Reactivation Time Constant Research Articles

Isoflurane and sevoflurane modulate inactivation kinetics of Ca2+ currents in single bullfrog atrial myocytes.

To clarify the mechanism(s) of anesthetic depression of myocardial Ca2+ currents, the effects of isoflurane and sevoflurane on the inactivation kinetics of Ca2+ current in single bullfrog atrial myocytes were studied. Freshly isolated bullfrog atrial myocytes were obtained with an enzymatic dispersion procedure. Ca2+ currents were recorded with a whole-cell voltage-clamp technique. Both isoflurane (1.25, 2.5, 5.0 vol%) and sevoflurane (2.5, 5.0 vol%) decreased the peak amplitude of Ca2+ current ICa with a minimal change in the time to peak and the reversal potential. The inactivation kinetics studies revealed that (1) isoflurane (2.5 vol%) and sevoflurane (5.0 vol%) markedly reduced the time constant of inactivation in ICa to 55% and 75% of control, respectively; (2) isoflurane (2.5 vol%) shifted the midpoint (V1/2) of steady-state inactivation curve of ICa toward negative by 2.3 mV; and (3) isoflurane (2.5 vol%) delayed the reactivation time constant of ICa to 119% of control. The further computer-simulation study demonstrated that the observed decrease of time constant by isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) can explain the reduction in amplitude of ICa. The depression of ICa by lower concentration of isoflurane (1.25, 2.5 vol%) and sevoflurane (2.5 vol%) mainly is due to the decrease of time constant and, at higher concentration, isoflurane and sevoflurane may affect the other membrane components.

Proton-activated currents in chick spinal motoneurons

Proton-activated currents were examined in patch-clamp recordings from embryonic chick motoneurons. Rapid application of protons evoked a large inward current that peaked and then decayed, presumably due to channel inactivation. A pH shift from 7.4 to 7.1 was sufficient to evoke detectable currents. The shift from pH 7.4 required for half-maximal current amplitude (EC50) was to pH 6.8. In single-channel recordings, activation was achieved within 6 ms at pH 7. The average channel open time was 1.4 ms; the closed-state time constants were 1.0 and 6.2 ms. At pH 6.5, the single-channel conductance was 22 pS, and the reversal potential was similar to the calculated Na+ equilibrium potential. Current amplitude declined by 49% following addition of Ni2+ and increased by 58% as Ca2+ was lowered from 2 to 0.1 mM. Inactivation time constants ranged from 90 to 200 ms as pH varied from 6 to 7; these values did not depend on membrane potential. The reactivation time constant was 22 s. Proton- and glutamate-activated currents summated. Thus, transient decreases in extracellular pH can evoke large inward currents that decay rapidly and reactivate slowly. These currents may occur under pathological conditions that affect extracellular pH.

A macro cell-attached patch-clamp study of the properties of the Na current in the vicinity of the motor endplate region of frog single interosseal skeletal muscle fibres.

A macro cell-attached patch-clamp technique using large (16 microns) electrodes was developed to study the properties of the Na current (INa) in the motor endplate region of frog interosseal muscle fibres (resting potential approx.-99 mV). The fibre isolation procedure allows the formation of gigaOhm seals and thus a good voltage-clamp control of the patch. At 22 degrees C, in the presence of 110 mM external [Na] and of K channel blockers, INa activates at -49.6 +/- 1.9 mV, reaches a maximum of 4.33 +/- 0.66 mA/cm2 at -7.5 +/- 2.5 mV and reverses at Vrev equal to +63.2 +/- 1.9 mV. There is no evidence for the presence of a tubular INa. When the [Na] in the recording pipette is changed, Vrev exactly follows the Nernst equation for Na ions. An internal [Na] of 9.69 mM is determined. The Na conductance is maximum (63.56 +/- 7.03 mS/cm2) at +8.9 +/- 3.0 mV and markedly decreases for potentials positive to +25 mV. In contrast, the Na permeability (maximum of 6.13 +/- 0.95 x 10(-4) cm/s at +51.4 +/- 5.6 mV) remains more constant at positive potentials. The Na channels are half-inactivated at -68.9 mV and half-activated at -37.9 mV. At the potential at which INa is maximum, the half-time of activation is 223.9 +/- 10.5 microseconds, the time to peak 365.7 +/- 13.5 microseconds and the time constant of inactivation 260.7 +/- 11.2 microseconds. The time constant of reactivation at -100 mV is 1.44 +/- 0.19 ms. These and other results show that INa can be adequately studied with this technique.

Blockage of the fast sodium current by dimetindene in frog auricular fibres

The effect of dimethindene (DMI) on action potential and fast inward Na current (INa) of frog atrial fibres was studied using double sucrose gap voltage-clamp technique. DMI reduced the amplitude and maximum rate of rise of the action potentials without altering the resting membrane potential. The drug inhibited the fast Na conductance in a concentration-dependent manner, without changing the reversal potential. The shape of the current-voltage curve along the voltage axis remained unchanged in the presence of DMI. The time to peak of the INa was not significantly altered by the drug whereas the rate of the INa inactivation (tau h) was slowed. DMI shifted the steady-state inactivation curve of INa (h infinity) to more negative potentials, and increased the reactivation time constant of the sodium system (tau r). The inhibition of INa was use-dependent. The results suggest that DMI interacts with the inactivation mechanism of the sodium channel of the frog myocardium, and explain its favourable antiarrhythmic activity.

Effects of moderate hypoxia on premature action potentials of rabbit papillary muscle.

Experiments were performed to study the effects of hypoxia on the characteristics of premature action potentials of rabbit papillary muscles. At normal resting potential, the duration of the premature action potential at the shortest coupling intervals was always greater than that of the control response. As the coupling interval was increased beyond 150 ms, the duration of the premature action potential regained control values. In cells depolarized to -70 mV by KCl, early lengthening of the premature response was attenuated. After 60 min of hypoxia, recovery of action potential duration at normal and reduced resting potentials was accelerated. The maximum rate of depolarization and its reactivation time constant were not affected by 60 min of hypoxia. It is suggested that intracellular free Ca is important in the control of action potential duration via the outward background potassium current.

Characterization of the fast sodium current in isolated rat myocardial cells: simulation of the clamped membrane potential.

1. The fast sodium inward current of freshly isolated single rat myocardial cells was studied by means of the internal perfusion-voltage clamp method. 2. The voltage dependence of this current did not differ from the current-voltage characteristics of the fast sodium inward current described for other excitable cells and tissues. 3. The time constant of inactivation of the Na+ current of the isolated myocardial cells ranged between 5.2 msec at -58 mV and 0.5 msec at +18 mV. The activation time constant ranged from 0.3 msec at -55 mV to 70 microseconds at +10 mV. 4. The reactivation time constant of the maximum sodium current at a holding potential of -100 mV was found to be 21 +/- 5 msec. 5. A mathematical model was developed for the simulation and analysis of the influence of the series and shunt resistances on the time response of the membrane potential. The results of the modelling make it clear that control of the series and shunt resistances in any given experiment is a conditio sine qua non for a valid analysis of the kinetic parameters of the sodium inward current. 6. Sodium currents with delayed activation kinetics must be regarded as an indication of insufficient control of the membrane potential.

Effect of RP 30356 on the fast inward Na current in frog atrial fibres

The effect of a new drug: RP 30356, on action potential and fast inward Na current of frog atrial fibres was studied using the double sucrose gap voltage clamp technique. The drug (10 −4 M) blocked the action potential without noticeably altering the resting membrane potential. RP 30356 inhibited the fast sodium conductance without changing the selectivity of the Na channel. The time to peak of the inward current was not significantly altered by the drug whereas the rate of the Na current inactivation τ h was slowed. The steady state inactivation membrane potential relationship of the Na system was shifted toward negative membrane potentials by the drug. It was also shown that in Ringer solution the reactivation time constant of the Na system (τ re) was faster at more negative membrane potentials than at more positive ones. The drug increased τ re; the increase was more marked when the membrane was depolarized. The block of Na conductance by the drug was partially removed by increasing the membrane potential to values more negative than −70 mV. The drug did not leave the channel or left it very slowly in the absence of clamp stimulation. The inhibition of the Na conductance by RP 30356 was also frequency-dependent. The data suggest that RP 30356 might be effective in the control of cardiac arrhythmias since it mainly decreased the excitability of depolarized fibres.

Comparison of two-pulse sodium inactivation with reactivation in Myxicola giant axons

Values for the time constant of reactivation of the sodium conductance following depolarization sufficient to completely inactivate GNa have been compared over a 15 mV range of membrane potential with the time constants of inactivation during a depolarization prepulse. Over this range the reactivation time constants were consistently 30-50% larger than the inactivation time constants determined simultaneously at the same potential in the same axon. The data suggests that inactivation and reactivation do not occur by identical mechanisms, and therefore implies that there are at least three kinds of experimental procedures necessary to fully characterize the sodium inactivation process in any particular system.

Slow recovery from inactivation of inward currents in mammalian myocardial fibres.

1. Reactivation kinetics of the rapid and slow inward currents in ventricular fibres have been assessed by studying the maximum rate of rise ((dV/dt)max) of the action potential upstroke and the duration of the plateau in progressively earlier premature responses. Reactivation of the slow inward current was also studied by voltage clamp technique in sheep and pig ventricular trabeculae.2. The time constant of recovery of (dV/dt)max was voltage dependent and increased from less than 20 msec when the resting membrane potential was more negative than -80 mV to more than 100 msec when the resting membrane potential was between -65 and -60 mV. Similar results were obtained in Purkinje fibres. These results suggest that the time constant for reactivation is slower than the time constant for inactivation of the rapid inward current system by at least one order of magnitude.3. The time constant of recovery of plateau duration was also voltage dependent and increased from 30 to 70 msec as the membrane potential was changed from -85 to -60 mV.4. The reactivation time constant of the slow inward current determined by voltage clamp experiments were similar to the results obtained by analysis of plateau duration. At potentials less negative than -60 mV the time constant of reactivation became progressively longer. Unlike reactivation time constants of (dV/dt)max, the time constants of reactivation of the slow inward current were similar to the time constants of inactivation.5. Our results indicate that (a) in premature action potentials, time as well as voltage are important determinants of (dV/dt)max in myocardial and Purkinje fibres, (b) the kinetics of reactivation of the rapid inward current in cardiac fibres are different from those in nerve and (c) plateau duration of premature action potentials in ventricular myocardial fibres is largely determined by the kinetics of reactivation of the slow calcium inward current.