期刊
JOURNAL OF GENERAL PHYSIOLOGY
卷 124, 期 3, 页码 239-258出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200409071
关键词
sarcoplasmic reticulum; excitation-contraction coupling; Ca channels; channel gating; channel modulation
类别
资金
- NIAMS NIH HHS [AR32808, R01 AR049184, R37 AR032808, AR049184, R01 AR032808] Funding Source: Medline
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 Delta[H+] and Delta[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 Cas, 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.
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