Calcium Sparks in Intact Skeletal Muscle Fibers of the Frog

Abstract

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.

Effects of Azumolene on Ca2+ Sparks in Skeletal Muscle Fibers

“…To determine whether there was an azumolene-dependent change in the amount of Ca 2ϩ released from SR by the individual sparks, spark mass at time of spark peak was calculated for each spark using the equation (Hollingworth et al, 2001):…”

Ca 2+ blinks: Rapid nanoscopic store calcium signaling

“…Fitting of blink time courses and spatial profiles used the nonlinear functions described previously for sparks (28,29). Blink parametric measurement was based on the fitting results.…”

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

“…Description of the confocal laser scanning microscope and its calibration We constructed a confocal laser scanning microscope (CLSM) based on the physical design and C++ image acquisition software of Hollingworth et al (Hollingworth et al, 2001), using a TE300 inverted microscope (Nikon) fitted with a CFI60 (60×, 1.2 NA), waterimmersion objective. The 3D point-spread function of the CLSM was measured with 0.1 µm diameter fluorescent microspheres.…”