scholarly journals Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers.

1991 ◽  
Vol 98 (2) ◽  
pp. 365-378 ◽  
Author(s):  
G Szücs ◽  
Z Papp ◽  
L Csernoch ◽  
L Kovács
Keyword(s):  
Skeletal Muscle ◽  
Voltage Dependence ◽  
Slow Component ◽  
Muscle Fibers ◽  
Ionic Current ◽  
Kinetic Properties ◽  
Constant Field ◽  
Charge Movement ◽  
Skeletal Muscle Fibers ◽  
Intramembrane Charge Movement

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.

scholarly journals A four-electrode method to study dynamics of ion activity and transport in skeletal muscle fibers

2019 ◽  
Vol 151 (9) ◽  
pp. 1146-1155 ◽  
Author(s):  
Judith A. Heiny ◽  
Stephen C. Cannon ◽  
Marino DiFranco
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Ionic Current ◽  
Transport Mechanisms ◽  
Electrical Stability ◽  
Ion Concentrations ◽  
Cell Functions ◽  
Skeletal Muscle Fibers ◽  
Intracellular Ion Concentrations ◽  
Ion Movements

Ion movements across biological membranes, driven by electrochemical gradients or active transport mechanisms, control essential cell functions. Membrane ion movements can manifest as electrogenic currents or electroneutral fluxes, and either process can alter the extracellular and/or intracellular concentration of the transported ions. Classic electrophysiological methods allow accurate measurement of membrane ion movements when the transport mechanism produces a net ionic current; however, they cannot directly measure electroneutral fluxes and do not detect any accompanying change in intracellular ion concentrations. Here, we developed a method for simultaneously measuring ion movements and the accompanying dynamic changes in intracellular ion concentrations in intact skeletal muscle fibers under voltage or current clamp in real time. The method combines a two-microelectrode voltage clamp with ion-selective and reference microelectrodes (four-electrode system). We validate the electrical stability of the system and the viability of the preparation for periods of ∼1 h. We demonstrate the power of this method with measurements of intracellular Cl−, H+, and Na+ to show (a) voltage-dependent redistribution of Cl− ions; (b) intracellular pH changes induced by changes in extracellular pCO2; and (c) electroneutral and electrogenic Na+ movements controlled by the Na,K-ATPase. The method is useful for studying a range of transport mechanisms in many cell types, particularly when the transmembrane ion movements are electrically silent and/or when the transport activity measurably changes the intracellular activity of a transported ion.

scholarly journals Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers.

1989 ◽  
Vol 93 (3) ◽  
pp. 565-584 ◽  
Author(s):  
C L Huang ◽  
L D Peachey
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Fiber Surface ◽  
Membrane Capacitance ◽  
Hypertonic Solution ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Transverse Tubule ◽  
Skeletal Muscle Fibers ◽  
Voltage Dependent

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.

scholarly journals Tracking Voltage-dependent Conformational Changes in Skeletal Muscle Sodium Channel during Activation

2002 ◽  
Vol 120 (5) ◽  
pp. 629-645 ◽  
Author(s):  
Baron Chanda ◽  
Francisco Bezanilla
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Slow Component ◽  
Fast Component ◽  
Lag Phase ◽  
Kinetic Properties ◽  
Charge Movement ◽  
Voltage Dependent ◽  
Domain Iii ◽  
Domain I

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

scholarly journals Effects of sulfhydryl inhibitors on nonlinear membrane currents in frog skeletal muscle fibers.

1993 ◽  
Vol 101 (3) ◽  
pp. 425-451 ◽  
Author(s):  
A Gonzalez ◽  
P Bolaños ◽  
C Caputo
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Membrane Conductance ◽  
Membrane Currents ◽  
Potential Range ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Sh Reagents ◽  
Nonlinear Membrane

The effect of sulhydryl reagents on nonlinear membrane currents of frog skeletal muscle fibers has been studied using the triple Vaseline gap voltage-clamp technique. These compounds, which are known to interfere with depolarization contraction coupling, also appear to diminish intramembranous charge movement recorded with fibers polarized to -100 mV (charge 1). This effect, however, is accompanied by changes in the fiber membrane conductance and in most cases by the appearance of an inwardly directed current in the potential range between -60 and +20 mV. This current is reduced by both cadmium and nifedipine and does not occur in Ca-free solution, suggesting that it is carried by calcium ions flowing through regular calcium channels that are more easily activated in the presence of SH reagent. These changes in the membrane electrical active and passive properties decrease the quality and reliability of the P/n pulse subtracting procedure normally used for charge movement measurements. These effects can be substantially reduced by cadmium ions (0.1 mM), which has no effect on charge movement. When SH reagents are applied in the presence of cadmium, no effects are observed, indicating that this cation may protect the membrane from the reagent effects. The effects of -SH reagents can be observed by applying them in the absence of cadmium, followed by addition of the cation. Under these conditions the conductance changes are reversed and the effects of the SH reagents on charge movement can be measured with a higher degree of confidence. Maximum charge is reduced by 32% in the presence of 1.5 mM PCMB and by 31% in the presence of 2 mM PHMPS. These effects do not occur in the presence of DTT and in some cases they may be reversed by this agent. Charge 2, recorded in depolarized muscle fibers, is also reduced by these agents.

scholarly journals The relationship between Q gamma and Ca release from the sarcoplasmic reticulum in skeletal muscle.

1991 ◽  
Vol 97 (5) ◽  
pp. 913-947 ◽  
Author(s):  
G Pizarro ◽  
L Csernoch ◽  
I Uribe ◽  
M Rodríguez ◽  
E Ríos
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Voltage Dependence ◽  
Time Derivative ◽  
Specific Model ◽  
Feedback Mechanism ◽  
Kinetic Properties ◽  
Charge Movement ◽  
Asymmetric Membrane ◽  
Additional Release

Asymmetric membrane currents and fluxes of Ca2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca2+ release and the "hump" component of charge movement current (I gamma), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 microM tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property of I gamma, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump", (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca2+ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I gamma described in the previous papers. It also accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I gamma accompanies activating transitions at the same set of voltage sensors as I beta. Therefore it should open additional release channels, which in turn should cause more I gamma, providing a positive feedback mechanism in the regulation of calcium release.

scholarly journals A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers.

1994 ◽  
Vol 104 (3) ◽  
pp. 449-476 ◽  
Author(s):  
N Shirokova ◽  
G Pizarro ◽  
E Ríos
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Muscle Fibers ◽  
Major Effect ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
High Concentration ◽  
Asymmetric Membrane ◽  
Average Value ◽  
Skeletal Muscle Fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)

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