Calcium Sparks in Intact Skeletal Muscle Fibers of the Frog

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be ∼0.21 μm in x and y and ∼0.51 μm in z. Observations were made at 17–20°C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K⁺] = 2.5 mM; estimated membrane potential, −80 to −90 mV) and elevated [K⁺] Ringer's (most frequently, [K⁺] = 13 mM; estimated membrane potential, −60 to −65 mV). The frequency of sparks was 0.04–0.05 sarcomere⁻¹ s⁻¹ in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K⁺] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, ∼4 ms; peak amplitude, ∼1 ΔF/F (change in fluorescence divided by resting fluorescence); decay time constant, ∼5 ms; full duration at half maximum (FDHM), ∼6 ms; late offset, ∼0.01 ΔF/F; full width at half maximum (FWHM), ∼1.0 μm; mass (calculated as amplitude × 1.206 × FWHM³), 1.3–1.9 μm³. Although the rise time is similar to that measured previously in frog cut fibers (5–6 ms; 17–23°C), cut fiber sparks have a longer duration (FDHM, 9–15 ms), a wider extent (FWHM, 1.3–2.3 μm), and a strikingly larger mass (by 3–10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca²+ buffering power of myoplasm in cut fibers and an increase in the flux of Ca²+ during release.