Fast calcium removal during single twitches in amphibian skeletal muscle fibres (original) (raw)
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The Journal of General Physiology, 1989
The mechanism(s) of the decay of slow calcium current (/ca) in cut twitch skeletal muscle fibers of the frog were studied in voltage-clamp experiments using the double vaseline-gap technique./ca decay followed a single exponential in 10 mM external Ca 2 § and 20 mM internal EGTA solutions in all pulse protocols tested: single depolarizing pulses (activation protocol), two pulses (inactivation protocol), and during a long pulse preceded by a short prepulse (400 ms) to 80 mV (tail protocol). In single pulses the rate constant oflc~ decay was ~0.75 s -~ at 0 mV and became faster with larger depolarizations. Ic~ had different amplitudes during the second pulses of the inactivation protocol (0 mV) and of the tail protocol (-20 to 40 mV) and had similar time constants of decay. The time constant of decay did not change significantly at each potential after replacing 10 mM Ca 2 § with a Ca2+-buffered solution with malate. With 70 mM intracellular EGTA and 10 mM external Ca 2+ solutions,/ca also decayed with a single-exponential curve, but it was about four times faster (~3.5 s -~ at 0 mV pulse). In these solutions the rate constant showed a direct relationship with Ic~ amplitude at different potentials. With 70 mM EGTA, replacing the external 10 mM Ca ~+ solution with the Ca ~+buffered solution caused the decay of Ic~ to become slower and to have the same relationship with membrane potential and/ca amplitude as in fibers with 20 mM EGTA internal solution. The mechanism of Ic~ decay depends on the intracellular EGTA concentration: (a) internal EGTA (both 20 and 70 mM) significantly reduces the voltage dependence of the inactivation process and (b) 70 mM EGTA dramatically increases the rate of tubular calcium depletion during the flow of ~c~.
Evidences for calcium-dependent inactivation of calcium current at the frog motor nerve terminal
Brain research bulletin, 2006
Assessment of calcium-dependent inactivation of calcium current in nerve terminals is not feasible due to technical reasons. Perineural measurement of calcium-flow, however, might be utilized as indirect means to evaluate synaptic currents. Using perineural recording from frog neuromuscular junction, supra-threshold stimuli applied to motor nerve in paired-pulse manner with varying inter-pulse intervals (5-50 ms) are demonstrated in this study to cause paired-pulse depression (PPD) of Ca(2+)-current. PPD of Ca(2+)-flow was reduced at lower extracellular Ca(2+) concentrations, in BAPTA-AM and EGTA-AM treated preparations and after replacing extracellular Ca(2+) with Sr(2+). Using perineural measurement of calcium current as an indirect model to investigate synaptic ionic activity, our findings demonstrate that PPD may be attributed to calcium-dependent inactivation of Ca(2+)-current, which may serve as negative feedback in response to massive Ca(2+) entry to motor nerve terminals. A ...
Fast gating kinetics of the slow Ca2+ current in cut skeletal muscle fibres of the frog
The Journal of physiology, 1990
1. Calcium currents and intramembrane charge movements were measured in cut twitch muscle fibres of the frog and the time course of activation of the current was studied using various conditioning pulse protocols. 2. When a conditioning activation was produced by a depolarizing pulse which ended before inactivation occurred, a subsequent depolarization led to a faster onset of activation, indicating that the system had not completely returned to the initial state during the interval between the two pulses. 3. The interval between conditioning and test pulse was varied at different subthreshold potentials to study the time course of restoring the steady-state conditions. Complete restoration required a waiting period of about 1 min at the holding potential of -80 mV due to a very slow process but partial recovery was reached within 100 ms. This initial recovery process was strongly voltage dependent and became considerably slower when the interval potential approached the threshold f...
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.
Biophysical Journal, 2003
In voltage-clamped frog skeletal muscle fibers, Ca 21 release rises rapidly to a peak, then decays to a nearly steady state. The voltage dependence of the ratio of amplitudes of these two phases (p/s) shows a maximum at low voltages and declines with further depolarization. The peak phase has been attributed to a component of Ca 21 release induced by Ca 21 , which is proportionally greater at low voltages. We compared the effects of two interventions that inhibit Ca 21 release: inactivation of voltage sensors, and local anesthetics reputed to block Ca 21 release induced by Ca 21. Holding the cells partially depolarized strongly reduced the peak and steady levels of Ca 21 release elicited by a test pulse and suppressed the maximum of the p/s ratio at low voltages. The p/s ratio increased monotonically with test voltage, eventually reaching a value similar to the maximum found in noninactivated fibers. This implies that the marked peak of Ca 21 release is a property of a cooperating collection of voltage sensors rather than individual ones. Local anesthetics reduced the peak of release flux at every test voltage, and the steady phase to a lesser degree. At variance with sustained depolarization, they made p/s low at all voltages. These observations were well-reproduced by the ''couplon'' model of dual control, which assumes that depolarization and anesthetics respectively, and selectively, disable its Ca 21-dependent or its voltage-operated channels. This duality of effects and their simulation under such hypotheses are consistent with the operation of a dual, two-stage control of Ca 21 release in muscle, whereby Ca 21 released through multiple directly voltage-activated channels builds up at junctions to secondarily open Ca 21-operated channels.
Calcium channel inactivation in frog (Rana pipiens and Rana moctezuma) skeletal muscle fibres
The Journal of Physiology, 1984
1. The decay of the Ca2+ current (ICa) during a maintained depolarization was studied in intact twitch skeletal muscle fibres of Rana pipiens and Rana moctezuma with the three-micro-electrode voltage-clamp technique. 2. ICa was recorded at 23 TC, after blocking K+ currents, in TEA methanesulphonate saline with 10 mM-Ca2+ made hypertonic by adding 350 mM-sucrose. 3. In two-pulse experiments, ICa during the test pulse was reduced to about 80 % (R. pipiens) or 50 % (R. moctezuma) of the control value, without any detectable inward ICa during 7 s conditioning pre-pulses. 4. The experimental points of the steady-state inactivation curve (h.) were fitted to hoo = (1 +exp ((Em-Vh)/kh))-, where Em is the membrane potential and with Vh =-33 + 3 mV and kh = 6 +1 mV for R. pipiens, and Vh =-44 + 3 mV and kh = 9-5 + ±10 mV for R. moctezuma. 5. The rate constant of decay for inactivated currents (range-8 to-47 mA cm3) and for control currents (range-23 to-62 mA cm-3), was independent of Ica amplitude. The average rate constant of decay at 0 mV was 118 + 002 s-1 (66). 6. These results indicate that in intact fibres under hypertonic solution Ica decay can be explained by a voltage-dependent inactivation process and not by depletion of tubular Ca2+. The absence of depletion could be due to a large fractional tubular volume or to the presence of a Ca2+ pump in the tubular system. INTRODUCTION Voltage-clamp experiments in frog skeletal muscle fibres have shown that Ca2+ current (Ica) spontaneously decays during a maintained depolarization (Beaty &
Characteristics of Ca2+ release induced by Ca2+ influx in cultured bullfrog sympathetic neurones
The Journal of Physiology, 1993
1. A rise in intracellular Ca21 ([Ca2+]i) and a Ca2+ current (ICa) induced by a depolarizing pulse were simultaneously recorded by fura-2 or indo-1 fluorescence and whole-cell patch clamp techniques in cultured bullfrog sympathetic ganglion cells. 2. [Ca2+]i (calculated from the ratio of fura-2 fluorescences excited at 380 and 340 nm and recorded with a photomultiplier at > 492 nm) rose regeneratively (in most cells) during a command pulse (from-60 to 0 mV, 100 ms), continued to rise thereafter, peaked at 666 ms (on average) and decayed slowly with a half-decay time of 22-8 s. 3. Scanning a single horizontal line across the cytoplasm with an ultraviolet argon ion laser (351 nm) and recording indo-1 fluorescences at two wavelengths (peaked at 410 and 475 nm) with a confocal microscope demonstrated that [Ca2+]i beneath the cell membrane rose much faster than that in the deeper cytoplasm. The time course of the spatial integral of [Ca2+]i, however, corresponded well with that recorded with fura-2 fluorescence using a photomultiplier. 4. [Ca21]i measured by fura-2 fluorescence ratio using a photomultiplier did not increase during a strong depolarizing pulse (-60 to + 80 mV), but sometimes rose after the pulse. A depolarization-induced rise in [Ca2+]i ([Ca2+]i transient) was blocked in a Ca2+-free, EGTA solution, reduced by lowering the extracellular Ca2+ concentration ([Ca2+]O) to 0 45 or 0-9 mm and enhanced by raising [Ca2+]o to 7-2 or 14-4 mM. 5. The extracellular Ca2+ dependence was non-linear when long depolarizing pulses (up to 500 ms) were applied; the amplitude of [Ca2+]i transient/Ca2+ entry (unit [Ca2+]i transient) increased with an increase in Ca2+ entry. 6. Increasing the duration of depolarization (-50 or-60 to 0 mV) from 20 to 500 ms enhanced asymptotically the integral of ICa (due to inactivation), and progressively the magnitude of [Ca2+]i transients, leading to the apparent non-linear dependence of unit [Ca2+]i transient on Ca2+ entry as well as on the duration of membrane depolarization. The peak time of [Ca2+]i transient was unchanged for pulse durations up to 300 ms, but prolonged with an increase in pulse duration to 500 ms. * To whom correspondence should be addressed. MS 1160 9 PHY 464 S.-Y. HUA, M. NOHMI AND K. KUBA 7. Inhibitors of Ca2" release from intracellular Ca"t reservoirs, dantrolene (10 saM) and ryanodine (50 AtM), blocked the [Ca2+]i transient to 56 and 30 %, respectively, of the control. 8. The higher the basal [Ca2+]i level, the greater was the magnitude of the [Ca2+]i transients. 9. When two [Ca2+]i transients were induced by a pair of pulses (-60 to 0 mV, 100 ms), the second transient was facilitated. The facilitation decreased in amplitude as the interval between the two pulses became longer, and was not seen when the second pulse was applied after the tail of the first transient. When the facilitation was induced by triple pulses, however, the facilitation of the third [Ca2+]i transient was greater at a relatively longer preceding interval. The facilitation was completely blocked by dantrolene (10 /aM). 10. The amplitudes of both ICa and [Ca2+]i transient showed a bell-shaped voltage dependence. There was a quantitative difference, however, in such a way that the unit [Ca2+]i transient was enhanced by membrane depolarization in the range-35 to-10 mV, and constant at more depolarized levels. 11. These results suggest that not only Ca2+ entry but also the resultant Ca2+ release from intracellular calcium stores are involved in a [Ca2+]i transient in cultured bullfrog sympathetic ganglion cells. Possible mechanisms for the graded activation of Ca2+-induced Ca2+ release are discussed.