Use-dependent block of sodium current by ethmozin in voltage-clamped internally perfused canine cardiac Purkinje cells

Block of sodium current (INa) by ethmozin (moricizine), an antiarrhythmic drug, was investigated in isolated, voltage-clamped, canine cardiac Purkinje cells. Initial block of INa by ethmozin (2 microM) in noninactivated cells (held at -150 mV) was 9.3 +/- 1.2% (S.D.). Additional "use-dependent" block developed in response to repetitive depolarization. This block was both frequency-dependent and dose-dependent with the fall in peak INa greater at increasing depolarization frequencies (0.625 to 4 Hz) and with increasing dose (2 microM to 20 microM). Use-dependent block was modeled according to the guarded receptor hypothesis assuming ingress to the channel binding site during the open state of the channel, and egress from the channel independent of the kinetic state of the channel. The rate constants (on-rate = 2100 +/- 100 (S.D.)/M/ms and off-rate = 1.7 +/- 0.3 (S.D.) 10(-5)/ms) were used to predict the time course of INa block in response to repeated depolarizations and the dose-response relationship of steady-state used-dependent block measured in independent experiments. We conclude that ethmozin blocks INa in Purkinje cells in both a non-use-dependent and a use-dependent manner and that the guarded receptor model is useful in describing the use-dependent block.     

Quinidine blocks cardiac sodium current after removal of the fast inactivation process with chloramine-T.

To determine if the fast sodium current inactivation process is necessary for sodium current (INa) blockade by quinidine, we studied the effects of quinidine on INa in guinea-pig ventricular myocytes treated with chloramine-T, which removes the fast inactivation process of INa. Following exposure to chloramine-T (2 mM), INa amplitude was reduced at all voltages and INa decay was irreversibly prevented. Quinidine (10 microM) produced resting block of INa of 36 +/- 2% (n = 5) at the peak potential of -30 mV in chloramine-T treated myocytes. Quinidine decreased INa in a dose-dependent manner. The half-blocking concentration (KD) was 1.9 +/- 0.2 x 10(-5) M (n = 4). The steady-state inactivation curve (hx) was shifted in the negative potential direction (-5.2 +/- 0.4 mV, n = 4). Even after removal of the fast inactivation process of INa, use-dependent block was observed in the presence of quinidine when various depolarizing pulse durations (5 ms approximately 200 ms) were applied repetitively at intervals of 300 ms approximately 2 s. Longer depolarizing pulses and higher frequency pulse trains produced greater use-dependent block. Use-dependent block was also enhanced at more positive holding potentials. These results suggest that quinidine produces both resting block and use-dependent block of sodium channels in the absence of the fast INa inactivation process.     

The cellular electropharmacology of mexiletine in papillary muscles of guinea pigs chronically treated with amiodarone.

OBJECTIVE: The combination of mexiletine and amiodarone has proved useful in the control of serious ventricular arrhythmias, but the electrophysiological basis for their effectiveness in combination is unknown. The objective of this study was to compare the effects of mexiletine on action potential parameters of papillary muscles taken from guinea pigs chronically treated with amiodarone, with tissue taken from a control group. DESIGN: The effects of 12.5 to 200 microM mexiletine on action potential parameters of papillary muscles taken from guinea pigs chronically treated with amiodarone were compared with tissue taken from a control group, at frequencies of 1.0, 1.5 and 3.0 Hz, using standard microelectrode techniques. Onset of use-dependent block was assessed by 30 beat trains, and recovery from block by extrastimuli at diastolic intervals ranging from 200 to 5000 ms at both 1.5 and 3.0 Hz. ANIMALS: Eighteen four-week-old guinea pigs were randomly divided into two groups. One group received amiodarone in a loading dose of 20 mg/kg/day by intraperitoneal injection for one week, followed by 10 mg/kg/day for 15 weeks. The control animals were given equivalent volumes of dextrose intraperitoneally for 16 weeks. MAIN RESULTS: Mexiletine depressed the maximum rate of depolarization of phase 0 of the action potential (Vmax) in a concentration- and use-dependent fashion. Whereas chronic amiodarone treatment did not alter steady-state Vmax values, the extent of tonic Vmax depression induced by mexiletine was decreased, while use-dependent depression was increased. Mexiletine combined with amiodarone increased effective refractory period prolongation from 306.7 +/- 27.2 to 348.8 +/- 37.2 ms, while action potential duration shortening of mexiletine was moderated from 134.0 +/- 19.8 to 151.0 +/- 8.3 ms (to 90% repolarization at 3 Hz in the presence of 200 microM mexiletine).     

Evidence for developmental changes in sodium channel inactivation gating and sodium channel block by phenytoin in rat cardiac myocytes.

The voltage-dependent properties of the voltage-activated sodium channel were studied in neonatal (1-2-day-old) and adult rat ventricular cardiac myocytes using the whole-cell variation of the patch-clamp technique (16 degrees C, [Na]i = 15 mM, [Na]o = 25 mM). The voltage dependence of the sodium conductance-membrane potential relation was similar in both neonatal and adult myocytes except for a difference in slope; the adult sodium conductance-membrane potential relation was slightly more steep. Neonatal cells also differed from adult cells by demonstrating a more negative voltage midpoint of their sodium availability curve, a slower rate of recovery from inactivation at hyperpolarized potentials, and a greater extent of slow inactivation development compared with adult cells. Phenytoin (40 microM) reduced the sodium current in a tonic and use-dependent manner in both adult and neonatal myocytes. However, phenytoin (40 microM) produced significantly more tonic block at negative holding potentials (e.g., -140 mV) in neonatal myocytes (22 +/- 5% [mean +/- SEM], n = 14) than in adult myocytes (10 +/- 2%, n = 11) (p less than 0.05). The amplitudes of use-dependent block obtained during trains of 1-second pulses to -20 mV were also significantly greater in neonatal myocytes than in adult myocytes when the diastolic interval was varied over a range of 0.1-1.5 seconds (p less than 0.05). Definition of the time courses of block development at -20 mV indicated that phenytoin had a slightly higher affinity for inactivated sodium channels in neonatal cells. In addition, the time constant of recovery from use-dependent block by phenytoin was found to be significantly longer in neonatal cells than in adult cells at membrane potentials between -160 and -100 mV (p less than 0.001). The marked differences in phenytoin effect on cardiac sodium channels in neonatal versus adult rat cardiac myocytes suggest that there may be significant developmental changes in the sodium channel blocking effects of class I antiarrhythmic drugs in cardiac tissue.     

Blockage of the sodium current in isolated single cells from rat ventricle with mexiletine and disopyramide

The blocking effects of local anesthetics, mexiletine and disopyramide on the sodium currents (INa) of enzymatically isolated, single cells from rat ventricle were studied under voltage clamp conditions. A suction pipette technique was used for voltage clamp and internal perfusion. Potassium currents were blocked by replacing K+ with Cs+ in the internal and external solutions; calcium currents were blocked by replacing Ca2+ with Co2+ in the external solution to isolate INa. When the cells were stimulated infrequently (less than 1 Hz), both drugs produced dose-dependent depression of INa, which was correlated with one-to-one binding to sodium channel. A half-blocking concentration (KD) of 2.8 X 10(-5) M was observed for both agents. The shape of the current-voltage curve along the voltage axis remained unchanged in the presence of either drug. Both drugs shifted the inactivation curve of INa to more negative potentials. Mexiletine produced a marked use-dependent blockage of INa, whereas disopyramide did not produce significant use-dependent block under similar experimental conditions. Both drugs prolonged the recovery of INa from inactivation. The results suggested that both drugs interact with the inactivation mechanism of the sodium channels of rat myocardial cells.     

Two components of use-dependent block of Na+ current by disopyramide and lidocaine in guinea pig ventricular myocytes.

We studied the kinetics of the use-dependent block of the Na+ current (INa) by disopyramide and lidocaine. INa was recorded from isolated guinea pig ventricular myocytes by using the whole-cell patch-clamp technique. The use-dependent block of INa by disopyramide with 20- and 200-msec depolarizing pulses developed in two exponential functions. The degree of the use-dependent block and the amplitude of the fast (Af) and slow (As) components with the short (20-msec) pulse protocol were comparable to those with the long (200-msec) pulse protocol. When pH was raised from 7.3 to 8.0, disopyramide increased Af without a change in As. At pH 6.5, INa block developed with a single exponential function revealing only the slow component. The fast and slow components of INa block by disopyramide could be explained by binding of the uncharged and charged forms, respectively, to the activated state of the channel. Development of INa block by lidocaine also was expressed by two exponentials at all pulse durations (5-200 msec). As pulse durations were prolonged or holding potentials were depolarized, the degree of the use-dependent block and Af increased. When pH was lowered to 6.5, the short pulse produced only the slow component, whereas the long pulse caused two exponentials with decreased Af and increased As. Internal application of QX-314, a permanently charged lidocaine analogue, produced a single exponential block of INa with a very slow onset rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for voltage-dependent block of cardiac sodium channels by tetrodotoxin

The effects of tetrodotoxin (TTX) on cardiac sodium channels in guinea-pig ventricular muscle were investigated. Membrane potential was controlled using a single sucrose gap voltage clamp method, and the maximum upstroke velocity of the ventricular action potential (Vmax) was used as an indicator of drug-free sodium channels. Reduction of Vmax by TTX was found to be both voltage- and time-dependent, similar to the effects of many local anesthetic drugs, with the exception that TTX concentrations high enough to produce significant use-dependent block (e.g. 2 microM), also produced significant tonic block, even at potentials negative to -85 mV. The mechanism underlying use-dependent block was determined by defining the time course of block development at potentials between -40 and +20 mV, and the time course of recovery at -85 mV. In 2 microM TTX, the time course of block development at +20 mV contained two phases, a fast phase (tau less than 3 ms) having a mean amplitude of 8.1 +/- 3.2% of control Vmax, and a slow phase (tau = 429 +/- 43 ms) having an amplitude of 35 +/- 2% of control Vmax (n = 5). Recovery from use-dependent block at -85 mV occurred with a time constant of 324 +/- 58 ms (n = 5). The effects of TTX could be well-described by a modulated receptor model with an estimated 12 mV drug-induced shift of inactivation, and state-dependent dissociation constants of 10, 4 and 0.3 microM for rested, activated and inactivated channels. These same drug rate constants could also be used to adequately simulate the reported effects of TTX on plateau sodium currents in a variant model with slow inactivation kinetics.     

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.     

Voltage- and use-dependent modulation of cardiac calcium channels by the dihydropyridine (+)-202-791

The modulation of L-type voltage sensitive calcium channels in isolated guinea pig ventricular myocytes by the dihydropyridine (+)-202-791 was examined with the whole-cell voltage-clamp technique with 1.8 mM Ba or Ca as the charge carrier. Striking voltage- and use-dependent effects of the dihydropyridine calcium channel "agonist" (+)-202-791 were revealed. From a holding potential of -60 mV, depolarizing test pulses in the presence of (+)-202-791 demonstrated a concentration-dependent (EC50, 177 nM) increase in the measured peak inward barium current compared to control. In contrast, more depolarized holding potentials (greater than or equal to -30 mV) (+)-202-791 caused a biphasic effect on the peak inward current resulting in a transient enhancement followed by a steady-state block. A saturable, concentration-dependent hyperpolarizing shift in the voltage dependence of current inactivation was observed in the presence of (+)-202-791 with an EC50 of 10.2 nM. The voltage dependence of current activation was also shifted in the hyperpolarizing direction in the presence of (+)-202-791. A use-dependent relative block by (+)-202-791 was observed after repetitive depolarizing test pulses at a frequency of 2 Hz. Thus, the single enantiomer (+)-202-791 can result in either an increase in the whole cell calcium channel current (favored by hyperpolarized holding potentials and low rates of stimulation) or block of calcium channel current (favored by depolarized holding potentials and high rates of stimulation). Various combinations of (-)-202-791, a reported calcium channel antagonist, and (+)-202-791 resulted in intermediate effects on voltage sensitive calcium or barium currents compared with the presence of either enantiomer alone, and no clear cooperative interactions between the enantiomers were observed in contrast to a previous single channel study (Kokuban S, Prod'ham B, Becker C, Porzig H, Reuter H: Studies on Ca channels in intact cardiac cells: Voltage-dependent effects and cooperative interaction of dihydropyridine enantiomers. Mol Pharmacol 1986;30:571-584). The results are discussed in relation to the possible presence of multiple dihydropyridine receptors associated with the voltage sensitive calcium channel.     

Block of sodium current by heptanol in voltage-clamped canine cardiac Purkinje cells.

Heptanol blocks sodium current (INa) in nerve, but its effects on cardiac INa have not been well characterized. Block of INa by heptanol was studied in 16 internally perfused voltage-clamped cardiac Purkinje cells at reduced Na+ (45 mM outside, 0 mM inside). Heptanol block of peak sodium conductance was well described by a single-site binding curve with half block at 1.3 mM (20 degrees C) and showed no "use dependence." With 1.5 mM heptanol, block increased slightly by 0.7%/degrees C from 10 degrees C to 27 degrees C. With 3.0 mM heptanol, steady-state availability shifted by 9.4 +/- 1.3 mV (n = 6) in the hyperpolarizing direction, and steady-state activation shifted by 8.3 +/- 2.2 mV (n = 5) in the depolarizing direction, thus closing off the INa "window current." Heptanol also decreased the time to peak and accelerated the decay of INa. Similar results were found with octanol at lower concentrations. These alcohols have important effects on cardiac INa at concentrations used in studies for cellular uncoupling in heart.     

Blockade of cardiac sodium channels by lidocaine. Single-channel analysis

The mechanism of interaction of lidocaine with cardiac sodium channels during use-dependent block is not well defined. We examined the blockade of single cardiac sodium channels by lidocaine and its hydrophobic derivative RAD-242 in rabbit ventricular myocytes. Experiments were performed in cell-attached and inside-out patches. Use-dependent block was assessed with trains of ten 200-msec pulses with interpulse intervals of 500 msec and test potentials of -60 to -40 mV. Single-channel kinetics sometimes showed time-dependent change in the absence of drug. During exposure to 80 microM lidocaine, use-dependent block during the trains was associated with a decrease in the average number of openings per step. At -60 mV, mean open time was not significantly changed (control, 1.4 +/- 0.6 msec; lidocaine, 1.2 +/- 0.3 msec, p greater than 0.05). Greater block developed during trains of 200-msec pulses compared with trains of 20-msec pulses at the same interpulse interval at test potentials during which openings were uncommon later than 20 msec (-50 and -40 mV). Prolonged bursts of channels showing slow-gating kinetics were observed both in control and the presence of 80 microM lidocaine. However, lidocaine may decrease the late sodium current by altering the kinetics of slow gating. The hydrophobic lidocaine derivative RAD-242, which has a 10-fold greater lipid solubility than lidocaine, decreased the peak averaged current during pulse train stimulation by 60% without a change in the mean open time. Our results suggest that the major effect of lidocaine during use-dependent block involves the interaction with a nonconducting state of the sodium channel followed by a failure to open during subsequent depolarization.     

Cell-attached patch clamp measurement of macroscopic rapid inward sodium current in cultured heart cell reaggregates

A megaohm seal, cell-attached patch clamp technique utilizing plastic suction pipettes for measuring macroscopic rapid inward sodium current (INa) was applied to cell membrane patches 30 to 100 micron2 in area at the periphery of reaggregates of myocardial cells from newborn rats cultured for 3 to 7 days. The reaggregates were composed of about 20 to several thousand of electrically well-coupled cells, the latter forming spheroidal reaggregates with a diameter of maximally 300 microns. This system was shown to guarantee satisfactory voltage-clamp control during ionic current measurements. The time and voltage dependence of the INa recorded during periods of up to 90 min was similar to that observed in single cardiac cell preparations from adult rats. INa inactivation was described by two time constants (tau h1, and tau h2). For maximal INa tau h1 and tau h2 had values of 1.5 +/- 0.25 ms and 8.7 +/- 3.9 ms (n = 4), respectively. The time course for recovery of INa from inactivation at the reaggregate resting potential exhibited also two time constants (tau re1 = 9.8 +/- 3 ms, tau re2 = 193 +/- 50 ms, n = 5). Estimated current density was about 10 pA/micron2. The concentration of tetrodotoxin needed to reduce maximal INa by 75% was 85 times lower in the reaggregates than it was in freshly isolated heart muscle cells from adult rats. The present system offers a combination of features that should make it well-suited for the study of both short- and long-term effects on the sodium channels in neonatal heart cells: Easy handling of the object, great electric stability, high time and amplitude resolution during ionic current measurements, and viability for at least one week.     

Action potentials that mimic fibrillation activate sodium current.

Ventricular fibrillation (VF) has brief action potentials (50-70 ms) with short diastolic intervals (10-30 ms). Under these conditions ion channel activity may be grossly different to normal sinus rhythm (NSR). In particular, sodium channel activation may not contribute to the generation and propagation of action potentials during VF. This study determined if sodium channels can be activated when action potentials mimic VF. Isolated chick ventricular myocytes (n=7) were voltage-clamped to quantitate fast inward sodium current. The voltage clamp protocol simulated VF with a 10 pulse train at 10 Hz (100 ms cycle length (CL)) and depolarization interval (action potential duration) ranging from 90 to 20 ms. After each train a test pulse was delivered from holding (-80 mV) in 10-ms steps. The train preceded each step pulse. Peak sodium current for control and each VF protocol occurred at a membrane potential (V(m)) of -10 mV. Sodium current was evident during brief resting intervals as short as 20 ms, albeit 10-20% of baseline. Resting intervals less than 60 ms shifted the sodium conductance activation curve from Vm(0.5)-30 mV to -22 mV membrane potential. Similar findings occurred when resting potential was at -65 mV, although there was less sodium current with all tested protocols. There was significantly less inactivation of sodium current when the prepulse was shorter (100 v 1000 ms). There was approximately 20% greater sodium current when the test pulse followed a short v long depolarized (>-80 mV) prepulse. Although the longer depolarization pulses produce approximately 20% greater sodium current at membrane potentials more negative than -80 mV. Lastly the time for half recovery of sodium current from activation was significantly less when the inactivating prepulse was short v long (45.9+/-9 v 118+/-20 ms, P<0.05). In conclusion, sodium current is evident when the diastolic rest interval is as brief as 10-20 ms. Rest interval, length of membrane depolarization and membrane potential interact to affect sodium channel activation, inactivation and recovery from inactivation. These data demonstrate that the brief action potentials at more depolarized membrane potentials seen during VF allow for inward sodium current upon depolarization, less sodium channel inactivation, and a faster recovery from inactivation, thereby compensating for a short diastolic rest interval. Therefore, it is likely that the inward sodium channel contributes to wave front propagation during ventricular fibrillation.     

The cellular electropharmacology of mexiletine combined with sotalol in porcine papillary muscle and Purkinje fibre. I. Alteration of mexiletine-induced Vmax depression

Using standard microelectrode techniques, the authors compared the effects of 15 to 125 microM concentrations of mexiletine, 31 to 500 microM concentrations of sotalol and 15 to 125 microM of mexiletine combined with 125 microM sotalol, on the beat-to-beat maximum rate of depolarization of phase 0 of the action potential (Vmax) of porcine papillary muscles and Purkinje fibres stimulated by 30 beat trains at a frequency of 1 Hz. Sotalol alone had no effect on Vmax. Mexiletine caused both tonic and use-dependent depression of Vmax in papillary muscle. In the presence of sotalol, tonic Vmax depression was exaggerated, while use-dependent depression was attenuated. In Purkinje fibres, mexiletine exposure resulted in tonic Vmax depression, but no use-dependence could be demonstrated at this frequency. These results are best explained in the context of the modulated receptor hypothesis with the added consideration of two receptors--one within and one external to the sodium channel.     

The effects of mexiletine on action potential duration and its restitution in guinea pig ventricular muscles

To clarify the mechanism of mexiletine-induced changes in action potential duration (APD), we studied the effects of mexiletine (2 micrograms/ml) on APD at 0 mV (APD0mV) and 90% (APD90%) repolarization and on restitution of premature responses in guinea pig ventricular muscle at three extracellular potassium concentrations [( K]0) and three stimulation rates using standard microelectrode techniques. The rates at which APD0mV and APD90% were shortened by mexiletine (S-APD0mV and S-APD90%) expressed as percent change from control were most pronounced at [K]0 = 5.4 mM. The percent changes at the three concentrations were 5.0 +/- 2.1% at a [K]0 of 2.7 mM, 6.0 +/- 3.2% at 5.4 mM and 1.8 +/- 1.2% at 10.0 mM for S-APD0mV and 2.0 +/- 1.9%, 4.7 +/- 2.0% and 1.3 +/- 1.5% for S-APD90% at the same concentrations, respectively. S-APD0mV and S-APD90% were more markedly affected when stimulated at a frequency of 1 Hz than when stimulated at 0.2 or 0.5 Hz. Mexiletine failed to produce any additional APD shortening beyond that produced by the introduction of tetrodotoxin (2.5 X 10(-6) M). Mexiletine and tetrodotoxin did not influence APD restitution that fitted a single exponential curve. We conclude that the shortening of APD by mexiletine results from inhibition of a tetrodotoxin-sensitive sodium current.     

Interactions of flecainide with guinea pig cardiac sodium channels. Importance of activation unblocking to the voltage dependence of recovery

Effects of flecainide, a potent antiarrhythmic agent, on sodium channel availability was investigated in guinea pig single cardiac cells by the whole-cell voltage-clamp technique. Sodium current (INa) experiments were performed at 17 degrees C, and maximum upstroke velocity (Vmax) experiments were performed at 37 degrees C. Flecainide (3 microM) caused little tonic block, but reduced sodium channel availability in a use-dependent manner. The latter effect was accentuated by depolarization and attenuated by hyperpolarization. Long (200-msec) and short (10-msec) depolarizations yielded similar use-dependent block. These results indicate that flecainide has a low affinity for rested (R) and inactivated (I) channels but a high affinity for activated ones (A). In each of these states, the channels can bind to drug to form the corresponding RD, ID, and AD states. Recovery from flecainide block consisted of two components. An initial fast component was strongly voltage dependent: with increasing hyperpolarization, recovery developed more quickly and to a larger extent. At 17 degrees C, the mean time constant shortened from 132 +/- 81.6 msec at -120 mV to 46.9 +/- 34.1 msec at +/- 160 mV (kinetics were too fast for accurate measurement at 37 degrees C). A later slow component was largely voltage independent: at 37 degrees C, the mean time constant was 9.8 +/- 3.2 seconds at -100 mV and 10.7 +/- 3.8 seconds at -75 mV. The slow component of recovery was similarly independent of voltage at 17 degrees C. In terms of the modulated-receptor theory, our results indicate that the fast recovery depends on availability for unblocking (RD) but occurs during activation (AD----A). Indeed, when the RD state is maximized by strong hyperpolarization, activation unblock was also maximized. However, during depolarization to -100 mV, availability for activation unblock declined with a time constant of 98 +/- 12 msec (RD----ID). Therefore, the voltage-dependent fast unblocking is mostly due to priming of the RD state (RD----ID), and the voltage-independent slow unblock reflects dissociation of flecainide from closed states (RD----R and ID----I). We conclude that flecainide interacts with sodium channels preferentially in the activated state, whereas unblocking occurs via two separate pathways: activated and closed states. Furthermore, drug association with channels shifts the voltage dependence of closed-state transitions (RD in equilibrium ID) and their kinetics toward more negative potentials.     

Voltage- and use-dependent effects of lidocaine on sodium current in rat single ventricular cells

We compared the blocking effects of the local anesthetics, lidocaine and benzocaine, on the sodium current, using single rat ventricular cells to obtain further information about the voltage dependence and kinetics of local anesthetic interaction with cardiac sodium channels. We used a hybrid voltage clamp system which employed a suction pipette for passing current and internal perfusion, and a microelectrode for membrane potential measurement. Lidocaine (20 microM) and benzocaine (100 microM) produced qualitatively similar effects on sodium current when test pulses were applied infrequently. Both of these agents decreased the peak sodium current without producing a shift of the current-voltage curve. They did, however, shift the inactivation curves of sodium current to hyperpolarized potentials; the V0.5 was shifted by -9.5 mV for lidocaine and by -5 mV for benzocaine. Lidocaine produced a significant use-dependent effect that was proportional to the duration of the voltage step. Benzocaine produced only minimal use-dependent effects. The characteristics of the lidocaine block suggest that this agent binds preferentially to inactivated sodium channels and that dissociation from resting channels is voltage-dependent. The differences in lipid solubility and molecular weight between lidocaine and benzocaine may explain the differences in their use-dependent blocking effects on sodium current.     

The slow component of the delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVE: The purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (I(Ks)) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37 degrees C. Nisoldipine (1 microM) was used to block inward calcium current (I(Ca)) and E-4031 (1-5 microM) was applied to inhibit the rapid component of the delayed rectifier potassium current (I(Kr)). RESULTS: In 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of I(K) (I(Ks)). Activation of I(Ks) was slow (tau=903+/-101 ms at 50 mV, n=14), but deactivation of the current was relatively rapid (tau=122.4+/-11.7 ms at -40 mV, n=19). The activation of I(Ks) was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at -50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was -81.6+/-2.8 mV on an average which is close to the K(+) equilibrium potential suggesting K(+) as the main charge carrier. CONCLUSION: In undiseased human ventricular myocytes, I(Ks) exhibits slow activation and fast deactivation kinetics. Therefore, in humans I(Ks) differs from that reported in guinea pig, and it best resembles I(Ks) described in dog and rabbit ventricular myocytes.     

Effects of amiodarone and dronedarone on voltage-dependent sodium current in human cardiomyocytes

Effect of Dronedarone on Cardiac Na Current. INTRODUCTION: Amiodarone (AM) is a highly effective antiarrhythmic agent used in the management of both atrial and ventricular arrhythmias. Its noniodinated analogue dronedarone (SR) may have fewer side effects than AM. In this study, we compared the effects of AM and SR on the sodium current I(Na) in human atrial myocytes. METHODS AND RESULTS: INa was studied with the whole-cell, patch clamp technique. Both AM and SR induced a dose-dependent inhibition of I(Na) recorded at -40 mV from a holding potential of -100 mV. AM inhibited I(Na) by 41%+/- 11% (n = 4) at 3 microM, and by 80%+/- 7% (n = 5) at 30 microM. SR produced more potent block, inhibiting INa significantly at only 0.3 microM (23%+/- 10%, n = 4) and completely (97%+/- 4%, n = 4) at 3 microM. Both AM and SR had only moderate effects on voltage-dependent properties of I(Na) (current-voltage relationship, availability for activation) and had no effect on the current decay kinetics. CONCLUSION: Both AM and SR inhibit I(Na) significantly in single human atrial cells, showing that the two drugs have Class I antiarrhythmic properties. The acute effects of SR are more potent than those of AM. The study supports the idea that the iodinated form of the molecule has no part in the acute effect of AM on Na+ channels.     

Open-state unblock characterizes acute inhibition of I potassium current by amiodarone in guinea pig ventricular myocytes

INTRODUCTION: The aim of the present study was to investigate the acute action of amiodarone on the slow component of delayed rectifier K+ current (IKs) under basal conditions and during beta-adrenoceptor stimulation in guinea pig ventricular myocytes. METHODS AND RESULTS: Using the whole-cell patch-clamp method, IKs was evoked by depolarizing voltage-clamp steps, during superfusion with the Na+-, K+-, and Ca2+-free solution supplemented with 0.4 microM nisoldipine and 5 microM E-4031. The acute effect of amiodarone was evaluated, within approximately 10 minutes after starting the bath application, by the amplitude of deactivating tail currents at -50 mV. Amiodarone concentration dependently blocked I(Ks) and exerted a more potent effect on IKs when activated by shorter pulse durations; the degree of block by 30 microM amiodarone on IKs activated by 200 ms, 500 ms, and 2000 ms depolarizing pulses to +30 mV was 55.9 +/- 5.8%, 38.6 +/- 6.0%, and 27.1 +/- 4.0% (n = 5 each), respectively. An envelope of tails test conducted at +10, +30, and +60 mV demonstrated that the degree of IKs block by amiodarone was gradually attenuated during membrane depolarization, which can be described by a monoexponential function, thus supporting the presence of open channel unblock. Amiodarone also blocked IKs maximally stimulated by 1 microM isoprenaline, to an extent similar to control, when IKs was activated by pulse durations of < or =2000 ms. CONCLUSION: We propose that amiodarone acutely blocks native IKs with characteristics associated with open channel unblock, and that the protein kinase A-mediated phosphorylation of channel proteins only minimally affects the amiodarone block.