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 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 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.

scholarly journals Longitudinal impedance of single frog muscle fibers.

1975 ◽  
Vol 65 (1) ◽  
pp. 97-113 ◽  
Author(s):  
B A Mobley ◽  
J Leung ◽  
R S Eisenberg
Keyword(s):  
Skeletal Muscle ◽  
Phase Shift ◽  
Muscle Fibers ◽  
Mean Value ◽  
Sartorius Muscle ◽  
Semitendinosus Muscle ◽  
Skeletal Muscle Fibers ◽  
Longitudinal Impedance ◽  
Small Phase ◽  
The Mean

The longitudinal impedance of single skeletal muscle fibers has been measured from1 to 10,000 Hz in an oil gap apparatus which forces current to flow longitudinally down the fiber. The impedance observed is purely resistive in some fibers from the semitendinosus muscle and in two fibers from the sartorius muscle. In other fibers from the semitendinosus muscle a small phase shift is observed. The mean value of the maximum phase shift observed from all fibers is 1.07 degrees. The artifacts associated with the apparatus and method are examined theoretically and it is shown that one of the likely artifacts could account for the small phase observed. It is concluded that the longitudinal impedance of skeletal muscle fibers is essentially resistive and that little, if any, longitudinal current crosses the membranes of the sarcoplasmic reticulum.

beta-Adrenergic modulation of Ba2+ currents and K+ contractures in frog slow skeletal muscle fibers

1997 ◽  
Vol 272 (1) ◽  
pp. C77-C81 ◽  
Author(s):  
M. Huerta ◽  
X. Trujillo ◽  
C. Vasquez
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Isometric Tension ◽  
External Application ◽  
Beta Adrenergic ◽  
Cyclic Monophosphate ◽  
Slow Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Adrenergic Modulation ◽  
Ca2 Channels

beta-Adrenergic modulation of the Ba2+ current (IBa) and K+ contracture in slow skeletal muscle fibers of the frog (Rana pipiens) were investigated in intact fibers with the three-microelectrode voltage-clamp technique and isometric tension measurements. Application of epinephrine (10(-6) to 10(-5) M) to the bath increased the amplitude of IBa. This increase was blocked by the beta-antagonist propranolol (3 microM), and a similar increase was observed with the beta-specific agonist isoproterenol (1 microM). Thus the epinephrine effect was mediated mainly by beta-adrenergic receptors. External application of permeable 8-bromoadenosine 3',5'-cyclic monophosphate (0.5 mM) increased the amplitude of both IBa and K+ contractures. The present results suggest that beta-adrenergic modulation of IBa in slow skeletal muscle fibers could reflect a modulation of Ca2+ channels via adenosine 3',5'-cyclic monophosphate (cAMP). cAMP (0.5 mM) also potentiated the K(+)-evoked tension in these slow fibers. The physiological contribution made by the modulation of slow skeletal muscle Ca2+ channels to the increase in tension is still not completely understood.

scholarly journals Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers.

1986 ◽  
Vol 87 (2) ◽  
pp. 289-303 ◽  
Author(s):  
P Volpe ◽  
G Salviati ◽  
A Chu
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Isometric Force ◽  
Muscle Fibers ◽  
Ruthenium Red ◽  
Scattering Method ◽  
Tension Development ◽  
Maximal Force ◽  
Skeletal Muscle Fibers ◽  
Ca2 Channels

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.

Effects of denervation on Ca2+ channels in slow skeletal muscle fibers of the frog

2001 ◽  
Vol 126 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Clemente Vásquez ◽  
Miguel Huerta ◽  
Xóchitl Trujillo ◽  
José Luis Marı́n ◽  
Leonardo Hernández
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Slow Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Ca2 Channels

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.

Blockade by cAMP of native sodium channels of adult rat skeletal muscle fibers

1998 ◽  
Vol 275 (6) ◽  
pp. C1465-C1472 ◽  
Author(s):  
Jean-François Desaphy ◽  
Annamaria De Luca ◽  
Diana Conte Camerino
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channels ◽  
Single Channel ◽  
Muscle Fibers ◽  
Rat Skeletal Muscle ◽  
Patch Clamp Technique ◽  
Cyclic Monophosphate ◽  
Adult Rat ◽  
Skeletal Muscle Fibers ◽  
Steady State Inactivation

Although the skeletal muscle sodium channel is a good substrate for cAMP-dependent protein kinase (PKA), no functional consequence was observed for this channel expressed in heterologous systems. Therefore, we investigated the effect of 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on the native sodium channels of freshly dissociated rat skeletal muscle fibers by means of the cell-attached patch-clamp technique. Externally applied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by ∼75% with no change in kinetics. Single-channel conductance and normalized activation curves were unchanged by CPT-cAMP. In contrast, steady-state inactivation curves showed a reduction of the maximal available current and a negative shift of the half-inactivation potential. Similar effects were observed with dibutyryl adenosine 3′,5′-cyclic monophosphate but not with cAMP, which does not easily permeate the cell membrane. Incubation of fibers for 1 h with 10 μM H-89, a PKA inhibitor, did not prevent the effect of CPT-cAMP. Finally, the β-adrenoreceptor agonist isoproterenol mimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM. These results indicate that cAMP inhibits native skeletal muscle sodium channels by acting within the fiber, independently of PKA activation.

Patch clamp of sarcolemmal spheres from stretched skeletal muscle fibers

1989 ◽  
Vol 256 (2) ◽  
pp. C434-C440 ◽  
Author(s):  
P. Stein ◽  
P. Palade
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Proteolytic Enzymes ◽  
Muscle Fibers ◽  
Low Noise ◽  
Skeletal Muscle Fibers ◽  
Direct Recording ◽  
Rapid Formation ◽  
Voltage Dependent ◽  
Selective Channel

Stretching frog skeletal muscle fibers to the breaking point results in the rapid formation of numerous large spheres of membrane (5-80 microns diam). The surface of the spheres readily forms gigaohm (G omega) seals against patch pipettes, allowing low-noise single-channel recording. Currents recorded from patches isolated from these spheres indicate that they contain a variety of channels including 1) a small Na+-selective channel seen in the presence of veratridine, 2) a K+-selective channel which is blocked by millimolar Mg-ATP, and 3) a relatively large voltage-dependent Cl- channel which is blocked by Zn2+ and limited in selectivity over other anions [PCl/PMOPS = 3.7; MOPS, 3-(N-morpholino)propanesulfonic acid]. These channels have been described previously and have been identified as markers for sarcolemmal (SL) membrane. Accordingly, this method allows rapid and direct recording of channels in the SL membrane without first having to pretreat fibers with proteolytic enzymes to render the SL accessible to patch pipettes.

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.

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