Effect of membrane polarization on contractile threshold and time course of prolonged contractile responses in skeletal muscle fibers (original) (raw)
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Depolarization-contraction coupling in short frog muscle fibers. A voltage clamp study
The Journal of General Physiology, 1984
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 >_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.
Ultraslow contractile inactivation in frog skeletal muscle fibers
The Journal of General Physiology, 1990
After a contracture response, skeletal muscle fibers enter into a state of contractile refractoriness or inactivation. Contractile inactivation starts soon after membrane depolarization, and causes spontaneous relaxation from the contracture response. Here we demonstrate that contractile inactivation continues to develop for tens of seconds if the membrane remains in a depolarized state. We have studied this phenomenon using short (1.5 mm) frog muscle fibers dissected from the Lumbricalis brevis muscles of the frog, with a two-microelectrode voltage-clamp technique. After a contracture caused by membrane depolarization to 0 mV, from a holding potential of -100 mV, a second contracture can be developed only if the membrane is repolarized beyond a determined potential value for a certain period of time. We have used a repriming protocol of 1 or 2 s at -100 mV. After this repriming period a fiber, if depolarized again to 0 mV, may develop a second contracture, whose magnitude and time course will depend on the duration of the period during which the fiber was maintained at 0 mV before the repriming process. With this procedure it is possible to demonstrate that the inactivation process builds up with a very slow time course, with a half time of ~35 s and completion in >100 s. After prolonged depolarizations (>100 s), the repriming time course is slower and the inactivation curve (obtained by plotting the extent of repriming against the repriming membrane potential) is shifted toward more negative potentials by >30 mV when compared with similar curves obtained after shorter depolarizing periods (10-30 s). These results indicate that important changes occur in the physical state of the molecular moiety that is responsible for the inactivation phenomenon. The shift of the inactivation curve can be partially reversed by a low concentration (50 #M) of lanthanum ions. In the presence of 0.5 mM caffeine, larger responses can be obtained even after prolonged depolarization periods, indicating that the fibers maintain their capacity to liberate calcium.
Proceedings of the National Academy of Sciences, 1997
Applying a brief repolarizing pre-pulse to a depolarized frog skeletal muscle fiber restores a small fraction of the transverse tubule membrane voltage sensors from the inactivated state. During a subsequent depolarizing test pulse we detected brief, highly localized elevations of myoplasmic Ca 2؉ concentration (Ca 2؉ "sparks") initiated by restored voltage sensors in individual triads at all test pulse voltages. The latency histogram of these events gives the gating pattern of the sarcoplasmic reticulum (SR) calcium release channels controlled by the restored voltage sensors. Both event frequency and clustering of events near the start of the test pulse increase with test pulse depolarization. The macroscopic SR calcium release waveform, obtained from the spark latency histogram and the estimated open time of the channel or channels underlying a spark, exhibits an early peak and rapid marked decline during large depolarizations. For smaller depolarizations, the release waveform exhibits a smaller peak and a slower decline. However, the mean use time and mean amplitude of the individual sparks are quite similar at all test depolarizations and at all times during a given depolarization, indicating that the channel open times and conductances underlying sparks are essentially independent of voltage. Thus, the voltage dependence of SR Ca 2؉ release is due to changes in the frequency and pattern of occurrence of individual, voltage-independent, discrete release events.
Journal of General Physiology, 1994
A B S T R A C T Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (=-45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n ---18), and the time at which it occurred was 34 ms (SEM = 1.8, n --18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/~F charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/r (SEM = 0.8, n --6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre-and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in INTRODUCTION The mechanochemical reactions that generate contractile tension in muscle are triggered by an increase in myoplasmic [Ca2+], due to release of calcium from the sarcoplasmic reticulum (SR). The Ca 2+ release SR channels are controlled by the potential across the T tubule membrane, in a process that involves voltage sensor proteins that are now thought to be the high affinity DHP receptors of the T tubule Tanabe, Takeshima, Mikami, Flockerzi, Takahashi, Kangawa, Kojima, Matsuo, Hirose and Numa, 1987; Tanabe, Beam, Powell, and Numa, 1988). Changes of these sensors driven by voltage underlie intramembranous charge movement .
Kinetics of contractile activation in voltage clamped frog skeletal muscle fibers
Biophysical Journal, 1997
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 Ill 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, koff of TnC was calculated to be 78 s-1 for the cooperative model at 1 00C.
Effects of denervation on Ca channels in slow skeletal muscle fibers of the frog
Effects of denervation on calcium channels in slow skeletal muscle fibers in the frog (Rana pipiens) were studied using the 21 21 21 three-microelectrode voltage-clamp technique in intact fibers. Ca , Ba , and Sr currents were all significantly reduced in amplitude during the first 2 weeks after denervation. After nerve section the selectivity sequence Ba(Ca.Sr was changed to Ba.Sr.Ca and the 21 21 21 values for relative ratio increased from 1.04 to 2.65 for Ba and from 0.58 to 1.20 for Sr (with respect to Ca ). Barium current saturation was more obvious in denervated fibers than in non-denervated fibers. The values obtained with the Michaelis–Menten type 2 expression, I5I /(11K /[Ba] ) were K 52.7 mM and I 520 mA/cm in fibers 2 weeks after nerve section compared with the max d e d max 2 values K 54.4 mM and I 560 mA/cm obtained in non-denervated fibers. Additionally, the effects of two calcium channel blockers d max (cobalt and nifedipine) were greater by a factor of two in denervated fibers than in non-denervated fibers. Three weeks or so after nerve section, all the biophysical properties studied began to show a tendency to recover toward the values obtained in non-denervated muscles (controls). These results suggest that calcium channels are modified or that there is a change in the types of calcium channels present in frog slow skeletal muscle fibers after denervation. Ó 2001 Elsevier Science B.V. All rights reserved.
The Journal of Physiology, 1997
1. Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea-pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions. 2. The voltage dependence of intramembrane charge movement followed a two-state Boltzmann distribution with maximal available charge of 26-1 + 1P5 and 26-1 + 1P3 nC ,uF-, mid-point voltage of -35'1 + 5'0 and -42-2 + 1P2 mV and steepness of 16-7 + 2'2 and 17-0 + 1-9 mV (means + S.E.M., n = 7 and 4) in rats and guinea-pigs, respectively. 3. Intracellular calcium concentration ([Ca2+]1) was monitored using the calcium-sensitive dyes antipyrylazo III, fura-2 and mag-fura-5. Resting [Ca2+]i was similar in rats and guinea-pigs with 125 + 18 and 115 + 8 nm (n = 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6'3 + 1 0 /SM; n = 7), than in guinea-pigs (2-8 + 0 3; n= 4). 4. The rate of calcium release (Rrei) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrei to the estimated SR calcium content (1P2 + 0 1 and 0 9 + 0.1 mM in rats and guinea-pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2-50 + 0-08 and 0-81 + 0 06 % ms-' in rats and 2-43 + 0-25 and 0-88 + 0 01 % ms-' in guinea-pigs. The voltage dependence was
The Journal of General Physiology, 1993
The effects of high intracellular concentrations of various calcium buffers on the myoplasmic calcium transient and on the rate of release of calcium (Rrel) from the sarcoplasmic reticulum (SR) were studied in voltage-damped frog skeletal muscle fibers. The changes in intracellular calcium concentration (A[Ca2+]) for 200-ms pulses to 0-20 mV were recorded before and after the injection of the calcium buffer and the underlying Rrel was calculated. If the buffer concentration after the injection was high, the initial rate of rise of the calcium transient was slower after injection than before and was followed by a slow increase of [Ca 2+] that resembled a ramp. The increase in myoplasmic [Mg 2+] that accompanies the calcium transient in control was suppressed after the injection and a slight decrease was observed instead. After the injection the buffer concentration in the voltageclamped segment of the fiber decreased as the buffer diffused away toward the open ends. The calculated apparent diffusion coefficient for fura-2 (D~pp = 0.40 -0.03 x 10 -6 cm2/s, mean -SEM, n = 6) suggests that ~65-70% of the indicator was bound to relatively immobile intracellular constituents. As the concentration of the injected buffer decreased, the above effects were reversed. The changes in A[Ca z+] were underlined by characteristic modification of Rrel. The early peak component was suppressed or completely eliminated; thus, Rre I rose monotonically to a maintained steady level if corrected for depletion. If Rrel was expressed as percentage of SR calcium content, the steady level after injection did not differ significantly from that before. Control injections of anisidine, to the concentration that eliminated the peak of Rr~l when high affinity buffers were used, had only a minor effect on Rrel , the peak was suppressed by 26 -+ 5% (mean -+ SE, n = 6), and the steady level remained unchanged. Thus, the peak component of Rr,t is dependent on a rise Address reprint requests to Dr. in myoplasmic [Ca~+], consistent with calcium-induced calcium release, whereas the steady component of Rret is independent of myoplasmic [Ca2+].
The elementary events of Ca2+ release elicited by membrane depolarization in mammalian muscle
2004
Cytosolic [Ca 2+ ] transients elicited by voltage clamp depolarization were examined by confocal line scanning of rat skeletal muscle fibres. Ca 2+ sparks were observed in the fibres' membranepermeabilized ends, but not in responses to voltage in the membrane-intact area. Elementary events of the depolarization-evoked response could be separated either at low voltages (near −50 mV) or at −20mV in partially inactivated cells. These were of lower amplitude, narrower andofmuchlongerdurationthansparks,similarto'loneembers'observedinthepermeabilized segments. Their average amplitude was 0.19 and spatial half-width 1.3 µm. Other parameters depended on voltage. At −50 mV average duration was 111 ms and latency 185 ms. At −20 mV duration was 203 ms and latency 24 ms. Ca 2+ release current, calculated on an average of events, was nearly steady at 0.5-0.6 pA. Accordingly, simulations of the fluorescence event elicited by a subresolution source of 0.5 pA open for 100 ms had morphology similar to the experimental average. Because 0.5 pA is approximately the current measured for single RyR channels in physiological conditions, the elementary fluorescence events in rat muscle probably reflect opening of a single RyR channel. A reconstruction of cell-averaged release flux at −20 mV based on the observed distribution of latencies and calculated elementary release had qualitatively correct but slower kinetics than the release flux in prior whole-cell measurements. The qualitative agreement indicates that global Ca 2+ release flux results from summation of these discrete events. The quantitative discrepancies suggest that the partial inactivation strategy may lead to events of greater duration than those occurring physiologically in fully polarized cells.