Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2

Cut muscle fibers from Rana temporaria (sarcomere length, 3.4-4.2 microns) were mounted in a double Vaseline-gap chamber (14-15 degrees C) and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2- 4 ms. Immediately after an action potential or a 10-15 ms prepulse to - 20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was approximately 25 ms in the action- potential experiments and approximately 50 ms in the voltage-clamp experiments. Fura-2, which is able to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a greater amount, did not prevent Ca inactivation of Ca release, even at concentrations as large as 6-8 mM. These effects of Ca inactivation of Ca release can be simulated by the three-state, two-step model proposed by Schneider, M. F., and B. J. Simon (1988, Journal of Physiology. 405:727-745), in which SR Ca channels function as a single uniform population of channels. (ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium release in frog cut twitch fibers exposed to different ionic environments under voltage clamp.

Calcium release was measured in highly stretched frog cut twitch fibers mounted in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 20 mM EGTA plus 0.4 or 1.8 mM added calcium. Rise in myoplasmic [Ca(2+)] was monitored with antipyrylazo III as the indicator at a temperature of 13 to 14 degrees C. The waveform of calcium release rate (Rel) computed from the absorbance change showed an early peak (Rel(p)) followed by a maintained phase (Rel(m)). Each Rel(p)-versus-V plot was fitted with a Boltzmann distribution function. The maximum value of Rel(p) (Rel(p,max)) was compared in various calcium-containing external solutions. The average value in a Cl(-) solution was about one-third larger than those in a CH(3)SO(3)(-) or gluconate solution, whereas the values in the CH(3)SO(3)(-) and gluconate solutions had no statistically significant difference. In external solutions containing CH(3)SO(3)(-) or gluconate, a replacement of the Ca(2+) with Mg(2+) reduced Rel(p,max) by 30 to 50%, on average. The values of Rel(p, max) also had no statistically significant difference among calcium-free external solutions containing different impermeant anions. An increase of the nominal free [Ca(2+)] in the end-pool solution from a reduced to the normal physiological level increased the value of Rel(p,max), and also slowed the decay of the maintained phase of the Rel waveform. The Rel waveforms in the Cl(-) and CH(3)SO(3)(-) solutions were compared in the same fiber at a fixed potential. CH(3)SO(3)(-) increased the time to peak, reduced Rel(p), and increased Rel(m), and the effects were partially reversible. Under the hypothesis that the decay of the peak was due to calcium inactivation of calcium release, the inactivation was larger in Cl(-) than in CH(3)SO(3)(-), in qualitative agreement with the ratio of Rel(p) in the two solutions. Under the alternative hypothesis that the peak and the maintained phase were separately gated by calcium and depolarization, respectively, then CH(3)SO(3)(-) appeared to decrease the calcium-gated component and increase the voltage-gated component.     

Effects of Partial Sarcoplasmic Reticulum Calcium Depletion on Calcium Release in Frog Cut Muscle Fibers Equilibrated with 20 mM EGTA

Resting sarcoplasmic reticulum (SR) Ca content ([Ca_SR]_R) was varied in cut fibers equilibrated with an internal solution that contained 20 mM EGTA and 0–1.76 mM Ca. SR Ca release and [Ca_SR]_R were measured with the EGTA–phenol red method (Pape et al. 1995. J. Gen. Physiol. 106:259–336). After an action potential, the fractional amount of Ca released from the SR increased from 0.17 to 0.50 when [Ca_SR]_R was reduced from 1,200 to 140 μM. This increase was associated with a prolongation of release (final time constant, from 1–2 to 10–15 ms) and of the action potential (by 1–2 ms). Similar changes in release were observed with brief stimulations to −20 mV in voltage-clamped fibers, in which charge movement (Q _cm) could be measured. The peak values of Q _cm and the fractional rate of SR Ca release, as well as their ON time courses, were little affected by reducing [Ca_SR]_R from 1,200 to 140 μM. After repolarization, however, the OFF time courses of Q _cm and the rate of SR Ca release were slowed by factors of 1.5–1.7 and 6.5, respectively. These and other results suggest that, after action potential stimulation of fibers in normal physiological condition, the increase in myoplasmic free [Ca] that accompanies SR Ca release exerts three negative feedback effects that tend to reduce additional release: (a) the action potential is shortened by current through Ca-activated potassium channels in the surface and/or tubular membranes; (b) the OFF kinetics of Q _cm is accelerated; and (c) Ca inactivation of Ca release is increased. Some of these effects of Ca on an SR Ca channel or its voltage sensor appear to be regulated by the value of [Ca] within 22 nm of the mouth of the channel.     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool solutions. The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1.75 x 10(-6) cm2/s at 18 degrees C. The shape of the resting absorbance spectrum suggests that a fraction 0.11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca. After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign. The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 microM. These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator. Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum. The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution. The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum. A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal. The estimated myoplasmic free [Ca] transient has an average peak value of 26 microM at 18 degrees C. Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143).     

Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca- induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.     

Calcium influx in contracting and paralyzed frog twitch muscle fibers

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.     

Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-81). Like arsenazo III, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18 degrees C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca:indicator complexation and, for indicator concentrations less than or equal to 0.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 microM at 18 degrees C. If only freely diffusible indicator can react, the estimate is 42 microM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42-microM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief; its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-microM transient and 0.93 for the 42-microM transient. During a 100-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J. R., R. Rudel, and S. R. Taylor. 1978. Journal of Physiology. 277:291-323; Lopez J. R., L. A. Wanck, and S. R. Taylor. 1981. Science. 214:47-82).     

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.- S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.     

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

The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.     

Simulation of calcium sparks in cut skeletal muscle fibers of the frog

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.     

Calcium signals recorded from two new purpurate indicators inside frog cut twitch fibers

Two new Ca indicators, purpurate-3,3'diacetic acid (PDAA) and 1,1'-dimethylpurpurate-3,3'diacetic acid (DMPDAA), were synthesized and used to measure Ca transients in frog cut muscle fibers. These indicators are analogues of the purpurate components of murexide and tetramethylmurexide, in which two acetate groups have been incorporated into each molecule to render it membrane impermeant. The apparent dissociation constant for Ca is 0.95 mM for PDAA and 0.78 mM for DMPDAA. One of the indicators was introduced into a cut fiber, which was mounted in a double Vaseline-gap chamber, by diffusion from the end-pool solutions. The time course of indicator concentration, monitored optically in the middle of the fiber in the central-pool region, suggests that 19% of the PDAA or 27% of the DMPDAA became bound or sequestered inside the fiber. In resting fibers, the absorbance spectrum of either indicator was well fitted by the indicator's [Ca] = 0 mM cuvette absorbance spectrum, which is consistent with the idea that PDAA and DMPDAA do not enter the sarcoplasmic reticulum as tetramethylmurexide appears to be able to do (Maylie, J., M. Irving, N.L. Sizto, G. Boyarsky, and W. K. Chandler, 1987. Journal of General Physiology. 89:145-176). After an action potential, the absorbance of either indicator underwent a rapid and transient change that returned to the prestimulus baseline within 100-200 ms. The amplitude of this change had a wavelength dependence that matched the indicator's Ca-difference spectrum. The average amplitude of peak free [Ca] was 21 microM (PDAA or DMPDAA) if all the indicator inside a fiber was able to react with Ca as in cuvette calibrations, and was 26 (PDAA) or 28 microM (DMPDAA) if only freely diffusible indicator could so react. These results suggest that PDAA and DMPDAA are the first Ca indicators that provide a reliable estimate of both the amplitude and time course of (the spatial average of) free [Ca] in a twitch muscle fiber after an action potential.     

Calcium sparks in intact skeletal muscle fibers of the frog.

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release.     

Kinetics of contractile activation in voltage clamped frog skeletal muscle fibers.

Excitation-contraction coupling events leading to the onset of contraction were studied in single skeletal frog muscle fibers. This entailed the simultaneous measurement of the changes in intracellular calcium concentration using antipyrylazo III and fura-2, isometric force, and clamp voltage in a modified single vaseline gap chamber for the first time. The calcium transients were incorporated into an analysis of calcium binding to regulatory sites of troponin C (TnC) that permitted both a linear and a cooperative interaction. The analysis assumed that the onset of mechanical activation corresponds with a particular TnC saturation with calcium setting constraints for the calcium binding parameters of the regulatory sites. Using a simple model that successfully reproduced both the time course and the relative amplitudes of the measured isometric force transients over a wide membrane potential range, k(off) of TnC was calculated to be 78 s(-1) for the cooperative model at 10 degrees C. Together with the above constraints this gave a dissociation constant of 8.8 +/- 2.5 microM and a relative TnC saturation at the threshold (Sth) that would cause just detectable movement of 0.17 +/- 0.03 (n = 13; mean +/- SE). The predictions were found to be independent of the history of calcium binding to the regulatory sites. The observed delay between reaching Sth and the onset of fiber movement (8.7 +/- 1.0 ms; mean +/- SE, n = 37; from seven fibers) was independent of the membrane potential giving an upper estimate for the delay in myofilament activation. We thus emerge with quantitative values for the calcium binding to the regulatory sites on TnC under maintained structural conditions close to those in vivo.     

Depolarization-contraction coupling in short frog muscle fibers. A voltage clamp study

Short muscle fibers (1.5 mm) were dissected from hindlimb muscles of frogs and voltage clamped with two microelectrodes to study phenomena related to depolarization-contraction coupling. Isometric myograms obtained in response to depolarizing pulses of durations between 10 and 500 ms and amplitudes up to 140 mV had the following properties. For suprathreshold pulses of fixed duration (in the range of 20-100 ms), the peak tension achieved, the time to peak tension, and contraction duration increased as the internal potential was made progressively more positive. Peak tension eventually saturates with increasing internal potentials. For pulse durations of greater than or equal to 50 ms, the rate of tension development becomes constant for increasing internal potentials when peak tensions become greater than one-third of the maximum tension possible. Both threshold and maximum steepness of the relation between internal potential and peak tension depend on pulse duration. The relation between the tension-time integral and the stimulus amplitude-duration product was examined. The utility of this relation for excitation-contraction studies is based on the observation that once a depolarizing pulse configuration has elicited maximum tension, further increases in either stimulus duration or amplitude only prolong the contractile response, while the major portion of the relaxation phase after the end of a pulse is exponential, with a time constant that is not significantly affected by either the amplitude or the duration of the pulse. Hence, the area under the tension-response curve provides a measure of the availability to troponin of the calcium released from the sarcoplasmic reticulum in response to membrane depolarization. The results from this work complement those obtained in experiments in which intramembrane charge movements related to contractile activation were studied and those in which intracellular Ca++ transients were measured.     

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.