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¹ Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612
² Departamento de Biofísica, Universidad de la República, Facultad de Medicina y Facultad de Ciencias, Montevideo, Uruguay

Address correspondence to Eduardo Ríos, Dept. of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 W. Harrison St., Suite 1279JS, Chicago, IL 60612. Fax: (312) 942-8711; email: erios{at}rush.edu

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 [H⁺] and [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 (Ca_SR). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca_SR was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca_SR (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca_SR. 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.

Key Words: sarcoplasmic reticulum • excitation–contraction coupling • Ca channels • channel gating • channel modulation

¹ This conclusion is inconsistent with the exponential fit analysis illustrated in Fig. 7 A, which implies a constant P. To clarify this issue, we fitted exponentials to separate sections of the depletion transient [H⁺](t), and found different rate constants. In the example shown, the rate constant of the fit to the entire transient was 2.4 s^–1, while for the last 450 ms it was 3.5 s^–1. The fits indicate an increase in permeability with increasing depletion, and are therefore in agreement with the time course of the corrected release waveform.

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