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.

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 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.

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.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

Effect of External Calcium and other Divalent Cations on K+ Contractures in Denervated Slow Skeletal Muscle Fibers of the Frog

2019 ◽  
Author(s):  
Leonardo Hernández
Keyword(s):  
Skeletal Muscle ◽  
Binding Capacity ◽  
Divalent Cations ◽  
Muscle Fibers ◽  
Free Calcium ◽  
Slow Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Free Calcium Concentration ◽  
Chronic Denervation ◽  
Denervated Muscles

The influence of Ca2+ and other divalent cations on contractile responses of slow skeletal muscle fibers of the frog (Rana pipiens) under conditions of chronic denervation was investigated.Isometric tension was recorded from slow bundles of normal and denervated cruralis muscle in normal solution and in solutions with free calcium concentration solution or in solutions where other divalent cations (Sr2+, Ni2+, Co2+ or Mn2+) substituted for calcium. In the second week after nerve section, in Ca2+-free solutions, we observed that contractures (evoked from 40 to 80 mM-K+) of non-denervated muscles showed significantly higher tensions (p<0.05), than those from denervated bundles. Likewise, in solutions where calcium was substituted by all divalent cations tested, with exception of Mn2+, the denervated bundles displayed lower tension than non-denervated, also in the second week of denervation. In this case, the Ca2+ substitution by Sr2+ caused the higher decrease in tension, followed by Co2+ and Ni2+, which were different to non-denervated bundles, as the lowest tension was developed by Mn2+, followed by Co2+, and then Ni2+ and Sr2+. After the third week, we observed a recovery in tension. These results suggest that denervation altering the binding capacity to divalent cations of the voltage sensor.

scholarly journals Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Kazuki Yamamoto ◽  
Nao Yamaoka ◽  
Yu Imaizumi ◽  
Takunori Nagashima ◽  
Taiki Furutani ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Microfluidic Device ◽  
Motor Neurons ◽  
Muscle Fibers ◽  
Three Dimensional ◽  
Tissue Model ◽  
Skeletal Muscle Fibers

A three-dimensional human neuromuscular tissue model that mimics the physically separated structures of motor neurons and skeletal muscle fibers is presented.

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