus, these values indicate that our kinetic constants for parvalbumin were appropriate for frog fibres at 10 'C. Our values were also consistent with the time course of changes in myoplasmic [Mg2+] determined from the AP III absorbance change at 590 nm (Irving et al. 1989; V. Jacquemond, M. G. Klein & M. F. Schneider, unpublished observations). In contrast, Ogawa & Tanokura (1986b) concluded that the on and off rate constants for calcium and magnesium were about 5 to 10 times smaller than used in our calculations, which would alter our calculations of [Ca]T. These values were obtained from simulations of the measured time course of decay of [Ca2+] after addition of calcium to parvalbumin at various levels of [Mg2+] in a stopped-flow apparatus (Ogawa & Tanokura, 1986b). The basis for the discrepancy between these values and the above reports is not apparent.

Calcium dependence of the SR calcium pump rate A second major new result in the present paper is the determination of the steadystate [Ca2+] dependence of the calcium transport rate of the SR calcium pump in a functionally and structurally intact skeletal muscle fibre preparation. Using the rate of decline of [Ca]T as a measure of the net rate of removal of calcium from the cytosol by the SR and assuming the pump to be in the steady state from 3 to 16 s after cessation of calcium release, the [Ca2+] and - d[Ca]T/dt data during this interval were used to determine the steady-state [Ca2+] dependence of the pump. The resulting pump rate was found to vary with the fourth power of [Ca2+]. A fourth power relationship between pump rate and [Ca2+] during the slow recovery indicates co-operative binding of calcium ions at four or more interacting binding sites. Since there are two calcium binding sites per pump monomer (Martonosi & Beeler, 1983; Inesi, 1985), our results indicate that at least two pump monomers must interact to form the basic functional unit of the SR calcium pump in frog skeletal muscle fibres. The simplest interpretation is that pump dimers constitute the basic functional unit. There is considerable precedent for the idea that pump dimers constitute the basic unit of the SR calcium pump. Close physical association of pairs of pump monomers is indicated by several observations. The molecular weight of a pump dimer corresponds to the target size of the pump determined from radiation inactivation measurements on isolated SR vesicles (Hymel, Maurer, Berenski, Jung & Fleischer, 1984). Fluorescence energy transfer occurs between fluorophore molecules bound to lysine residues at the ATP binding sites of different pump monomers in SR vesicles (Papp, Pikula & Martonosi, 1987). Pump dimers are the basic structural unit of crystallized SR calcium pump protein (Taylor, Ho & Martonosi, 1986). Although close physical association of pump monomers does not necessarily indicate functional interaction, calcium binding studies have provided evidence for functional interaction between pump monomers. In studies using SR vesicles prepared from


667 rabbit skeletal muscle, measurements and analysis of the [Ca2+] dependence of equilibrium calcium binding in the absence of ATP have indicated the likely presence of four interacting calcium binding sites (Watanabe, Lewis, Nakamoto, Kurzmack, Fronticelli & Inesi, 1981; Hill & Inesi, 1982). Finally, inactivation of SR ATPase by chemical modification exhibited a dependence on extent of fractional reaction consistent with functional pump dimers (Squier, Hughes & Thomas, 1988). An important consideration in the interpretation of the measured [Ca2+] dependence of steady-state calcium pumping is the extent to which the calcium binding step is reflected in the overall pump rate. In our experiments we have determined the pump rate under conditions of relatively low [Ca2+] and, consequently, at relatively low cycling rates. If the pump rate were sufficiently slow that the calcium bindings steps were effectively at equilibrium with the cytosolic [Ca2+], the pump rate would simply be proportional to the fraction of pump units having the required number of bound calcium ions. In order to investigate this possibility we have employed a detailed kinetic model for the pump (Inesi & de Meis, 1989). Although the model does not consider interactions between pump monomers, it does serve as a useful starting point for examining the relationship between calcium binding and the calcium transport rate at low [Ca2+]. Using the model with cytosolic [Ca2+] below 100 nm and with 1 mm SR luminal free [Ca2+], 1 mm [ATP], 100 ,um [ADP] and 100 /IM-inorganic phosphate, the predicted rate of calcium transport followed the 1 9 power of [Ca2+] (our calculations from the model of Inesi & de Meis, 1989). This is very close to the predicted value of 2 for equilibrium calcium binding at two infinitely co-operative calcium binding sites. Thus, for the model in which pump monomers do not interact the steady-state pump rate at low [Ca2+] follows the [Ca2+] dependence of the assumed highly co-operative calcium binding steps. It therefore seems plausible that a modified model that did include pump dimers could also give a rate of calcium transport that followed the equilibrium [Ca2+] dependence of calcium binding. The observed fourth power dependence of the transport rate of [Ca21] would then be accounted for in the modified model by assuming that calcium binding at any one of four interacting sites would greatly increase the affinity of the remaining three sites for Ca2+.

Time course of recovery of releasable calcium The final major result in the present paper was the determination of the time course of recovery of releasable calcium in the SR and its comparison with the time course and [Ca2+] dependence of the decline of [Ca]T. From 1 to 16 s after cessation of calcium release the recovery of releasable calcium occurred with two components. The faster component had a time constant of about 1 5 s, which was about the same as the time constant of 1 7 s for the faster of the two slow components of decline of [Ca]T. However, the slower phase of recovery of releasable calcium had a time constant of 32 s, considerably longer than the time constant of 14 s for the slower phase of recovery of [Ca]T. Furthermore, the slower phase of recovery of releasable calcium may have represented a larger fractional contribution to the total recovery than the fractional contribution of the slower phase of decline of [Ca]T. Both results arise from the prolonged final recovery of releasable calcium compared to the final decline of [Ca]T. Although the reason for the slowed final recovery of release is not established, it could be due to the existence of a pool of calcium removed from the

668


myoplasm by the SR but not immediately available for release. Alternatively, the slowed final recovery of release could be due to a slow phase of recovery from suppression of release due to some other mechanism such as some slow inactivation process. It is interesting to note in this regard that the initial study of the time course of the recovery of calcium release (Schneider et al. 1987) also contained some indication for a slow final phase of recovery of release that was not reflected in the decline of calcium bound to myoplasmic sites. Figure 6 of that report indicates that the resting rate of release was slightly but consistently higher than that expected from the recovery of unoccupied myoplasmic sites, but this slight discrepancy was not pointed out in the original report (Schneider et al. 1987). Excluding the return to resting conditions at times after 16 s of recovery, the [Ca2"] dependence of the rate of recovery of releasable calcium observed here exhibited a [Ca2"] dependence that was similar to the [Ca2+] dependence of the rate of decline of ICa]T. This is as expected if both measures reflect the [Ca2+] dependence of the SR calcium pump rate. In conclusion, the present results indicate the presence of two slow components in the decline of both [Ca21] and [Ca]T and in the recovery of releasable calcium. [Ca2+] and ICa]T followed the same time course of decline, whereas the releasable calcium exhibited a slower final phase of recovery than the final phase of decline of either [Ca2+] or [Ca]T. However, both the decline of [Ca]T and the recovery of releasable calcium exhibited a high power dependence on [Ca2+], consistent with dimers being the functional unit of the SR calcium pump in voltage-clamped muscle fibres. APPENDIX

Assuming the rate of efflux of calcium from the SR during slow recovery to be proportional to the SR calcium content, the magnitude of leak efflux would be given by the product fLr where f is the fraction of the resting SR content actually present in the SR at any time and Lr is the rate of leak efflux in the resting fibre. If [Ca]SR is the SR calcium content at any time expressed as an equivalent concentration that would be produced if the entire content at that time were free in the myoplasmic volume and if [Ca]SR r is the value of [Ca]sR in the resting fibre, f would be given by (A 1) f = [Ca]SR/[Ca]SR, rAssuming calcium fluxes across the external membranes to be negligible, any change in the total myoplasmic calcium [Ca]T would be produced by an opposite change in [Ca]sR so that

[Ca]SR, r-[Ca]SR

=

[Ca]T-[Ca]T, r,

(A 2)

where [Ca]T r is the value of [Ca]T in the resting fibre. Assuming the change in [Ca]T to be proportional to the change in [Ca2+] during slow recovery (present results), (A 3) [Ca]sR r-[Ca]sR = R([Ca2+]-[Ca2+]r), where [Ca2+]r is the level of [Ca21] in the resting fibre and the constant R is the ratio of the change in [CaIT to the change in [Ca2+], which is the total myoplasmic expansion for calcium (Kovacs et al. 1983). Solving eqn (A 3) for [Ca]SR and substituting in eqn (A 1), (A 4) f I -R*([Ca 2+] [Ca 2+ Ir), =

SR CALCIUTM PUTMP IN FROG MUTSCLE where R* is R/[Ca]SR r. Using fLr in place of L in eqn (4), - d[Ca]T/dt = A [Ca2"] -Lr +L1R*([Ca2+] -[Ca2+]r).

669

(A 5)

The first term on the right-hand side of eqn (A 5) represents the rate of calcium influx via the pump, the second term represents the leak efflux in the resting fibre and the third term gives the change in leak efflux with changes in myoplasmic [Ca2+] from [Ca2+]r due to calcium release from the SR. We thank Gerard Vaio for excellent technical assistance, Gabe Sinclair and Walt Knapik for custom modification of optical and mechanical apparatus, and Jeff Michael and Chuck Leffingwell for construction of electronic equipment. This work was supported by grants from the National Institutes of Health (PO1-HL27867) and the National Science Foundation (DCB-8544787). REFERENCES

BAYLOR, S. M., CHANDLER, W. K. & MARSHALL, M. W. (1982). Use of metallochromic dyes to measure changes in myoplasmic Ca2' during activity in frog skeletal muscle fibres. Journal of Physiology 331, 139-178. 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, 625-666. BAYLOR, S. M. & HOLLINGWORTH, S. (1988). Fura-2 Ca2+ transients in frog skeletal muscle fibres. Journal of Physiology 403, 151-192. BAYLOR, S. M., HOLLINGWORTH, S., Hui, C. S. & QuINTA-FERREIRA, M. E. (1986). Properties of the metallochromic dyes arsenazo III, antipyrylazo III and azo 1 in frog skeletal muscle fibres at rest. Journal of Physiology 377, 89-141. BAYLOR, S. M., QUINTA-FERREIRA, M. E. & Hui, C. S. (1985). Isotropic components of antipyrylazo III signals from frog skeletal muscle fibres. In Calcium in Biological Systems. ed. RUBIN, R. P., WEISS, G. & PUTNEY, J. W., pp. 339-349. Plenum Publishing Corp., New York. BLINKS, J. R., WIER, W. G., HESS, P. & PRENDERGAST, F. G. (1982). Measurement of Ca2+ concentrations in living cells. Progress in Biophysics and Molecular Biology 40, 1-114. BRUM, G., RIos, E. & SCHNEIDER, M.F. (1988a). A quantitative model of calcium removal from the myoplasmic solution (Appendix to G. BRUM, E. RIos & E. STEFANI, Effects of extracellular calcium on the calcium movements of excitation-contraction coupling in skeletal muscle fibres). Journal of Physiology 398, 441-473. BRUM, G., RIos, E. & STEFANI, E. (1988b). Effects of extracellular calcium on calcium movements of excitation-contraction coupling in frog skeletal muscle fibres. Journal of Physiology 398, 441-473. CANNELL, M. B. (1986). Effect of tetanus duration on the free calcium during the relaxation of frog skeletal muscle fibres. Journal of Physiology 376, 203-218. GILLIS, J. M., PIRONT, A. & GoSSELIN-REY, C. (1979). Parvalbumins. Distribution and physical state inside the muscle cell. Biochimica et Biophysica Acta 585, 444-450. GILLIS, J. M., THOMASON, D., LEFEVRE, J. & KRETSINGER, R. H. (1982). Parvalbumins and muscle relaxation: a computer simulation study. Journal of Muscle Research and Cell Motility 3, 377-398. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. HILL, T. L. & INESI, G. (1982). Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase. Proceedings of the National Academy of Sciences of the USA 79, 3978-3982. Hou, T. T., JOHNSON, J. D. & RALL, J. A. (1990). Effects of temperature on Ca2+ and Mg2+ dissociation rates from frog parvalbumin. Biophysical Journal 57, 550a. HYMEL, L., MAURER, A., BERENSKI, C., JUNG, C. Y. & FLEISCHER, S. (1984). Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum. Journal of Biological Chemistry 259, 4890-4895.

670


INESI, G. (1985). Mechanism of calcium transport. Annual Review of Physiology 47, 573-601. INESI, G. & DE MEIS, L. (1989). Regulation of steady state filling in sarcoplasmic reticulum. Roles of back-inhibition, leakage, and slippage of the calcium pump. Journal of Biological Chemistry 264, 5929-5936. IRVING, M., MAYLIE, J., SIZTO, N. L. & CHANDLER, W. K. (1989). Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibres containing Antipyrylazo III. Journal of General Physiology 93, 585-608. JOHNSON, J. D., ROBINSON, D. E., ROBERTSON, S. P., SCHWARTZ, A. & POTTER, J. (1981). Ca++ exchange with troponin and the regulation of muscle contraction. In Regulation of Muscle Contraction: Excitation-Contraction Coupling, ed. GRINNELL, A. D. & BRAZIER, M. A. B., pp. 241-257. Academic Press, New York. KLEIN, M. G., SIMON, B. J. & SCHNEIDER, M. F. (1990). Effects of caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle fibres. Journal of Physiology 425, 599-626. KLEIN, M. G., SIMON, B. J., Sztcs, G. & SCHNEIDER, M. F. (1988). Simultaneous recording of calcium transients in skeletal muscle using high and low affinity calcium indicators. Biophysical Journal 55, 971-988. KONISHI, M., OLSON, A., HOLLINGWORTH, S. & BAYLOR, S. M. (1988). Myoplasmic binding of FURA-2 investigated by steady-state fluorescence and absorbance measurements. Biophysical Journal 54, 1089-1104. KOVACS, L., KLEIN, M. G., SIMON, B. J. & SCHNEIDER, M. F. (1989). The calcium dependence of calcium uptake by sarcoplasmic reticulum in voltage clamped skeletal muscle fibres. Biophysical Journal 55, 94a. 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, 161-196. MARTONOSI, A. & BEELER, T. J. (1983). Mechanism of Ca2" transport by sarcoplasmic reticulum In Handbook of Physiology: Skeletal Muscle, ed. PEACHEY, L. D., pp. 417-485. American Physiological Society, Bethesda, MD, USA. MAYLIE, J., IRVING, M., SIZTO, N. L. & CHANDLER, W. K. (1987). Calcium signals recorded from cut frog twitch fibres containing antipyrylazo III. Journal of General Physiology 89, 83-143. MELZER, W., RIoS, E. & SCHNEIDER, M. F. (1984). Time course of calcium release and removal in skeletal muscle fibres. Biophysical Journal 45, 637-641. MELZER, W., RIos, E. & SCHNEIDER, M. F. (1986). The removal of myoplasmic free calcium following calcium release in frog skeletal muscle. Journal of Physiology 372, 261-292. MELZER, W., RIoS, E. & SCHNEIDER, M. F. (1987). A general procedure for determining calcium release from the sarcoplasmic reticulum in skeletal muscle fibres. Biophysical Journal 51, 849-863. MILEDI, R., PARKER, I. & ZHU, P. H. (1983). Calcium transients on frog skeletal muscle fibres following conditioning stimuli. Journal of Physiology 339, 223-242. OGAWA, Y. & TANOKURA, M. (1986a). Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle. Journal of Biochemistry 99, 81-89. OGAWA, Y. & TANOKURA, M. (1986b). Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature. Journal of Biochemistry 99, 73-80. PAPP, S., PIKULA, S. & MARTONOSI, A. (1987). Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum. Biophysical Journal 51, 205-220. PECHERE, J.-F., DEMAILLE, J. & CAPONY, J.-P. (1971). Muscular parvalbumins: preparative and analytical methods of general applicability. Biochimica et Biophysica Acta 236, 391-408. POTTER, J. D., JOHNSON, J. D., DEDMAN, J. R., SCHREIBER, W. E., MANDEL, F., JACKSON, R. L. & MEANS, A. R. (1977). Calcium-binding proteins: relationship of binding, structure, conformation and biological function. In Calcium-binding Proteins and Calcium Function, ed. WASSERMAN, R. H., CORRADINO, R. A., CAROFOLI, E., KRETSINGER, R. H., MAcLENNAN, D. H. & SIEGEL, F. L., pp. 239-250. North-Holland, Amsterdam. SCHNEIDER, M. F. & SIMON, B. J. (1988). Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle. Journal of Physiology 405, 727-745. SCHNEIDER, M. F., SIMON, B. J. & KLEIN, M. G. (1989). Decline of calcium release from the sarcoplasmic reticulum in skeletal muscle cells due to inactivation and calcium depletion. In


671

Physiology and Pharmacology of Transmembrane Signaling, ed. SEGAWA, T., ENDO, M., UI, M. & KURIHARA, K., pp. 253-260. Elsevier Science Publishers B.V. (Biomedical Division), Amsterdam. SCHNEIDER, M. F., SIMoN, B. J. & Szucs, G. (1987). Depletion of calcium from the sarcoplasmic reticulum during calcium release in frog skeletal muscle. Journal of Physiology 392, 167-192. SIMON, B. J., KLEIN, M. G. & SCHNEIDER, M. F. (1991). Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers. Journal of General Physiology 97, 437-471. SQUIER, T. C., HUGHES, S. E. & THOMAS, D. D. (1988). Rotational dynamics and protein-protein interactions in the Ca-ATPase mechanism. Journal of Biological Chemistry 263, 9162-9170. TAYLOR, K. A., Ho, M.-H. & MARTONOSI, A. (1986). Image analysis of the Ca2+-ATPase from sarcoplasmic reticulum. Annals of the New York Academy of Sciences 483, 31-42. WATANABE, T., LEWIS, D., NAKAMOTO, R., KURZMACK, M., FRONTICELLI, C. & INESI, G (1981). Modulation of calcium binding in sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry 20, 6617-6625.

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles.

Activation of inositol trisphosphate-sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

Effects of procaine and caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle.

Involvement of sarcoplasmic reticulum 'Ca2+ release channels' in excitation-contraction coupling in vertebrate skeletal muscle.

Effects of caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle fibres.

Properties of immunoaffinity purified 106-kDa Ca2+ release channels from the skeletal sarcoplasmic reticulum.

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle.

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel.

A Calsequestrin-1 Mutation Associated with a Skeletal Muscle Disease Alters Sarcoplasmic Ca2+ Release.

Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels.

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

Photooxidation of skeletal muscle sarcoplasmic reticulum induces rapid calcium release.

Effects of type 1 diabetes, sprint training and sex on skeletal muscle sarcoplasmic reticulum Ca2+ uptake and Ca2+-ATPase activity.

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The effects of Suramin on Ca2+ activated force and sarcoplasmic reticulum Ca2+ release in skinned fast-twitch skeletal muscle fibers of the rat.

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.

Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers.

Effect of perfusate [Ca2+] on cardiac sarcoplasmic reticulum Ca2+ release channel in isolated rat hearts.

The effects of dihydropyridine derivatives on force and Ca2+ current in frog skeletal muscle fibres.

Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.

Ca2+ release from the sarcoplasmic reticulum of barnacle myofibrillar bundles initiated by photolysis of caged Ca2+.