scholarly journals Comparison of Simulated and Measured Calcium Sparks in Intact Skeletal Muscle Fibers of the Frog

2002 ◽  
Vol 120 (3) ◽  
pp. 349-368 ◽  
Author(s):  
S.M. Baylor ◽  
S. Hollingworth ◽  
W.K. Chandler
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Muscle Fibers ◽  
Diffusion Constant ◽  
Calcium Sparks ◽  
Binding Reaction ◽  
Skeletal Muscle Fibers ◽  
Apparent Diffusion ◽  
Intact Muscle ◽  
Regulatory Sites

Calcium sparks in frog intact skeletal muscle fibers were modeled as stereotypical events that arise from a constant efflux of Ca2+ from a point source for a fixed period of time (e.g., 2.5 pA of Ca2+ current for 4.6 ms; 18°C). The model calculates the local changes in the concentrations of free Ca2+ and of Ca2+ bound to the major intrinsic myoplasmic Ca2+ buffers (troponin, ATP, parvalbumin, and the SR Ca2+ pump) and to the Ca2+ indicator (fluo-3). A distinctive feature of the model is the inclusion of a binding reaction between fluo-3 and myoplasmic proteins, a process that strongly affects fluo-3′s Ca2+-reaction kinetics, its apparent diffusion constant, and hence the morphology of sparks. ΔF/F (the change in fluo-3′s fluorescence divided by its resting fluorescence) was estimated from the calculated changes in fluo-3 convolved with the microscope point-spread function. To facilitate comparisons with measured sparks, noise and other sources of variability were included in a random repetitive fashion to generate a large number of simulated sparks that could be analyzed in the same way as the measured sparks. In the initial simulations, the binding of Ca2+ to the two regulatory sites on troponin was assumed to follow identical and independent binding reactions. These simulations failed to accurately predict the falling phase of the measured sparks. A second set of simulations, which incorporated the idea of positive cooperativity in the binding of Ca2+ to troponin, produced reasonable agreement with the measurements. Under the assumption that the single channel Ca2+ current of a ryanodine receptor (RYR) is 0.5–2 pA, the results suggest that 1–5 active RYRs generate an average Ca2+ spark in a frog intact muscle fiber.

Calcium sparks in skeletal muscle fibers

Cell Calcium ◽  
2005 ◽  
Vol 37 (6) ◽  
pp. 513-530 ◽  
Author(s):  
Stephen M. Baylor
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Calcium Sparks ◽  
Skeletal Muscle Fibers

scholarly journals Block of single L-type Ca2+ channels in skeletal muscle fibers by aminoglycoside antibiotics.

1996 ◽  
Vol 107 (3) ◽  
pp. 421-432 ◽  
Author(s):  
C M Haws ◽  
B D Winegar ◽  
J B Lansman
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Muscle Fibers ◽  
External Solution ◽  
Bimolecular Reaction ◽  
Aminoglycoside Antibiotics ◽  
Ion Concentration ◽  
Ion Flow ◽  
Skeletal Muscle Fibers ◽  
Ca2 Channels

The activity of single L-type Ca2+ channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were concerned with the mechanism by which aminoglycoside antibiotics inhibit ion flow through the channel. Aminoglycosides produced discrete fluctuations in the single-channel current when added to the external solution. The blocking kinetics could be described as a simple bimolecular reaction between an aminoglycoside molecule and the open channel. The blocking rate was found to be increased when either the membrane potential was made more negative or the concentration of external permeant ion was reduced. Both of these effects are consistent with a blocking site that is located within the channel pore. Other features of block, however, were incompatible with a simple pore blocking mechanism. Hyperpolarization enhanced the rate of unblocking, even though an aminoglycoside molecule must dissociate from its binding site in the channel toward the external solution against the membrane field. Raising the external permeant ion concentration also enhanced the rate of unblocking. This latter finding suggests that aminglycoside affinity is modified by repulsive interactions that arise when the pore is simultaneously occupied by a permeant ion and an aminoglycoside molecule.

scholarly journals Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

10.3791/50898 ◽  
2014 ◽  
Author(s):  
Ki Ho Park ◽  
Noah Weisleder ◽  
Jingsong Zhou ◽  
Kristyn Gumpper ◽  
Xinyu Zhou ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Calcium Sparks ◽  
Skeletal Muscle Fibers

scholarly journals Calcium Sparks in Intact Skeletal Muscle Fibers of the Frog

2001 ◽  
Vol 118 (6) ◽  
pp. 653-678 ◽  
Author(s):  
S. Hollingworth ◽  
J. Peet ◽  
W.K Chandler ◽  
S.M. Baylor
Keyword(s):  
Skeletal Muscle ◽  
Membrane Potential ◽  
Rise Time ◽  
Rana Temporaria ◽  
Muscle Fibers ◽  
Fluorescence Decay ◽  
Half Maximum ◽  
Calcium Sparks ◽  
Mean Values ◽  
Skeletal Muscle Fibers

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.

Depression of force by phosphate in skinned skeletal muscle fibers of the frog

1990 ◽  
Vol 259 (2) ◽  
pp. C349-C357 ◽  
Author(s):  
G. J. Stienen ◽  
M. C. Roosemalen ◽  
M. G. Wilson ◽  
G. Elzinga
Keyword(s):  
Skeletal Muscle ◽  
Atp Hydrolysis ◽  
Isometric Force ◽  
Muscle Fibers ◽  
Diffusion Constant ◽  
Phosphate Concentration ◽  
Cross Sectional ◽  
Average Value ◽  
A Value ◽  
Skeletal Muscle Fibers

The relation between isometric force and phosphate concentration in skinned skeletal muscle fibers of the frog is found to depend on fiber size. Force decreased with increasing phosphate concentration, but depression of force in thick fibers was smaller than in thin segments. When the external phosphate concentration was abruptly altered during a sustained contracture, force changed. The half-time of the force change was proportional to the cross-sectional area of the preparation. From this relation, a value for the diffusion constant of phosphate in skinned fibers of 0.9 x 10(-10) m2/s was derived. The rate of phosphate production was determined photometrically via the enzymatic coupling of the resynthesis of ATP to the oxidation of nicotinamide adenine dinucleotide. The average value (+/- SE) of the rate of ATP hydrolysis (at 4 degrees C) was 2.7 +/- 0.3 mumol.s-1.g dry wt-1, which corresponds to 0.34 mmol.l-1.s-1. From a calculation based on the diffusion constant and the rate of phosphate production determined, it follows that the dependency of the force-phosphate relation on fiber diameter is due to phosphate accumulation inside the fiber.

The SR of skeletal muscle: Its structure after quick-freezing and freeze-etching, following field stimulation of single intact muscle fibers

1984 ◽  
Vol 42 ◽  
pp. 300-301
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
N.R. Wallace
Keyword(s):  
Skeletal Muscle ◽  
Striated Muscle ◽  
Muscle Fibers ◽  
Freeze Fracture ◽  
Electron Gun ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing ◽  
Intact Muscle ◽  
Similar Images ◽  
Freeze Etching

It is known that the P faces of freeze-fractured SR of fixed and cryoprotected striated muscle fibers are studded with particles, whereas the E faces remain smooth, except for two staggered rows of pits in the junctional SR (JSR) which face transverse tubules (junctional pits). Freeze-fracture after quick-freezing of native skeletal muscle provides similar images (1). We have used freeze-etching to look at the SR's structure in single intact skeletal muscle fibers (r.temporaria) without stimulation, following varied post-stimulation intervals, and in tetanus. Single intact skeletal muscle fibers were isolated and quick-frozen as previously reported (2). After quick-freezing, the fibers were transferred to a Balzers 301 device and etched for 3 minutes at -100°C, followed by unidirectional Pt evaporation with an electron gun and carbon coating.

scholarly journals Measurement of force and calcium release using mechanically skinned fibers from mammalian skeletal muscle

2018 ◽  
Vol 125 (4) ◽  
pp. 1105-1127 ◽  
Author(s):  
Graham D. Lamb ◽  
D. George Stephenson
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fiber ◽  
Calcium Release ◽  
Muscle Fibers ◽  
Mammalian Skeletal Muscle ◽  
Ec Coupling ◽  
Skeletal Muscle Fibers ◽  
Intact Muscle ◽  
Key Variables ◽  
Coupling Sequence

The mechanically skinned (or “peeled”) skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the “intracellular” conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

scholarly journals Effects of Tetracaine on Voltage-activated Calcium Sparks in Frog Intact Skeletal Muscle Fibers

2006 ◽  
Vol 127 (3) ◽  
pp. 291-307 ◽  
Author(s):  
Stephen Hollingworth ◽  
W. Knox Chandler ◽  
Stephen M. Baylor
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Muscle Fibers ◽  
Mean Value ◽  
Half Maximum ◽  
Mean Values ◽  
Skeletal Muscle Fibers ◽  
The Mean ◽  
Ca2 Channels

The properties of Ca2+ sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K+] Ringer's are well described by a computational model with a Ca2+ source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 °C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca2+ current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1–5 RyR Ca2+ release channels open during a voltage-activated Ca2+ spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K+] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75–100 μM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca2+ channels per spark, and (b) the single-channel Ca2+ current of an RyR channel is ≤1.2 pA under physiological conditions. The results support the conclusion that some normal voltage-activated sparks (i.e., in the absence of tetracaine) are produced by two or more active RyR Ca2+ channels. The question of how the activation of multiple RyRs is coordinated is discussed.

scholarly journals Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics.

1996 ◽  
Vol 107 (3) ◽  
pp. 433-443 ◽  
Author(s):  
B D Winegar ◽  
C M Haws ◽  
J B Lansman
Keyword(s):  
Skeletal Muscle ◽  
Open Channel ◽  
Single Channel ◽  
Muscle Fibers ◽  
Bimolecular Reaction ◽  
Aminoglycoside Antibiotics ◽  
Ion Flow ◽  
Channel Current ◽  
Skeletal Muscle Fibers ◽  
Drug Concentrations

The activity of single mechanosensitive channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.

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