Search

1 The possibility that the class III antiarrhythmic drugs clofilium and d-sotalol might affect delayed rectifier potassium channels at the level of their gating currents was assessed with the whole-cell patch-clamp technique in guinea-pig isolated ventricular heart cells. 2 Clofilium (up to 20 microM) and d-sotalol (1 microM) did not decrease the Na current, the L-type Ca current or the background K current iKl, but significantly depressed the time-dependent delayed outward K current iK. 3 Clofilium partially decreased in a dose-dependent manner (1-20 microM) QON of intramembrane charge movements (ICM) elicited by a depolarizing pulse applied from a holding potential of -110 mV or following a 100 ms inactivating prepulse to -50 mV. D-sotalol (1 microM) also decreased QON. Channel density estimated from the clofilium-sensitive ICM closely matched that of the delayed rectifier channels. 4 Clofilium and d-sotalol decreased QOFF seen on repolarization in a dose- and voltage-dependent manner. The kinetics of the decay of the OFF gating currents were not affected, and only the fast phase was depressed. 5 In control conditions, QON availability with voltage was most of the time well described by two inactivating components. In the presence of clofilium and d-sotalol, a complex behaviour of QON availability was observed, unmasking additional components. The reactivation kinetics of QON after a 500 ms inactivating pulse to 0 mV was not affected. 6 We conclude that delayed rectifier K channels significantly contribute to QON and QOFF of ICM in guinea-pig ventricular heart cells, besides Na and Ca channels, and that clofilium and d-sotalol directly interact with these K channels proteins by affecting their gating properties.

1. The effect of ruthenium red on calcium and sodium currents was studied in guinea-pig isolated ventricular heart cells with the whole cell patch-clamp technique. 2. Ruthenium red very efficiently blocked the L-type calcium current in a dose-dependent manner. A significant block was observed for concentrations as low as 0.3 microM. Analysis of the dose-response curve with the logistic equation indicated an EC50 of 0.8 microM, a maximum inhibition of 85% reached at 5 microM, and a coefficient of 2.37. 3. There was no shift in the voltage-dependence of the Ca current activation, nor in that of its steady-state inactivation determined with a 1 s prepulse. However, removal of Ca current inactivation at positive voltage was considerably reduced in the presence of concentrations of ruthenium red above 1 microM. A slowing of the time-course of inactivation of the Ca current was also observed. 4. At 10 microM, a concentration generally used to block the sarcoplasmic Ca release channels or the mitochondrial Ca uptake, ruthenium red blocked 26.7+/-4.3% (n=8) of the sodium current, and slowed its inactivation time-course. No effect was observed on the voltage-dependence of the current activation or inactivation. The peak sodium current was also decreased at a 10 times lower concentration by 7.6+/-2.7% (n=3). 5. Thus, at concentrations used to assess intracellular Ca movements, ruthenium red induced in heart cells a significant block of both Ca and Na channels.

Cyclosporin A (CsA), an immunosuppressive agent used to reduce rejection after organ transplantation, induces secondary effects in heart tissue. We have studied the effects in vivo and in vitro of CsA on L-type Ca2+ current (ICa) and the associated gating currents of isolated guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. For in vivo experiments, a group of animals (n=28) was treated for 21 days by subcutaneous injection of CsA (15 mg/kg/day). Blood level of CsA was 1191+/-221 ng/ml (n=9). In cells from these animals (n=65, 19 animals), ICa was reduced to about 75% of that recorded from control cells (n=32, six animals). CsA decreased the availability of Ca2+ channels at potentials more positive than +30 mV. Isoproterenol (100 nM) was still able to increase ICa but only by 30+/-6% (n=9), whereas in control it increased ICa by 290+/-22% (n=5). Gating currents related to L-type Ca2+ channels were not altered in cells from CsA-treated animals. In in vitro experiments, CsA reduced ICa when applied directly to cardiomyocytes. CsA affected the kinetics of ICa inactivation, slowing down the rapid phase and accelerating the slow phase (n=4). The steady-state inactivation curve of ICa was shifted to more negative voltages and the degree of availability at -80 mV decreased by in vitro application of CsA. The half inactivation potential (V1/2) changed from -23+/-0.6 mV in control to -31+/-2 mV, -48+/-0.6 mV and -49+/-0.6 mV, in 1, 50 and 80 microM CsA, respectively. In these cells, the gating currents related to L-type Ca2+ channels were also not altered by CsA. CsA does not modify the Ca2+ channel density, although it induces a decrease in the beta1-adrenergic stimulation of ICa. The results are explained by a direct effect on the calcium channel inactivation of CsA and a non specific indirect effect.

The effects of the benzodiazepine midazolam were studied on frog skeletal muscle fibres held under current- or voltage-clamp conditions. Midazolam induced a concentration-dependent (10(-5) mol/l to 10(-3) mol/l) block of the action potential and of the underlying Na current. Block of the Na current occurred without any changes in its voltage dependence or in its activation and inactivation kinetics. An apparent dissociation constant of 223 mumol/l was determined for midazolam from the rested Na channels of well polarized fibres. The blocking effect of a threshold concentration (10(-5) mol/l) could be greatly enhanced (up to the complete suppression of the current) by predepolarizations, positive holding potentials or high stimulation frequencies. This apparent voltage- and frequency-dependent block (no use dependence, i.e., no activation block) could be ascribed to a blockade of inactivated Na channels. From the apparent shift towards negative potentials of the steady-state inactivation curve, a dissociation constant of 6.0 mumol/l was calculated for midazolam from the inactivated Na channels, according to the modulated-receptor model. These results show that midazolam preferentially blocks inactivated rather than rested Na channels, and suggest that this mechanism of action might contribute to the well-known myorelaxant effect of the benzodiazepines.

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.

The effects of synthetic poneratoxin (PoTX), a new toxin isolated from the venom of the ant Paraponera clavata, were studied under current- and voltage-clamp conditions in frog skeletal muscle fibres. PoTX induces a concentration-dependent (10(-9) M-5 x 10(-6) M) prolongation of the action potentials and, at saturating concentration, a slow repetitive activity developing at negative potentials. PoTX specifically acts on voltage-dependent Na channels by decreasing the peak Na current (INa) and by simultaneously inducing a slow INa which starts to activate at -85 mV and inactivates very slowly. Both the fast and the slow components of INa are suppressed by tetrodotoxin and reverse at the same potential corresponding to the equilibrium potential for Na ions. The fast component of INa has voltage dependence, activation and steady-state inactivation almost similar to those of the control INa. The voltage dependence of the slow Na conductance is 40 mV more negative than that of the fast one. The results suggest that PoTX affects all the Na channels and that the fast and the slow INa components originate from a possible PoTX-induced interconversion between a fast and a slow operating mode of the Na channels.