Gonzalo Pizarro

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.

How Source Content Determines Intracellular Ca2+ Release Kinetics. Simultaneous Measurement of [Ca2+] Transients and [H+] Displacement in Skeletal Muscle

In skeletal muscle, the waveform of Ca2+ release under clamp depolarization exhibits an early peak. Its decay reflects an inactivation, which locally corresponds to the termination of Ca2+ sparks, and is crucial for rapid control. In cardiac muscle, both the frequency of spontaneous sparks (i.e., their activation) and their termination appear to be strongly dependent on the Ca2+ content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca2+ release and a way to reliably modify the SR content, we combined in the same cells the “EGTA/phenol red” method (Pape et al., 1995) to evaluate Ca2+ release, with the “removal” method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Δ[H+] and Δ[Ca2+] from which the amount of released Ca2+ and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (CaSR). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as CaSR was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting CaSR (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low CaSR. The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca2+ release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca2+ on the activity of Ca2+ release channels.

Differential effects of tetracaine on two kinetic components of calcium release in frog skeletal muscle fibres.

1. Intramembrane charge movements and changes in intracellular calcium concentration were recorded simultaneously in voltage clamped cut skeletal muscle fibres of the frog in the presence and absence of tetracaine. 2. Extracellular application of 20 microM tetracaine reduced the increase in myoplasmic [Ca2+]. The effect on the underlying calcium release flux from the sarcoplasmic reticulum was to suppress the peak of the release while sparing the steady level attained at the end of 100 ms clamp depolarizations. 3. While the peak of the release flux at corresponding voltages was reduced by 62% after the addition of tetracaine, the rate of inactivation was the same when the pulses elicited release fluxes of similar amplitude. 4. Higher concentrations of tetracaine, 0.2 mM, abolished the calcium signal in stretched fibres whereas in slack fibres this concentration left a non‐inactivating calcium release flux. 5. Lowering the extracellular pH antagonized the effect of the drug both on charge movements and on calcium signals. The permanently charged analogue tetracaine methobromide lacked effects on excitation‐contraction coupling. 6. These results imply that the two kinetic components of calcium release flux have very different tetracaine sensitivities. They are also consistent with an intracellular site of action of the drug at low concentration. Taken together they strongly suggest that the inactivating and non‐inactivating components of calcium release correspond to different pathways: one that inactivates, is sensitive to tetracaine and is controlled by calcium, and another that does not inactivate, is much less sensitive to tetracaine and is directly controlled by voltage.

‘Quantal’ calcium release operated by membrane voltage in frog skeletal muscle

1 Ca2+ transients and Ca2+ release flux were determined optically in cut skeletal muscle fibres under voltage clamp. ‘Decay’ of release during a depolarizing pulse was defined as the difference between the peak value of release and the much lower steady level reached after about 100 ms of depolarization. Using a double‐pulse protocol, the inactivating effect of release was measured by ‘suppression’, the difference between the peak values of release in the test pulse, in the absence and presence of a conditioning pulse that closely preceded the test pulse. 2 The relationship between decay and suppression was found to follow two simple arithmetic rules. Whenever the conditioning depolarization was less than or equal to the test depolarization, decay in the conditioning release was approximately equal to suppression of the test release. Whenever the conditioning depolarization was greater than that of the test, suppression was complete, i.e. test release was reduced to a function that increased monotonically to a steady level. The steady level was the same with or without conditioning. 3 These arithmetic rules suggest that inactivation of Ca2+ release channels is strictly and fatally linked to their activation. More than a strict linkage, however, is required to explain the arithmetic properties. 4 The arithmetic rules of inactivation result in three other properties that are inexplicable with classical models of channel gating: constant suppression, incremental inactivation and increment detection. These properties were first demonstrated for inositol trisphosphate (IP3)‐sensitive channels and used to define IP3‐induced release as quantal. In this sense, it can now be stated that skeletal muscle Ca2+ release is activated by membrane voltage in a quantal manner. 5 For both classes of intracellular Ca2+ channels, one explanation of the observations is the existence of subsets of channels with different sensitivities (to voltage or agonist dose). In an alternative explanation, channels are identical, but have a complex repertoire of voltage‐ or dose‐dependent responses.

The Voltage Sensor of Skeletal Muscle Excitation-Contraction Coupling: A Comparison with Ca 2+ Channels

A recent development in the study of Ca2+ channels is the realization [2, 44] that skeletal muscle Ca2+ channels have a number of properties in common with the voltage sensor of excitation-contraction (Ee) coupling, the molecule or structure of skeletal muscle membrane that has the role of sensing the action potential to control the opening of the Ca2+ release channels of the sarcoplasmic reticulum (SR).

Effects of 2,3-butanedione monoxime on excitation-contraction coupling in frog twitch fibres.

Abstract10 and 30mm 2,3-butanedione monoxime (BDM) applied extracellularly to voltage-clamped frog skeletal muscle twitch fibres suppressed both Ca2+ release flux and intramembranous charge movement. Both effects could be clearly separated. The early peak of the Ca2+ release flux was suppressed at every test voltage. The steady level attained at the end of a 100ms clamp depolarization was relatively spared for lower depolarizing pulses, but was as suppressed as the peak at voltages above −20mV. The intramembranous charge movement was affected mainly in the Iγ component. The drug had a distinct effect on the kinetics of the intramembranous charge movement current around the threshold for Ca2+ release. The three kinetic components of Iγ were simultaneously affected. For more positive depolarizations where the kinetic effect was not evident, the oxime had no significant effect on the charge moved. Under conditions in which Iγ was absent (i.e. stretched fibres, intracellular solutions containing 6 to 10mm BAPTA), treatment with 10mm BDM had a small, not significant suppressive effect on the maximum charge moved (Qmax), while it affected Ca2+ release significantly. When 10mm BDM was applied in the presence of 0.2mm tetracaine, the local anaesthetic-resistant Ca2+ release flux was not further suppressed by the oxime.

A Third Role for Calcium in Excitation-Contraction Coupling

Two important roles for calcium ions in excitation-contraction coupling are traditionally described. First is the second-messenger role—Ca2+ undergoes a transient increase in the myoplasm and triggers the mechanochemical reaction of contraction. (1) This transient increase in myoplasmic free calcium concentration is usually called the Ca transient; it is the result of a flux of calcium release from the sarcoplasmic reticulum (SR). In this chapter we discuss the mechanisms by which the SR is made to open its release channels during normal muscle function. In regard to this question a second role of Ca2+ has been identified: Ca2+ is an effective agonist for opening the Ca channels of the SR. The increase in [Ca2+] i in the vicinity of the SR release channels constitutes the normal mechanism of triggering release in the heart (Ca-induced Ca release)(2,3)

Effects of Calcium Release from the Sarcoplasmic Reticulum on Intramembrane Charge Movement in Skeletal Muscle

Skeletal muscle contraction is initiated by the increase in the intracellular calcium concentration ([Ca2+]) following the depolarization of the surface- and transverse-(T-) tubular membranes (see the review by Ebashi, 1991). Calcium ions are released into the myoplasm from an intracellular store, the sarcoplasmic reticulum (SR), through the ryanodine receptor calcium release channel (Imagawa et al., 1987; Inui et al., 1987). The voltage sensitive step that links channel opening to depolarization is the displacement of permanent charges (charge movement, Schneider and Chandler, 1973) found in the T-tubular membrane and localized in the DHP receptor (Rios and Pizarro, 1991).

COMPARISON OF THE EFFECTS OF BDM ON L-TYPE Ca CHANNELS OF CARDIAC AND SKELETAL MUSCLE

Effects of the compound 2,3 Butanedione monoxime (BDM) on force development have been described in skeletal muscle (Fryer et al., 1988), cardiac muscle (Bergey et al., 1981; West & Stephenson 1989) as well as in smooth muscle (Osterman et al., 1993; Watanabe, 1993). It inhibits contraction acting at different levels: on the contractile mechanism as was shown by Horiuti et al. (1988) and Osterman et al. (1993) and on the excitation-contraction coupling process (Hui & Maylie, 1991; De Armas et al., 1993; Li et al., 1985). In addition to these effects on contractility the drug reduces Ca“ current through L-type Ca2+ channels in cardiac (Coulombe et al., 1990; Chapman, 1992; Ferreira et al., 1993), skeletal muscle (Fryer et al., 1988) and smooth muscle (Lang & Paul, 1991). This reduction obeys to an enhanced voltage dependent inactivation of the channel (Chapman 1992, 1993; Ferreira et al., 1993). Since BDM is a chemical phosphatase, member of a group of oximes with the ability to reactivate cholinesterase after exposure to organo-phosphorous compounds (Wilson & Grinsberg, 1955), it has been suggested that dephosphorylation is the mechanism of action of the drug. Several experimental evidences recently provided are in line with this hypothesis (Chapman, 1993a; Chapman, 1995).