Factors affecting the appearance of the hump charge movement component in frog cut twitch fibers.

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.

Comparison of charge movement components in intact and cut twitch fibers of the frog. Effects of stretch and temperature.

Charge movements were measured in frog intact fibers with the three-microelectrode technique and in cut fibers with the double Vaseline gap technique. At 13-14 degrees C, the ON segments of charge movement records from both preparations showed an early I beta component and a late I gamma hump component. When an intact fiber was cooled to 4-7 degrees C, the time-to-peak of I gamma (tp,gamma) was prolonged, but I gamma still appeared as a hump. Q-V plots from intact fibers at 4-7 degrees C were fitted with a sum of two Boltzmann distribution functions (method 1). The more steeply voltage-dependent component, identified with Q gamma, accounted for 32.1% (SEM 2.2%) of the total charge. This fraction was larger than the 22.6% (SEM 1.5%) obtained by separating the ON currents with a sum of two kinetic functions (method 2). The total charge in cut fibers stretched to a sarcomere length of 3.5 microns at 13-14 degrees C was separated into Q beta and Q gamma by methods 1 and 2. The fraction of Q gamma in the total charge was 51.3% (SEM 1.7%) and 53.7% (SEM 1.8%), respectively, suggesting that cut fibers have a larger proportion of Q gamma:Q beta than intact fibers. When cut fibers were stretched to a sarcomere length of 4 microns, the proportion of Q gamma:Q beta was unchanged. Between 4 and 13 degrees C, the Q10 of l/tp,gamma in intact fibers was 2.33 (SEM 0.33) and that of 1/tau beta was less than 1.44 (SEM 0.04), implying that the kinetics of I gamma has a steeper temperature dependence than the kinetics of I beta. When cut fibers were cooled from 14 to 6 degrees C, I gamma in the ON segment generally became too broad to be manifested as a hump. In a cut fiber in which I gamma was manifested as a hump, the Q10 of l/tp,gamma was 2.08 and that of l/tau beta was less than 1.47. Separating the Q-V plots from cut fibers at different temperatures by method 1 showed that the proportion of Q gamma:Q beta was unaffected by temperature change. The appearance of I gamma humps at low temperatures in intact fibers but generally not in cut fibers suggests an intrinsic difference between the two fiber preparations.

Q beta and Q gamma components of intramembranous charge movement in frog cut twitch fibers.

Intramembranous charge movement was measured in frog cut twitch fibers mounted in a double Vaseline-gap chamber with a TEA.Cl solution at 13-14 degrees C in the central pool. When a fiber was depolarized from a holding potential of -90 mV to a potential near -60 mV, the current from intramembranous charge movement was outward in direction and had an early, rapid component and a late, more slowly developing component, referred to as I beta and I gamma, respectively (1979. J. Physiol. [Lond.]. 289:83-97). When the pulse to -60 mV was preceded by a 100-600-ms pulse to -40 mV, early I beta and late I gamma components were also observed, but in the inward direction. The shape of the Q gamma vs. voltage curve can be estimated with this two-pulse protocol. The first pulse to voltage V allows the amounts of Q beta and Q gamma charge in the active state to change from their respective resting levels, Q beta (-90) and Q gamma (-90), to new steady levels, Q beta (V) and Q gamma (V). A second 100-120-ms pulse, usually to -60 mV, allows the amount of Q beta charge in the active state to change from Q beta (V) to Q beta (-60) but is not sufficiently long for the amount of Q gamma charge to change completely from Q gamma (V) to Q gamma (-60). The difference between the amount of Q gamma charge at the end of the second pulse and Q gamma (-60) is estimated from the OFF charge that is observed on repolarization to -90 mV. The OFF charge vs. voltage data were fitted, with gap corrections, with a Boltzmann distribution function plus a constant. The mean values of V (the potential at which, in the steady state, charge is distributed equally between the resting and active states) and k (the voltage dependence factor) were -59.2 mV (SEM, 1.1 mV) and 1.2 mV (SEM, 0.6 mV), respectively. The one-pulse charge vs. voltage data from the same fibers were fitted with a sum of two Boltzmann functions (1990. J. Gen. Physiol. 96:257-297). The mean values of V and k for the steeply voltage-dependent Boltzmann function, which is likely to be associated with the Q gamma component of charge, were -55.3 mV (SEM, 1.3 mV) and 3.3 mV (SEM, 0.6 mV), respectively, similar to the corresponding values obtained with the two-pulse protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane capacitance in frog cut twitch fibers mounted in a double vaseline-gap chamber.

In experiments on cut muscle fibers mounted in a double Vaseline-gap chamber, electrical measurements are usually made by measuring the voltage V1(t) in one end pool and by passing current I2(t) from the other end pool to the central pool, which is usually clamped to earth potential. The voltage in the current-passing end pool is denoted by V2(t). This article describes how the value of the holding current, Ih, and the values of delta V2(infinity)/delta V1(infinity) and delta I2(infinity)/delta V1(infinity) that are associated with a small change in V1(t) can be used to estimate the linear cable parameters rm, ri, and re in a cut fiber that has been equilibrated with a Cs-containing internal solution. rm, ri, and re represent, respectively, the resistance of the plasma membranes, the internal longitudinal resistance, and the external longitudinal resistance under the Vaseline seals, all for a unit length of fiber. The apparent capacitance, Capp, of the preparation is defined to equal integral of infinity 0 delta I2,tr(t) dt/delta V1(infinity), in which delta I2,tr(t) represents the transient component of current that is associated with a change in V1(t) of amplitude delta V1(infinity). A method is described to estimate cm, the capacitance of the plasma membranes per unit length of fiber, from Capp and the values of rm, ri, and re. In experiments carried out with a tetraethylammonium chloride (TEA.Cl) solution at 13-14 degrees C in the central pool, cm remained stable for as long as 3-4 h. The values of cm, 0.19 microF/cm on average, and their variation with fiber diameter are similar to published results from intact fibers. This article also describes the different pathways that are taken by the current that flows from the current-passing end pool to the central pool. Approximately two-thirds of delta I2,tr(t) flows across the capacitance of the plasma membranes in the central-pool region. The rest flows either across plasma membranes that are under the two Vaseline seals or directly from the current-passing end pool to the central pool, across the external longitudinal resistance under the Vaseline seal. [There is also a current that flows directly from the voltage-measuring end pool to the central pool but this does not contribute to delta I2,tr(t).]

Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber.

Intramembranous charge movement was measured in cut twitch fibers mounted in a double Vaseline-gap chamber with either a tetraethylammonium chloride (TEA.Cl) or a TEA2.SO4 solution (13-14 degrees C) in the central pool. Charge vs. voltage data were fitted by a single two-state Boltzmann distribution function. The average values of V (the voltage at which steady-state charge is equally distributed between the two Boltzmann states), k (the voltage dependence factor), and qmax/cm (the maximum charge divided by the linear capacitance, both per unit length of fiber) were V = -53.3 mV (SEM, 1.1 mV), k = 6.3 mV (SEM, 0.3 mV), qmax/cm = 18.0 nC/microF (SEM, 1.1 nC/microF) in the TEA.Cl solution; and V = -35.1 mV (SEM, 1.8 mV), k = 10.5 mV (SEM, 0.9 mV), qmax/cm = 36.3 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. These values of k are smaller than those previously reported for cut twitch fibers and are as small as those reported for intact fibers. If a correction is made for the contributions of currents from under the Vaseline seals, V = -51.2 mV (SEM, 1.1 mV), k = 7.2 mV (SEM, 0.4 mV), qmax/cm = 22.9 nC/microF (SEM, 1.4 nC/microF) in the TEA.Cl solution; and V = -34.0 mV (SEM, 1.9 mV), k = 10.1 mV (SEM, 1.1 mV), qmax/cm = 38.8 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. With this correction, however, the fit of the theoretical curve to the data is poor. A good fit with this correction can be obtained with a sum of two Boltzmann distribution functions. The first has average values V = -33.0 mV (SEM, 2.8 mV), k = 11.0 mV (SEM, 0.5 mV), qmax/cm = 10.6 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -20.0 mV (SEM, 3.3 mV), k = 17.0 mV (SEM, 2.0 mV), qmax/cm = 36.4 nC/microF (SEM, 2.3 nC/microF) in the TEA2.SO4 solution. The second has average values V = -56.5 mV (SEM, 1.3 mV), k = 2.9 mV (SEM, 0.4 mV), qmax/cm = 13.2 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -41.6 mV (SEM, 1.4 mV), k = 2.5 mV (SEM, 0.8 mV), qmax/cm = 11.8 nC/microF (SEM, 1.7 nC/microF) in the TEA2.SO4 solution. When a fiber is depolarized to near V of the second Boltzmann function, a slowly developing "hump" appears in the ON-segment of the current record.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of conditioning depolarization and repetitive stimulation on Q beta and Q gamma charge components in frog cut twitch fibers.

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. Steady-state inactivation of charge movement was studied by changing the holding potential from -90 mV to a level ranging from -70 to -30 mV. Q beta and Q gamma at each holding potential were separated by fitting the Q-V plot with a sum of two Boltzmann distribution functions. At -70 mV Q beta and Q gamma were inactivated to 54.0% (SEM 2.2) and 82.7% (SEM 3.0) of the amounts at -90 mV. At holding potentials greater than or equal to -60 mV, more Q gamma was inactivated than Q beta, and at -30 mV Q gamma was completely inactivated but Q beta was not. There was no holding potential at which Q beta was unaffected and Q gamma was completely inactivated. The differences between the residual fractions of Q beta and Q gamma are significant at all holding potentials (P less than 0.001-0.05). The plot of the residual fraction of Q beta or Q gamma versus holding potential can be fitted well by an inverted sigmoidal curve that is a mirror image of the activation curve of the respective charge component. The pair of curves for Q gamma correlates well with those for tension generation or Ca release obtained by other investigators. The time courses of the inactivation of Q beta and Q gamma were studied by obtaining several Q-V plots with conditioning depolarizations lasting 1-20 s and separating each Q-V plot into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. The inactivation time constant of Q beta was found to be 5-10 times as large as that of Q gamma. During repetitive stimulation, prominent I gamma humps could be observed in TEST-minus-CONTROL current traces and normal Q gamma components could be separated from the Q-V plots, whether 20 or 50 mM EGTA was present in the internal solution, whether 2 or 10 stimulations were used, and whether the stimuli were separated by 400 ms or 6 s. Repetitive stimulation slowed the kinetics of the I gamma hump and could shift the Q-V curve slightly in the depolarizing direction in some cases, resulting in an apparent suppression of charge at the potentials that fall on the steep part of the Q-V curve.

Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods.

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 microM tetracaine had very little effect on the maximum amounts of Q beta and Q gamma but slowed the kinetics of the I gamma humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V gamma 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/cm = 9.2 (SEM 1.5) nC/microF, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q gamma component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V gamma and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to greater than -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-V plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q gamma component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q gamma should be separated by more than one method to obtain more reliable results.

Possible origin of gating current in nerve membrane.

The present information about gating current observed in squid giant axons points towards the distinct possibility of the current arising from the Debye relaxation of the carboxyl groups in the side chains of the globular proteins enclosing the ionic channels. These carboxyl groups form dipole chains stretching across the membrane. A dipole model is constructed to study the relaxation process under the assumption that the relaxation time tau of the dipoles is modified by dipole-dipole interaction. This model explains qualitatively some of the features of the asymmetric gating current, but is not indicative of any specific mechanism leading to the opening of the gates in the ionic channels. We speculate that the conformational change in the protein globules as a result of dipole reorientation would be the key to the mystery.

Calcium release in frog cut twitch fibers exposed to different ionic environments under voltage clamp.

Calcium release was measured in highly stretched frog cut twitch fibers mounted in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 20 mM EGTA plus 0.4 or 1.8 mM added calcium. Rise in myoplasmic [Ca(2+)] was monitored with antipyrylazo III as the indicator at a temperature of 13 to 14 degrees C. The waveform of calcium release rate (Rel) computed from the absorbance change showed an early peak (Rel(p)) followed by a maintained phase (Rel(m)). Each Rel(p)-versus-V plot was fitted with a Boltzmann distribution function. The maximum value of Rel(p) (Rel(p,max)) was compared in various calcium-containing external solutions. The average value in a Cl(-) solution was about one-third larger than those in a CH(3)SO(3)(-) or gluconate solution, whereas the values in the CH(3)SO(3)(-) and gluconate solutions had no statistically significant difference. In external solutions containing CH(3)SO(3)(-) or gluconate, a replacement of the Ca(2+) with Mg(2+) reduced Rel(p,max) by 30 to 50%, on average. The values of Rel(p, max) also had no statistically significant difference among calcium-free external solutions containing different impermeant anions. An increase of the nominal free [Ca(2+)] in the end-pool solution from a reduced to the normal physiological level increased the value of Rel(p,max), and also slowed the decay of the maintained phase of the Rel waveform. The Rel waveforms in the Cl(-) and CH(3)SO(3)(-) solutions were compared in the same fiber at a fixed potential. CH(3)SO(3)(-) increased the time to peak, reduced Rel(p), and increased Rel(m), and the effects were partially reversible. Under the hypothesis that the decay of the peak was due to calcium inactivation of calcium release, the inactivation was larger in Cl(-) than in CH(3)SO(3)(-), in qualitative agreement with the ratio of Rel(p) in the two solutions. Under the alternative hypothesis that the peak and the maintained phase were separately gated by calcium and depolarization, respectively, then CH(3)SO(3)(-) appeared to decrease the calcium-gated component and increase the voltage-gated component.

A slow calcium-dependent component of charge movement in Rana temporaria cut twitch fibres.

1. Charge movement was studied in highly stretched frog cut twitch fibres in a double Vaseline-gap voltage-clamp chamber, with the internal solution containing either 0.1 mM EGTA or 20 mM EGTA plus 1. 8 mM total Ca2+. 2. Fibres were stimulated with TEST pulses lasting 100-400 ms. Replacement of the external Cl- with an 'impermeant' anion, such as SO42-, CH3SO3-, gluconate or glutamate, greatly reduced the calcium-dependent Cl- current in the ON segment and generated a slowly decaying inward OFF current in charge movement traces. 3. Application of 20 mM EGTA to the internal solution abolished the slow inward OFF current, implying that the activation of the current depended on the presence of Ca2+ in the myoplasm. The possibility that the slow inward OFF current was carried by cations flowing inwards or anions flowing outwards was studied and determined to be unlikely. 4. During a long (2000 ms) TEST pulse, a slowly decaying ON current was also observed. When the slow ON and OFF currents were included as parts of the total charge movement, ON-OFF charge equality was preserved. This slow capacitive current is named Idelta. 5. When Cl- was the major anion in the external solution, the OFF Idelta was mostly cancelled by a slow outward current carried by the inflow of Cl-. 6. The OFF Idelta component showed a rising phase. The average values of the rising time constants in CH3SO3- and SO42- were similar and about half of that in gluconate. 7. The OFF Idelta component in CH3SO3- had a larger magnitude and longer time course than that in SO42-. The maximum amount of Qdelta in CH3SO3- was about three times as much as that in SO42-, whereas the voltage dependence of Qdelta was similar in the two solutions. 8. Since the existence of Qdelta depends on the presence of Ca2+ in the myoplasm, it is speculated that Qdelta could be a function of intracellular calcium release.

Pharmacological dissection of charge movement in frog skeletal muscle fibers.

When charge movement is measured from muscle fibers bathed in a moderately hypertonic solution, a secondary hump appears in the decay phase of the signal during the "on" of the test pulse. The hump can be suppressed by the application of dantrolene sodium or tetracaine. The amount of charge associated with the hump is approximately 20-25% of the total charge. All the observed properties of the hump charge are consistent with the possibility that it is more closely associated with calcium release from the sarcoplasmic reticulum, and thus more relevant to excitation-contraction coupling, than the rest of the charge.

Differential properties of two charge components in frog skeletal muscle.

Charge movement in frog twitch muscle fibres bathed in a moderately hypertonic solution (containing 350 mM-sucrose) showed a main component (Q beta) and a secondary hump (Q gamma). Assuming that Q beta decays with a single exponential and Q gamma follows the time course of a symmetrical bell, a mathematical model was developed to separate Q gamma from Q beta. The activation curves of Q beta and Q gamma can be individually fitted by the two-state model of Schneider & Chandler (1973). The voltage distribution of Q gamma is approximately twice as steep as that of Q beta and the maximum moveable amount of Q beta about four times that of Q gamma. The decay rate constant of Q beta, expressed as a function of potential, follows the familiar U-shaped curve whereas the reciprocal of the time-to-peak of Q gamma rises linearly with increasing potentials. Application of the mathematical dissection technique to Qon values in solutions of varying concentrations of tetracaine yielded the dose dependence of the suppression of Q gamma by tetracaine. Q gamma inactivates more steeply than Q beta as the holding potential is made less negative. Each of the steady-state inactivation curves of Q beta and Q gamma forms a mirror image with the respective activation curve. It is speculated that Q gamma might be more directly involved than Q beta in triggering calcium release and activating contraction.

Pharmacological studies of charge movement in frog skeletal muscle.

Charge movements in frog twitch fibres were studied using the three-microelectrode voltage-clamp technique. In a solution made moderately hypertonic with 350 mM-sucrose, fibre contraction was effectively blocked and a secondary hump appeared in the decay phase of the 'on' part of charge movement. At small depolarizations, the hump (Q gamma) is small and slow. As depolarization is increased, Q gamma becomes larger in magnitude and faster in kinetics until it merges with the main part of charge movement (Q beta). As the fibre is perfused extracellularly with a test solution saturated with dantrolene sodium, Q gamma disappears in about 30 min whereas the kinetics of Q beta are slowed down. After equilibration in the dantrolene sodium solution, the total moveable charge is reduced by about 20%, which could very well be the charge carried by Q gamma. Tetracaine also suppresses Q gamma but does not seem to have any effect on the kinetics of Q beta. The suppression of Q gamma appears to be dose-dependent, with complete abolition occurring at about 4 mM-tetracaine. Dissection of charge movement with tetracaine indicates that Q gamma might be bell-shaped and capacitive in nature. Q beta and Q gamma might be two distinct species of charge and Q gamma would probably be more closely associated with calcium release from the sarcoplasmic reticulum.

D600 binding sites on voltage-sensors for excitation-contraction coupling in frog skeletal muscle are intracellular.

Charge movements were measured in frog cut twitch fibres mounted in a double Vaseline gap chamber at 14 degrees C with 30 microM D600 in the external solution. TEST-minus-CONTROL current traces appear normal with a hump current component (I gamma) embedded in the decay phase of the early current component (I beta) in the ON-segment and an exponentially decaying current transient in the OFF-segment. When a conditioning depolarization to 0 mV is applied at around 6 degrees C, charge movement is greatly reduced but not completely suppressed and no hump component can be visualized in the ON-segment. In addition, an extra capacitive component is generated having a time course slower than, and a polarity opposite to, that of the usual charge movement. This extra component makes the transients in both the ON- and OFF-segments appear bisphasic. When temperature is restored to 14 degrees C, the biphasic nature is greatly enhanced. After the application of a conditioning hyperpolarization, the shape of the TEST-minus-CONTROL current trace is converted back to that before paralysis, but the total amount of charge reprimed is less than 100% of control. In general, more Q beta is reprimed than Q gamma, and the amount of Q gamma reprimed varies over a wider range from fibre to fibre than that of Q beta. Extracellularly applied D890 cannot reproduce the blocking effect of D600 whereas intracellularly applied D890 can. As D890 is permanently charged and cannot permeate through the plasma membranes, it can be concluded that the binding sites for D600/D890 on the charge movement macromolecules must be on the myoplasmic side. This adds another parallelism between the charge movement entities and L-type calcium channels. However, the specific prerequisites for the blockage of the former not shared by the latter differentiates the two physiological units.

Contractile properties of frog twitch fibres after D600 paralysis.

Twitch and contracture tensions were measured in single intact fibres of semitendinosus muscles with a sensitive miniature transducer. After a fibre was paralysed by a conditioning depolarization in the presence of 30 microM D600 at low temperature (around 5 degrees C), no twitch tension could be detected. When a paralysed fibre was warmed, its ability to give potassium-contractures recovered almost completely but its twitch tension revived to a variable extent, ranging from fully to partially or not at all. This variable recovery of twitch tension appeared to correlate very well with the variable repriming of Q gamma observed previously in revived fibres. The optimal temperature at which twitch tension could be revived readily lay within a narrow window roughly between 8 to 16 degrees C, within which the rate and extent of revival of twitch tension were temperature-dependent. Removal of D600 from the bathing solution after conditioning depolarization facilitated the revival of twitch tension but was neither a necessary nor a sufficient condition for revival. A reduction of potassium concentration in the high K solution or an abbreviation of the duration of conditioning depolarization could bring a fibre to a partially paralysed state (or equivocally a partially revived state) without going through complete paralysis. The paralysing actions of submaximal condition depolarizations were additive. The partially revived state was unstable and affected by repetitive stimulations in a use-dependent manner. The effect of a 0.1 Hz train of action potentials on twitch tension was generally biphasic, with a small initial suppression followed by an enhancement. It is speculated that this use-dependent enhancement could be due to a competition between D600 molecules and intracellular Ca2+ ions.

Differential blockage of charge movement components in frog cut twitch fibres by nifedipine.

1. The effect of nifedipine on charge movement was studied in cut twitch fibres of Rana temporaria with a double Vaseline-gap voltage-clamp technique. The steady-state charge-voltage (Q-V) plot, in the absence or presence of nifedipine, was separated into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. 2. When a fibre was held at -90 mV, low concentrations (around 20 nM) of nifedipine suppressed a large fraction of Q beta than Q gamma. Higher concentrations of nifedipine suppressed Q gamma more effectively than Q beta, but even 2 microM-nifedipine did not suppress Q beta and Q gamma completely. Ten micromolar was required for complete suppression of Q gamma. Nifedipine thus suppressed Q beta and Q gamma with different dose dependencies. 3. When the holding potential was changed to -70 mV, some Q beta and Q gamma were inactivated. Low concentrations (around 20 nM) of nifedipine still suppressed a larger fraction of the mobile Q beta than the mobile Q gamma. Higher concentrations of nifedipine also suppressed Q gamma more effectively than Q beta, but 2 microM-nifedipine was sufficient to suppress Q gamma completely. Hence, at this slightly depolarized holding potential, nifedipine also suppressed Q beta and Q gamma with different dose dependencies. 4. A portion of Q beta appeared to be resistant to the action of nifedipine. At -70 mV, the blockage of the nifedipine-sensitive portion of Q beta appeared to saturate at 2 microM of the drug. At -90 mV, the nifedipine-resistant portion of Q beta was more difficult to identify, because the blockage of the nifedipine-sensitive portion of Q beta did not saturate at 2 microM. 5. Based on double-reciprocal plots for the dose-response relationships, the half-blocking concentration of nifedipine for Q beta was found to be 14-19 nM at -90 mV and less than 13 nM at -70 mV, whereas that for Q gamma was approximately 1.6 microM at -90 mV and 120 nM at -70 mV. Thus, nifedipine suppressed Q gamma in a voltage-dependent manner, but its suppression of Q beta was much less voltage dependent. 6. It was demonstrated that the enhancement in the blockage of charge movement by maintained depolarization could not be achieved by depolarizing pulses lasting up to hundreds of milliseconds. 7. The difference in the half-blocking concentrations of nifedipine for Q beta and Q gamma implies that Q beta and Q gamma cannot be tightly coupled.

Multiple actions of 2,3-butanedione monoxime on contractile activation in frog twitch fibres.

1. The effects of 2,3-butanedione monoxime (BDM) on various steps in the excitation-contraction coupling sequence, including action potential, charge movement and twitch tension, were studied in twitch fibres of Rana temporaria. 2. The resting potential of intact fibres in whole muscle bathed in 20 mM-BDM was the same as control. The resting potential also remained stable after more than 100 min in 20 mM-BDM. 3. The action potential was measured in intact fibres of fibre bundles with an intracellular microelectrode. Applications of 5 and 7.5 mM-BDM had no effect on its amplitude, whereas 10 and 20 mM suppressed its amplitude by about 4 and 10%, respectively. Increasing concentrations of BDM prolonged the half-width and elevated the after-potential of the action potential progressively. The action potential was also measured in cut fibres mounted in a double Vaseline-gap chamber. Results were similar to those in intact fibres. 4. Charge movement was measured in intact fibres of halved muscles with the three-microelectrode voltage-clamp technique. The steady-state Q-V plot of the total charge measured in isotonic tetraethylammonium (TEA) Ringer solution with 20 mM-BDM appeared to be shifted about 10 mV in the depolarizing direction and to be slightly more shallow when compared with the control Q-V plot measured in hypertonic TEA Ringer solution with 350 mM-sucrose. After allowing for the voltage shift, 20 mM-BDM did not appear to affect the kinetics of both components of charge movement, but suppressed the maximum amount of total charge by about one-quarter. 5. Charge movement was also measured in cut fibres with the double Vaseline-gap voltage-clamp technique. In the presence of 20 mM-BDM, charge movement traces resembled those from intact fibres. Twenty millimolar BDM suppressed the maximum amount of total charge by about one-quarter, as in intact fibres. The steady-state Q-V plots from cut fibres were separated into Q beta (early current) and Q gamma (late hump current) components by least-squares fitting with a sum of two Boltzmann distribution functions. On average, 20 mM-BDM suppressed Q beta and Q gamma in roughly equal proportion, but did not affect the individual voltage distributions of Q beta and Q gamma. 6. Twitch tension was measured in single intact fibres stimulated extracellularly. BDM effectively reduced the peak amplitude, the time-to-peak and the half-width of twitch tension. The interaction of BDM with receptors appeared to follow more or less a simple 1:1 binding in fibres stretched to sarcomere lengths of about 3.6 microns.(ABSTRACT TRUNCATED AT 400 WORDS)

Action of 2,3-butanedione monoxime on calcium signals in frog cut twitch fibres containing antipyrylazo III.

1. The effects of 2,3-butanedione monoxime (BDM) on the optical retardation and myoplasmic Ca2+ signal were studied in twitch fibres of Rana temporaria. The myoplasmic Ca2+ transient in response to action potential stimulation was monitored in cut fibres containing Antipyrylazo III under current clamp in a double Vaseline-gap chamber. 2. In fibres not stretched adequately to suppress all the contraction, BDM blocked the movement-related intrinsic optical signal at 810 nm very effectively. 3. In fibres stretched to sarcomere lengths greater than or equal to 4 microns to reduce the contraction to below detectable levels, the effect of BDM on the Ca(2+)-Antipyrylazo III signal was studied after correcting for the instrinsic signal unrelated to movement. With increasing concentrations of BDM, the peak of the Ca(2+)-Antipyrylazo III signal was suppressed progressively. Concomitantly, the half-width was prolonged somewhat. On average, 5, 10 and 20 mM-BDM reduced the peak amplitude to 88, 78 and 54% of control, respectively. 4. BDM had no effect on the rising phase or the peak amplitude of the retardation signal measured at 720 nm, but suppressed the undershoot in the decay phase of the signal in a dose-dependent manner. BDM also had no effect on the late pedestal level of the signal. 5. During repetitive stimulation by a train of ten action potentials, 10 mM-BDM suppressed the second to the tenth peaks of the Ca2+ signal and of the retardation signal more effectively than the first peak. Twenty millimolar BDM almost completely suppressed the later peaks of both signals such that the signals decayed with a time course similar to that elicited by a single action potential. 6. The effect of BDM on the Ca(2+)-Antipyrylazo III signal was also studied in fibres under voltage clamp; 10 mM-BDM lowered the threshold for the Ca(2+)-Antipyrylazo III transient by a few millivolts and reduced the steepness of the peak amplitude versus voltage plot near threshold. 7. Based on a model used by Baylor, Chandler & Marshall (1983) to estimate the net Ca2+ release from the sarcoplasmic reticulum, 10 and 20 mM-BDM were found to reduce the peak release to 75 and 52%, to prolong the half-width of the release waveform to 118 and 147%, and to reduce the peak uptake to 76 and 54% of control values, respectively. 8. It is concluded that BDM affects the optical retardation and myoplasmic Ca2+ signal monitored with Antipyrylazo III in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Existence of Q gamma in frog cut twitch fibers with little Q beta.

Charge movements were measured in frog cut twitch fibers with the double Vaseline-gap voltage-clamp technique. In most fibers, when a depolarizing pulse to -60 to -40 mV was applied at 13-14 degrees C, the ON segment of a charge movement trace showed an early I beta component and a late I gamma hump component. An ongoing controversy is whether the I gamma hump component triggers calcium release from the sarcoplasmic reticulum or arises as a consequence of the release. Interestingly, a number of cut fibers showed normal I gamma components but greatly diminished, or unresolvable, I beta components. When the amount of charge associated with the current transient was plotted against the membrane potential, the steeply voltage-dependent Q gamma component appeared normal whereas the less steeply voltage-dependent Q beta component was also greatly diminished or unresolvable. These results suggest that I gamma can flow in the absence of I beta, thereby ruling out the possibility that Q beta triggers calcium release which, in turn, causes Q gamma to move. The results, however, do not rule out the positive feedback of calcium release to activate Q gamma, if calcium release is not triggered by Q beta but by Q gamma itself or by some other signal.

Differential suppression of charge movement components by gluconate in cut twitch fibres of Rana temporaria.

1. Charge movement was studied in cut twitch fibres of Rana temporaria using a double Vaseline-gap voltage-clamp technique. 2. Replacement of Cl- by gluconate in the external solution reduced the magnitude of the early current component (I beta) substantially but affected the magnitude and slowed the kinetics of the hump current component (I gamma) slightly. 3. The early (Q beta) and hump (Q gamma) charge components in the gluconate solution were 11.8 +/- 2.3 and 88.0 +/- 4.8% (mean +/- S.E.M., n = 9), respectively, of those in the Cl- solution. 4. These results suggest that Q beta cannot be a precursor of Q gamma. Moreover, since fibres bathed in a gluconate solution can release calcium, Q beta is probably not involved in triggering calcium release.