scholarly journals An improved vaseline gap voltage clamp for skeletal muscle fibers.

1976 ◽  
Vol 67 (3) ◽  
pp. 265-293 ◽  
Author(s):  
B Hille ◽  
D T Campbell
Keyword(s):  
Skeletal Muscle ◽  
Voltage Clamp ◽  
Sodium Current ◽  
Muscle Fibers ◽  
Complex Impedance ◽  
Sodium Currents ◽  
Disk Model ◽  
Transverse Tubular System ◽  
Skeletal Muscle Fibers ◽  
Tubular System

A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the "disk" equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5 degrees C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with sodium permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and sodium currents. The peak inward sodium current density at 5 degrees C is 3.7 mA/cm2. At 5 degrees C the sodium currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular system. Comparison of recorded currents with simulations shows that while the transverse tubular system has regenerative sodium currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage clamp control.

scholarly journals Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers

2010 ◽  
Vol 137 (1) ◽  
pp. 21-41 ◽  
Author(s):  
Marino DiFranco ◽  
Alvaro Herrera ◽  
Julio L. Vergara
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Muscle Fibers ◽  
Optical Data ◽  
Mammalian Skeletal Muscle ◽  
Chloride Currents ◽  
Actual Distribution ◽  
Transverse Tubular System ◽  
Skeletal Muscle Fibers ◽  
Tubular System

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (ICl) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from TTS contributions.

scholarly journals The Capacitance of Skeletal Muscle Fibers in Solutions of Low Ionic Strength

1972 ◽  
Vol 59 (3) ◽  
pp. 347-359 ◽  
Author(s):  
P. C. Vaughan ◽  
J. N. Howell ◽  
R. S. Eisenberg
Keyword(s):  
Skeletal Muscle ◽  
Ionic Strength ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
External Solution ◽  
Rectangular Pulse ◽  
Bathing Solution ◽  
Skeletal Muscle Fibers ◽  
Tubular System ◽  
Low Ionic Strength

The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 µF/cm2 in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 µF/cm2. It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 µF/cm2 which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential—like mechanism in the tubular membrane.

scholarly journals Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367.

1982 ◽  
Vol 80 (2) ◽  
pp. 203-230 ◽  
Author(s):  
J A Heiny ◽  
J Vergara
Keyword(s):  
Skeletal Muscle ◽  
Voltage Clamp ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Optical Signal ◽  
Intensity Change ◽  
Optical Signals ◽  
Potential Control ◽  
Skeletal Muscle Fibers ◽  
T System

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.

Calcium Signaling in Isolated Skeletal Muscle Fibers Investigated Under "Silicone Voltage-Clamp" Conditions

2004 ◽  
Vol 40 (2) ◽  
pp. 225-236 ◽  
Author(s):  
Claude Collet ◽  
Sandrine Pouvreau ◽  
Laszlo Csernoch ◽  
Bruno Allard ◽  
Vincent Jacquemond
Keyword(s):  
Skeletal Muscle ◽  
Calcium Signaling ◽  
Voltage Clamp ◽  
Muscle Fibers ◽  
Skeletal Muscle Fibers

scholarly journals Arsenazo III and antipyrylazo III calcium transients in single skeletal muscle fibers.

1982 ◽  
Vol 79 (4) ◽  
pp. 679-707 ◽  
Author(s):  
P Palade ◽  
J Vergara
Keyword(s):  
Skeletal Muscle ◽  
Voltage Clamp ◽  
Spectral Characteristics ◽  
Muscle Fibers ◽  
Calcium Transients ◽  
Arsenazo Iii ◽  
Tension Development ◽  
Skeletal Muscle Fibers ◽  
Concentration Changes

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diffusion with binding from Ca++ release sites to filament overlap sites may be significant. The spectral characteristics of the absorbance transients recorded with arsenazo III suggest that in situ recorded signals cannot be easily interpreted in terms of Ca concentration changes. A more exhaustic knowledge of the dye chemistry and/or in situ complications in the use of the dye will be necessary.

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