Voltage-clamped Frog Muscle Fibres Research Articles

Involvement of a pertussis toxin-sensitive G-protein in excitation-contraction coupling of intact and cut-end voltage-clamped skeletal muscle fibres.

In voltage-clamped frog muscle fibres 10 ng/ml PTX induced a decrease (approximately 35%) of tension when applied externally. Internal application in cut-end fibres significantly depressed tension after 20 min. This effect increased with time to reach 65% after 60 min. PTX shifted the voltage-dependent inactivation curve of tension by 30 mV towards hyperpolarizations and this was counteracted by raising external calcium concentration. The toxin induced a parallel decrease in tension and voltage-sensitive charge movement (49 +/- 9% and 52 +/- 6% respectively; n = 6). This was not counteracted by prior impregnation with forskolin. Internally applied GTP gamma S (500 microM) induced a simultaneous increase in tension (57 +/- 5%) and charge amount displaced (40 +/- 7%). By contrast, GDP beta S decreased tension and charge movement by 35 +/- 5% and 36 +/- 6% respectively.

The correlation between the increase in slow outward current and in contraction induced by caffeine, ryanodine, and rapid cooling in voltage-clamped frog muscle fibers

The effects of caffeine, ryanodine, and rapid cooling were tested on the depolarization-induced contraction and the apamin-insensitive slow outward current (Iso) of voltage-clamped (double mannitol gap) single frog muscle fibers. Subthreshold caffeine concentrations (0.5-2 mM) induced a monotonic increase in contractile and Iso amplitude. Whatever the concentration, the increase in contraction was roughly twice the one in current. Similar results were obtained upon rapid cooling (20-4 degrees C) in the presence of 0.5 mM caffeine. In the absence of external Na+ (choline-substituted) 10(-5) M ryanodine induced a delayed increase (approximately 30 min) in contraction and in current, shortly before the development of a drastic and irreversible contracture. Here again, the increase in contraction was twice that in current. In the presence of 5 mM tetraethylammonium (TEA) and (or) 25 nM charybdotoxin, 2 mM caffeine still induced a strong facilitating effect on contraction but the parallel increase in current was strongly reduced. The linear relationship between the increase in current and contractile amplitude has a slope approximately 0.5 (whatever the drug used to increase contractility); it is approximately 0.1 in the presence of TEA and (or) charybdotoxin. In conclusion, provided the changes in contractile amplitude are caused by parallel changes in depolarization-induced sarcoplasmic reticulum Ca2+ release, about 50% of the apamin-insensitive Iso is controlled by internal Ca2+ release. The main part of this current corresponds to the TEA- and charybdotoxin-sensitive component of Iso.

The depressing effect of tetracaine and ryanodine on the slow outward current correlated with that of contraction in voltage-clamped frog muscle fibres

The effects of tetracaine (10-50 microM) and ryanodine (0.1-10 microM) were tested on the slow outward K+ current (Iso) and the mechanical tension of isolated frog muscle fibres in a voltage-clamp device (double mannitol-gap) connected to a mechanoelectric transducer. In the concentration range tested, both drugs induced a simultaneous inhibition of tension and current. In all cases the effect on tension was twice that on current. The tetracaine-induced current and tension blocks were fully reversible and dose-dependent. In contrast the ryanodine effects on current and tension were not reversible and did not exhibit a dose dependence except for the delay before the onset of the response, which was shortened when the concentration was raised. Linear regression analysis of the time-dependent and dose-dependent effects of both drugs indicated a strong correlation between the decreases in tension and current. It is concluded that the slow outward current is partly under the control of the Ca2+ release from sarcoplasmic reticulum during contraction.

The depressant effects of some amiloride analogues on the slow outward K + current and contraction of voltage-clamped frog muscle fibres

The effects of 4 derivatives of amiloride, which are known to block the Na +/H + antiporter, were studied on the slow outward current (I so) and on tension development of voltage-clamped single muscle fibres of the frog Rana ridibunda. Each compound tested induced a strong depressant effect on tension and I so in a voltage-, time- and dose-dependent (10 −8 to 10 −5 M) manner at physiological pH. The effects exhibited a strong pH dependence (the greater the pH in the range between 6.6 and 8.0, the greater the depressant action). This seems to exclude the involvement of the Na +/H + antiport and alterations of surface membrane charges as mediators. The current block was not observed after the release of calcium from the sarcoplasmic reticulum was blocked with 75 μM tetracaine. It is concluded that the primary effect of the 4 compounds used was to inhibit the internal release of Ca 2+ from the sarcoplasmic reticulum, and that this inhibition blocked the Ca 2+-dependent slow outward current.

Antamanide antagonizes the phalloidin-induced negative inotropic effect and blocks voltage dependently the fast outward K + current in voltage-clamped frog muscle fibres

The effects of antamanide (10 −14-10 −5M) and N-acetyl-secophalloidin (10 −7-5 × 10 −3 M) a neutral non-toxic derivative of phalloidin, were tested on voltage-clamped single frog muscle fibres. Antamanide protected muscle fibres against the negative inotropic effect of phalloidin but blocked the fast potassium permeability in the same concentration range and the same voltage-dependent manner as did phalloidin. N-Acetyl-secophalloidin exhibited a strongly attenuated blocking effect on K + permeability in a 1000-fold higher concentration range than phalloidin. Neither antamanide nor N-acetyl-secophalloidin affected the contractile properties. These results suggest the existence in the frog muscle membrane of a receptor with two sites for phalloidin and antamanide which acts on potassium conductance.

Effect of external ammonium on the kinetics of the sodium current in frog muscle

(1) The effect of externally applied 20 mM NH 4Cl on steady-state Na + inactivation h ∞ and other electrical parameters has been studied in voltage-clamped frog muscle fibres. (2) Exposure to Ringer with 20 mM NH 4Cl causes a small transient shift of the h ∞(E) curve to more positive potentials. Upon return to normal Ringer the h ∞(E) curve shifts in the opposite direction, overshooting its original position, and then returns slowly into the original position. (3) Similar but smaller shifts of the descending branch of the I Na (E) curve, of the P Na (E) curve and of the time to peak curve are also observed. (4) The shifts are thought to result from the changes in intracellular pH which occur during and after NH 4Cl application. The observations are compatible with the idea that intracellular pH affects the surface charge potential at the inner side of the membrane.

Endplate currents in sucrose solution.

Endplate currents were recorded from voltage-clamped frog muscle fibres bathed in an isotonic sucrose solution containing 2 mM K + . In this solution the major part of the current is carried by K + ions, and at negative potentials the membrane voltage–current amplitude relations of both miniature endplate currents and the single channel current estimated from noise analysis were linear, with smaller conductance than in normal Ringer solution. At positive potentials miniature endplate currents and currents induced by acetylcholine (ACh) showed a saturation, or sometimes even decline, with increasing potential. In contrast, the single channel current continued to increase linearly at these potentials. It is suggested that in sucrose solution the number of functional ACh receptors decreases as the endplate is depolarized to more positive potentials.

Decamethonium both opens and blocks endplate channels.

Miniature endplate currents, endplate current fluctuations ("membrane noise"), and voltage-jump current relaxations were studied in voltage-clamped frog muscle fibers during decamethonium action. All three types of experiments revealed two kinetic processes controlling the opening of endplate channels, one that reflects agonist action and another that reflects local anesthetic-like blocking activity. The kinetic constants for these two steps were evaluated from measurements of the fast and slow time constants as a function of decamethonium concentration. At -130 mV membrane potential and 13 degrees, the mean open time of decamethonium-activated channels is 2.8 msec. The forward and backward rate constants for channel blocking are 1.7 X 10(7) M-1 sec-1 and 10(3) sec-1. The voltage dependencies of the channel lifetime and of the blocking equilibrium are similar to those seen with pure agonists and local anesthetics, respectively.

Relaxation experiments using bath‐applied suberyldicholine.

1. The effect of step changes in membrane potential on the end-plate conductance change produced by bath-applied suberyldicholine was studied in voltage-clamped frog muscle fibres. 2. The suberyldicholine-induced conductance increased exponentially from its previous equilibrium level to a new equilibrium level following a step hyperpolarization. 3. For low suberyldicholine concentrations the time constant of this relaxation was independent of the concentration. 4. For low suberyldicholine concentrations the voltage dependence of equilibrium conductance and relaxation time constants was identical. 5. Bungarotoxin pretreatment did not affect the responses beyond a simple reduction in their amplitude. 6. The conductance evoked by high suberyldicholine concentrations was less voltage-sensitive than that evoked by low concentrations. 7. A new model for explaining noise and relaxation data is proposed. This postulates rate-limiting binding steps followed by a voltage-dependent isomerization.

Voltage-dependence of drug-induced conductance in frog neuromuscular junction.

Membrane currents from voltage-clamped frog muscle fibers were recorded during iontophoretic application of steady doses of carbachol, acetylcholine, and suberyldicholine to the endplate region. In the presence of these drugs, an exponentially relaxing current was observed after step changes of membrane potential. The time constant of relaxation was found to be voltage-dependent. It was equal to the time constant obtained from the autocorrelation function of drug-induced conductance fluctuations measured under similar conditions. Analysis of instantaneous current at the on- and offsets of voltageclamp pulses showed that there is no shift in equilibrium potential during the pulses.

The effect o f calcium on contraction and conductance thresholds in frog skeletal muscle.

1. The effect of extracellular calcium and magnesium on the contraction threshold and on the thresholds for an increase in sodium and potassium conductance with depolarization was studied in voltage-clamped frog muscle fibres.2. A larger depolarization was required to reach each of the three thresholds when the concentration of divalent cation was increased.3. The contraction and potassium conductance thresholds appeared to shift in parallel with alterations in calcium over the concentration range 0.2-10.0 mM and in magnesium over the concentration range 5.4-90.0 mM. The shift amounted to about 4 mV for a threefold change in concentration of divalent cation.4. The sodium conductance threshold was much more sensitive to alterations in divalent cation concentration than was either the contraction or the potassium conductance threshold.