N Shirokova

Local calcium release in mammalian skeletal muscle

1 Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres mechanically dissected from fast twitch muscle of rats or frogs, voltage clamped in a two Vaseline‐gap chamber. 2 Interventions that elicited Ca2+ sparks in frog skeletal muscle (low voltage depolarizations, application of caffeine) generated in rat fibres images consistent with substantial release from triadic regions, but devoid of resolvable discrete events. Ca2+ sparks were never observed in adult rat fibres. In contrast, sparks of standard morphology were abundant in myotubes from embryonic mice. 3 Depolarization‐induced gradients of fluorescence between triadic and surrounding regions (which are proportional to Ca2+ release flux) peaked at about 20 ms and then decayed to a steady level. Gradients were greater in frog fibres than in rat fibres. The ratio of peak over steady gradient (R) was steeply voltage dependent in frogs, reaching a maximum of 4.8 at −50 mV (n= 7). In rats, R had an essentially voltage‐independent value of 2.3 (n= 5). 4 Ca2+‐induced Ca2+ release, resulting in concerted opening of several release channels, is thought to underlie Ca2+ sparks and the peak phase of release in frog skeletal muscle. A diffuse ‘small event’ release, similar to that observed in these rats, is also present in frogs and believed to be directly activated by voltage. The present results suggest that in these rat fibres there is little contribution by CICR to Ca2+ release triggered by depolarization, and a lack of concerted channel opening.

Small event Ca2+ release: a probable precursor of Ca2+ sparks in frog skeletal muscle

1 Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres. Clamp depolarization to −65 or −60 mV elicited Ca2+ sparks with amplitudes and spatial widths distributed approximately normally, with mean values of 0.79 of resting fluorescence and 0.8 μm (S.D., 0.17 and 0.2 μm; n= 193), respectively. Given these distributions, events of amplitude less than 0.45 or width less than 0.4 μm are unlikely to be sparks. 2 Low voltage depolarization (−72 mV) elicited only one spark per triad every 6 s, but generated a relative increase in fluorescence at triads of 0.05. This increase must therefore have been due to events smaller than sparks. 3 The variance/mean ratio of triadic fluorescence gradients averaged 0.11 at low voltages and increased severalfold at the higher voltages at which sparks appeared, indicating the existence of at least two event amplitudes. 4 Tetracaine (200 μm) reversibly abolished sparks and the early peak of Ca2+ release at all voltages. In its presence, discrete events were smaller than the spark criterion, and triadic gradients had a variance/mean ratio of 0.11. 5 The phenylalkylamine D600 (2 μm) reduced release at all voltages, abolishing sparks and the peak of Ca2+ release at low but not at high voltages. 6 The parallel abolition of all sparks and the peak of Ca2+ release indicates that both phenomena are activated by Ca2+. The restoration of sparks by voltage in D600 suggests that release in small events provides the trigger Ca2+ for activation of sparks.

Spatially segregated control of Ca2+ release in developing skeletal muscle of mice.

1 Confocal laser scanning microscopy was used to monitor Ca2+ signals in primary‐cultured myotubes, prepared from forelimbs of wild‐type or ryanodine receptor type 3 (RyR3) knockout mice. Myotubes loaded with the acetoxymethyl ester (AM) form of fluo‐3 were imaged at rest or under whole‐cell patch clamp. 2 Discrete Ca2+ release events were detected in intact wild‐type and RyR3‐knockout myotubes. They showed almost no difference in amplitude and width, but were substantially different in duration. In wild‐type myotubes (660 events, 57 cells) the amplitude was 1.27 (0.85, 1.97) (median (25 %, 75 %)) units of resting fluorescence, the full width at half‐magnitude (FWHM) was 1.4 (0.9, 2.3) μm, and the full duration at half‐magnitude (FDHM) was 25.3 (9.6, 51.7) ms. In RyR3‐knockout myotubes (655 events, 83 cells) the amplitude was 1.30 (0.84, 2.08), FWHM was 1.63 (1.02, 2.66) μm, and FDHM was 43.6 (23.6, 76.9) ms. 3 Depolarization under voltage clamp of both wild‐type and RyR3‐knockout myotubes produced substantial Ca2+ release devoid of discrete Ca2+ events. Discrete events were still present but occurred without correlation with the applied pulse, largely at locations where the pulse did not elicit release. 4 The local correspondence between voltage control and absence of discrete events implies that the functional interaction with voltage sensors suppresses the mechanism that activates discrete events. Because it applies whether RyR3 is present or not, it is this exclusion by voltage of other control mechanisms, rather than isoform composition, that primarily determines the absence of discrete Ca2+ events in adult mammalian muscle.

A Preferred Amplitude of Calcium Sparks in Skeletal Muscle

In skeletal and cardiac muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in frog skeletal muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.

Activation of Ca2+ release by caffeine and voltage in frog skeletal muscle.

1. Using a fast flow, computer‐controlled, two‐Vaseline‐gap chamber, single muscle fibres were subjected to ‘pulses’ of caffeine at Ca2+ releasing concentrations, combined with voltage‐clamp depolarizations, while monitoring intracellular [Ca2+]. 2. Ca2+ release flux elicited by caffeine reached 2.5 mM s‐1, or less, after 3 s of exposure, then decayed to zero. The caffeine‐releasable pool of sarcoplasmic reticulum (SR) Ca2+ was 2.9 +/‐ 0.4 mM (mean +/‐ S.E.M., n = 10). 3. In parallel with release induced by caffeine, release induced by voltage pulses applied during a caffeine exposure increased in the first second of exposure, then decreased, to abolition after 5 s. 4. The amount of Ca2+ releasable by depolarizing pulses was always equal to the amount of Ca2+ in the caffeine‐releasable pool. Therefore, there is a single releasable Ca2+ pool. This pool is well stirred‐it takes much more time to lose its Ca2+ by release than to diffusionally homogenize its [Ca2+]. Its depletion explains quantitatively the decay of release induced by caffeine or voltage during an exposure to caffeine. 5. A 1.5 s pulse to 10 mV, applied during exposure to caffeine, resulted in large Ca2+ release and, upon repolarization, termination of the caffeine‐induced release. This is similar to repolarization‐induced stop of caffeine contracture (RISC) in embryonic murine myoballs. The permeability elicited by caffeine (ratio of flux to calcium in the releasable pool) was not affected by depolarizing pulses. Therefore, the mechanism of the RISC‐like effect was Ca2+ depletion. 6. Caffeine‐induced release did not depend on the holding potential. 7. Whether caffeine was present or not, release activated by voltage remained always under voltage control, ending rapidly upon repolarization. A depolarizing pulse induced a release permeability with an early peak, followed by decay to a steady level. Caffeine (10 mM) shifted the mid‐activation voltage of both peak and steady components by ‐15 mV and increased the steepness of their voltage dependence by 15%. The maximum permeability increased by 30% for the peak and 25% for the steady component (n = 5). These results neither support nor disprove the hypothesis that the peak of Ca2+ release is activated by Ca2+. 8. The similar potentiation by caffeine of both components of release, the continued ability of voltage to control release in the presence of caffeine, and its failure to alter caffeine‐induced permeability indicate that caffeine and the voltage sensor enhance independently the channels tendency to open.

Caffeine enhances intramembranous charge movement in frog skeletal muscle by increasing cytoplasmic Ca2+ concentration.

1. Currents of intramembranous charge movement were recorded, together with intracellular [Ca2+], in single muscle fibres subjected to voltage‐clamp depolarization and ‘pulses’ of extracellular solution with a Ca2+ release‐inducing concentration of caffeine (10 mM). 2. When caffeine was present prior to and during the voltage pulses, the charge transferred by pulses to between ‐60 and ‐40 mV increased by about 40%. 3. In fibres depleted of Ca2+ in the sarcoplasmic reticulum (SR), caffeine had no effect on charge transfer or kinetics. 4. Whenever the prior exposure to caffeine resulted in a large elevation in [Ca2+]i at the start of the depolarizing pulse, there was an increase in I beta, the monotonically decaying component of charge movement. When the presence of caffeine enhanced Ca2+ release induced by the pulse, there was increase in I gamma, the hump‐like component. 5. The charge transferred during a pulse to ‐50 mV increased with time of exposure to caffeine. Ca2+ release induced by the voltage pulse grew during the first second of caffeine exposure, then decreased with longer exposure time. The enhancement of charge transfer by caffeine was therefore not due to the increase in Ca2+ release caused by the drug. 6. The increase in charge transfer was a uniform, monotonically increasing function of the [Ca2+]i attained at the end of the voltage pulse. 7. Charge transfer, as a function of [Ca2+]i, pulse voltage and time, was simulated with a model, used previously, in which Ca2+ binds to intracellular sites and increases the electrical potential near the voltage sensors. Two sites were needed to fit the observations, with dissociation constants of 60 nM and 2 to 10 microM. 8. In the presence of caffeine, the voltage‐driven movement of a given amount of intra‐membranous charge resulted in greater activation of release permeability. 9. All effects of caffeine observed in this and the preceding paper could be explained assuming a single action: caffeine increases the tendency of the release channels to open. This results in opening of closed channels and an increase in their susceptibility to activation by the voltage sensors.

PROPERTIES AND ROLES OF AN INTRAMEMBRANOUS CHARGE MOBILIZED AT HIGH VOLTAGES IN FROG SKELETAL MUSCLE

1. Membrane Ca2+ currents (ICa), intramembranous charge movement currents and changes in intracellular Ca2+ concentrations were recorded in voltage clamped cut skeletal muscle fibres of the frog. Intra‐ and extracellular solutions, designed to prevent ionic current, and use of the saponin‐permeabilization procedure made possible the measurement of transfer of intramembranous charge up to high positive potentials. 2. Substantial charge moved at positive potentials. This charge was shown to be intramembranous in four tests of charge conservation, demonstrating that the total displacement of charge depended only on the initial and final voltages, and not on the history or pathway of intermediate voltages. 3. On average, in twenty‐three cells, the charge moved at 50 mV was 31 +/‐ 1.9 nC microF‐1 (mean +/‐ S.E.M.), and at 0 mV was 25 +/‐ 1.5 nC microF‐1. Approximately one‐fifth of the total charge moved above 0 mV. 4. The charge that moved at high voltage could be fitted, in most cases, with a Boltzmann distribution function. In twenty of twenty‐three cells, the total charge distribution could be fitted as the sum of two Boltzmann terms; the high voltage term was centred at 11 +/‐ 3.9 mV, with a steepness factor of 12 +/‐ 1.6 mV and a magnitude of 8.6 +/‐ 1.1 nC microF‐1. The low voltage term was centered at ‐43 +/‐ 2.1 mV, with a steepness factor of 7.7 +/‐ 0.6 mV and a magnitude of 22 +/‐ 1.8 nC microF‐1. Thus, the high voltage component comprised about one‐quarter of the mobile charge. In four cells it was possible to fit the sum of three Boltzmann terms to the distribution of mobile charge; the parameters of the high voltage term then were similar to those found by fitting the sum of two Boltzmann terms to the same data. 5. The voltage dependence of activation of ICa was determined in a buffered 2 mM Ca2+ external solution, from the tails of ionic current at ‐30 mV, after activating pulses to various voltages, the duration of which was sufficient to reach the peak of inward current. The voltage dependence was described by a Boltzmann function centred at 2.6 +/‐ 6.9 mV (n = 6), with a steepness factor of 20 +/‐ 1.4 mV. The voltages at which the high voltage charge moved were roughly the same as those at which ICa was activated. 6. Calcium release from the sarcoplasmic reticulum was determined from the Ca2+ transients. Calcium release continued to increase at potentials above 0 mV.(ABSTRACT TRUNCATED AT 400 WORDS)