Component Of Charge Movement Research Articles

Components of charge movement in rabbit skeletal muscle: the effect of tetracaine and nifedipine.

The effects of tetracaine and nifedipine on asymmetric charge movement in rabbit muscle fibres were examined to investigate whether mammalian charge movement could be subdivided into several components. Tetracaine (0.05-0.2 mM) stopped contraction in every sternomastoid fibre examined (n = 9) and reduced the asymmetric charge (moved by depolarizing steps to 0 mV) by 15% (S.E. of mean 3%). Tetracaine had little effect on the charge moved at potentials more negative than the threshold potential (established in the absence of the drug). Application of the Ca2+ channel blocker nifedipine (2 or 10 microM), reduced the mean maximum asymmetric charge to 50% (+/- 4) of the control value in twenty-three sternomastoid fibres and to 32% (+/- 5) in four soleus fibres. Increasing the concentration of nifedipine to 120 microM had little further effect. The charge moved at potentials more negative than -60 mV was unaffected by nifedipine. A similar result was found with 30 microM-D600 (two fibres). 10 microM-nifedipine completely blocked Ca2+ currents (external [Ca2+] = 8 mM), but 0.15 microM-nifedipine only had a small effect on either the Ca2+ current or charge movement in the four fibres examined. Contractions could no longer be elicited in eleven of eighteen fibres within 6 min of the application of 2 or 10 microM-nifedipine. However, in the remaining seven fibres contractions could be elicited with unchanged thresholds over 30 min, even in the presence of 50 microM-nifedipine. Nifedipine did not noticeably effect q gamma. It is suggested that nifedipine might prevent contraction only when, for other reasons, the normal release of Ca2+ from the sarcoplasmic reticulum has been disrupted and contraction is dependent on the inflow of external Ca2+. The amount of asymmetric charge moved by depolarizing steps was about 50% greater with a holding potential of -110 mV than with one of -90 mV. This 'extra' charge was not suppressed by nifedipine.

Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH.

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental analysis of the relationship between charge movement components in skeletal muscle of Rana temporaria.

Experiments were performed to ascertain whether the monotonic (q beta) and delayed (q gamma) components of non-linear charge in skeletal muscle membranes form a sequential system, or are the result of separate, independent processes. The non-linear capacitance studied in a large number of fibres increased with fibre diameter. This dependence was attributable to tetracaine-sensitive (q gamma) but not to tetracaine-resistant (q beta and q alpha) charge. The kinetics and total quantity of q gamma charge moving in response to voltage steps from varying pre-pulse potentials to a fixed probe potential remained constant despite variations in the size of the early q beta decay. The kinetics of the delayed (q gamma) charging current obtained from a single 20 mV depolarizing step were compared with the sum of the responses to two 10 mV steps adding to the same voltage excursion. The respective transients superimposed only if one of the 10 mV steps did not reach the voltage at which q gamma first appears. In the two preceding experiments, total charge was conserved. These results are consistent with separate and functionally independent q beta and q gamma systems of potential-dependent charge, with q gamma residing in the transverse tubules and q beta on surface membrane. The findings can be discussed in terms of a contractile 'activator' with a steep sensitivity to voltage that begins only with depolarization beyond a level close to the actual mechanical threshold.

Charge movement in skeletal muscle fibers paralyzed by the calcium-entry blocker D600.

We report measurements of nonlinear charge movement in frog skeletal muscle fibers paralyzed by the calcium-entry blocker [Schwartz, A. & Taira, N., eds. (1983) Circ. Res. 52, Part II, Number 2, 1-181.] D600 (methoxyverapamil, recently renamed gallopamil). Nonlinear charge movement was not seen in such fibers, suggesting that the drug severs the link between membrane depolarization and the main components of charge movement. This is the only pharmacological agent that blocks the main components of charge movement.

Sodium channel gating currents in frog skeletal muscle.

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.

Time domain spectroscopy of the membrane capacitance in frog skeletal muscle.

Dielectric spectra representing the frequency dependence of the complex permitivity at a range of depolarizations were obtained from voltage-clamped frog skeletal muscle membranes. This employed an analysis that derived the Fourier coefficients defining the capacitative transients to 10 mV steps as continuous functions of frequency, and so could examine closely the relevant frequencies at which non-linear components occurred. Non-linear capacitative components were identified through their appearance at lower frequencies than those of the linear components as obtained at the -85 mV control voltage, from spectra representing a logarithmic scale of frequencies. Permitivities from small depolarizing steps between about -75 and -50 mV gave single q beta dielectric loss peaks; the real permitivities declined monotonically with increasing frequency. Simple arc loci were obtained in the complex plane. With further depolarization, an additional q gamma loss peak at low frequencies and a resonant frequency in the real spectra occurred over a narrow voltage range around -45 mV. The complex loci then showed features implying an increased movement of charge not explicable through the simple effect of an electric field on a dielectric species. Spectra from small hyperpolarizing steps possessed only single dielectric loss peaks and real permitivities that declined monotonically with increasing frequency. However, in the complex plane, the loss tangents at the higher frequencies implied a population of two or more dielectric relaxations. The potential dependence of the frequency at maximum dielectric loss obtained from depolarizing steps showed a discontinuity at the onset of q gamma. In contrast, in hyperpolarizing responses, this dependence was smooth. The q beta relaxations obtained after q gamma was abolished by 1 mM-tetracaine gave dielectric spectra that were similar whether to depolarizing or hyperpolarizing potential steps. They gave single dielectric loss peaks and semicircular complex plane loci. The singularities in the dielectric spectra thus result from the q gamma charge movement component. They may reflect co-operative mechanisms that might also produce its steep voltage dependence and kinetics, and consequently those of the physiological processes it may control. These are discussed in terms of the mechanisms expected in allosteric proteins.

Pharmacological separation of charge movement components in frog skeletal muscle.

1. Charge movements to small 10 mV steps superimposed upon a wide range of closely spaced depolarizing voltage-clamp pulses were studied in frog skeletal muscles under different pharmacological conditions in hypertonic solutions.2. In control fibres, capacitance was strongly voltage-dependent, especially between potentials of -60 and -20 mV, confirming earlier work. There was a sharp increase in capacitance at around -50 mV. The dependence of non-linear charge on potential was asymmetrical and saturated at around 25 nC/muF.3. The presence of tetracaine abolished the ;hump' in the non-linear transients, which became simple monotonic decays. The dependence of capacitance upon potential was reduced. The maximum available amount of non-linear charge fell to 10 nC/muF.4. The presence of lidocaine abolished both the ;hump' as well as the monotonic part of the non-linear transients. This resulted in capacitance falling with depolarization from -85 mV.5. Comparing the steady-state properties of the non-linear charge under the different pharmacological conditions made it possible to deduce empirically the following components:(i) A lidocaine-resistant component (q(alpha)), which was responsible for the fall in observed capacitance with depolarization from the control voltage.(ii) A component resistant to tetracaine yet abolished by lidocaine (q(beta)). This possesses quasi-exponential kinetics, and a maximum charge of about 20 nC/muF.(iii) A component abolished by both lidocaine and tetracaine (q(gamma)), which possesses a maximum charge of 15 nC/muF. This has complex kinetics, and its steep dependence upon voltage resembles the potential-dependence of the development of tension in skeletal muscle.

Effects of local anaesthetics on the relationship between charge movements and contractile thresholds in frog skeletal muscle.

1. Charge movements to 10 mV steps at different potentials were studied in voltage-clamped frog skeletal muscle fibres in isotonic solutions that minimized ionic currents, under different pharmacological conditions. The earliest onset of detectable mechanical movement ('threshold') was assessed visually under magnification. 2. Charge movements in isotonic solutions were similar to those reported in hypertonic solutions, under identical pulse procedures. 3. In the absence of local anaesthetics, threshold occurred at a mean membrane potential of -55 mV, after the movement of 4.0 nC/muF of non-linear charge and when the membrane capacitance approximated values that corresponded to the onset of the 'Qgamma' charge movement component. 4. Lidocaine shifted the threshold in the hyperpolarizing direction to -62 mV, and reduced the amount of non-linear charge needed to reach threshold to 1.4 nC/muF. 5. The presence of tetracaine shifted threshold in the depolarizing direction to -39 mV, and more than doubled the amount of non-linear charge that had to move to reach threshold to 10.8 nC/muF. Much of this increase was attributed to the Qgamma component of charge movement. 6. It is concluded that more non-linear charge is required to initiate mechanical movement when calcium release by sarcoplasmic reticulum is inhibited by tetracaine, whereas less charge is so required when calcium re-uptake is being inhibited by lidocaine. Assuming earlier interpretations of the strength duration curve, this is consistent with charge movement preceding, rather than being a consequence of calcium release.

Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin

A voltage clamp technique was used to study sodium currents and gating currents in squid axons internally perfused with the membrane impermeant sodium channel blocker, QX-314. Block by QX-314 is strongly and reversibly enhanced if a train of depolarizing pulses precedes the measurement. The depolarization-induced block is antagonized by external sodium. This antagonism provides evidence that the blocking site for the drug lies inside the channel. Depolarization-induced block of sodium current by QX-314 is accompanied by nearly twofold reduction in gating charge movement. This reduction does not add to a depolarization-induced immobilization of gating charge normally present and believed to be associated with inactivation of sodium channels. Failure to act additively suggests that both, inactivation and QX-314, affect the same component of gating charge movement. Judged from gating current measurement, a drug-blocked channel is an inactivated channel. In the presence of external tetrodotoxin and internal QX-314, gating charge movement is always half its normal size regardless of conditioning, as it QX-314 is then permanently present in the channel.

Charge movement and membrane capacity in frog muscle.

1. The transient current required to impose a step charge of potential has a complex time course especially in the region of internal potential between -50 and -40 mV. 2. Examination of non-linear transient current in this voltage range suggests two components of charge movement: (a) an initial more-or-less exponential movement, and (b) a slower component with a complex time course. 3. Measurements of membrane capacity support such a division and confirm the steeper voltage dependence of the slower charge movement. 4. Permanent depolarization to 40 mV appears to immobilize the slowly moving charge. Depolarization to -20 mV immobilizes both charge movements, and uncovers the presence of a third charge which seems to correspond to Charge 2 (cf. Adrian & Almers, 1976b; Adrian, Chandler & Rakowski, 1976).