Amiodarone blocks calcium current in single guinea pig ventricular myocytes

Ca++ current (lca) block by amiodarone and the underlying mechanisms thereof were investigated in guinea pig single ventricular myocytes using the single suction pipette whole cell voltage clamp method. The dose-response curve revealed a 1:1 stoichiometry for binding of amiodarone to its receptor with an apparent dissociation constant of 5.8 microM in the resting state. Amiodarone, 5 microM did not significantly alter the time course of ICa decay, but did shift the steady-state inactivation curve for lca in the hyperpolarizing direction by 9.2 +/- 3.1 mV. Development of block at depolarized potentials was voltage-dependent between -20 and 10 mV with time constants of 112 +/- 33 and 755 +/- 212 msec at 10 mV. In the presence of 0.2 microM amiodarone, recovery from inactivation was fitted by a double exponential most likely indicating rapid recovery of the drug-free Ca++ channels and slow recovery of the drug-associated Ca++ channels with time constants of 44 +/- 12 and 108 +/- 403 msec, respectively, at -80 mV. The proportion of the current recovering via the slow phase was 36 +/- 7%. By using this value, we estimated the dissociation constant in the inactivated state to be 0.36 microM. Amiodarone's marked use-dependent block of lca is explicable in terms of its high affinity for, and slow dissociation from, Ca++ channels in the inactivated state. These results suggest that amiodarone blocks lca in both the resting and inactivated states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of linoleic Acid metabolites on cardiac sodium current

9,10-Epoxy-12-octadecenoic acid (EOA), a metabolite of linoleic acid, causes cardiac arrest in dogs. Other metabolites of linoleic acid also have toxic effects. This study investigates the mechanism of action of four of these compounds on cardiac Na(+) current (I(Na)). The whole-cell patch-clamp technique was used to investigate the effects of EOA, 9,10-dihydroxy-12-octadecenoic acid (DHOA), and their corresponding methyl esters (9,10-epoxy-12-octadecenoic methyl ester, EOM; and 9,10-dihydroxy-12-octadecenoic methyl ester, DHOM) on I(Na) in isolated adult rat ventricular myocytes. Extracellular application of each compound elicited a concentration-dependent inhibition of I(Na). The dose-response curve yielded 50% inhibition concentrations of 301 +/- 117 microM for DHOA, 41 +/- 6 microM for DHOM, 34 +/- 5 microM for EOA, and 160 +/- 41 microM for EOM. Although there was no effect on activation, 50 microM DHOM, EOA, and EOM significantly hyperpolarized the steady-state inactivation curve by approximately -6 mV. Furthermore, EOM significantly increased the slope of the steady-state inactivation curve. These compounds also seemed to stabilize the inactivated state because the time for recovery from inactivation was significantly slowed from a control value of 12.9 +/- 0.5 ms to 30.5 +/- 3.3, 31.4 +/- 1.4, and 20.5 +/- 1.0 ms by 50 microM DHOM, EOA, and EOM, respectively. These compounds have multiple actions on Na(+) channels and that despite their structural similarities their actions differ from each other. The steady-state block of I(Na) suggests that either the pore is being blocked or the channels are prevented from gating to the open state. In addition, these compounds stabilize the inactivated state and promote increased population of a slower inactivated state.     

Characterization of the block of sodium channels by phenytoin in mouse neuroblastoma cells

The interaction of phenytoin (DPH) with membrane ionic channels of cultured N1E-115 neuroblastoma cells was studied. The single suction pipette technique was used for voltage clamp and intracellular perfusion. When the cells were held at -80 mV for periods of 1 min or more, DPH (20-100 microM) inhibited inward sodium current in a dose-dependent manner (resting block); resting block was relieved by hyperpolarizing cells to -100 mV for 1 min. A hyperpolarizing shift of the slow inactivation curve for the Na current was induced by DPH and can explain the effect of holding potential on the resting block. The fast Na inactivation curve, however, was not affected. During repetitive pulsing the DPH-induced inhibition of Na current was enhanced (conditioned block). Conditioned block was both voltage- and frequency-dependent. Conditioning pulses to potentials which do not appreciably open Na channels also produced conditioned block; prolongation of conditioning pulses even to durations longer than the time for maximal steady-state inactivation of the Na current progressively increased the extent of conditioned block, suggesting that DPH can interact with inactivated and closed Na channels. The time course of recovery from voltage-dependent inactivation of sodium current during conditioned block was both slowed and exhibited voltage dependence. Recovery occurred faster when membrane potential during the recovery period was more negative. We conclude that DPH blocks Na channels both by increasing the fraction of channels in an inactivated state and by delaying the transition from inactivated to closed but available channels. This effect is enhanced by depolarizing membrane potential and increasing the frequency of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mibefradil on voltage-dependent gating and kinetics of T-type Ca(2+) channels in cortisol-secreting cells

We have studied the effect of the Ca(2+) antagonist mibefradil on low voltage-activated T-type Ca(2+) channels in whole-cell patch clamp recordings from bovine adrenal zona fasciculata (AZF) cells. AZF cells are distinctive in expressing only T-type Ca(2+) channels, allowing the mechanism of pharmacological agents to be explored without interference from other Ca(2+) channels. The inhibition of T-type Ca(2+) channels by mibefradil was voltage- and use-dependent. When Ca(2+) currents were activated from holding potentials of -80 and -60 mV, mibefradil inhibited currents with IC(50) values of 1.0 and 0.17 microM, respectively. When T-type Ca(2+) current (I(T)) was activated from a holding potential of -90 mV in the presence of 2 microM mibefradil, a single voltage step to -10 mV inhibited I(T) by 16.2% +/- 2.9% (n = 10). With subsequent voltage steps, applied at 10-s intervals, block reached a steady-state value of 51.9% +/- 5.0% (n = 5). Mibefradil (1 microM) produced a leftward shift of 5.7 mV (n = 4) in the voltage-dependent steady-state availability curve such that T-type Ca(2+) channels inactivated at more negative potentials, but this drug did not change the voltage-dependence of T channel opening. Mibefradil failed to alter the kinetics of T channel activation, inactivation, or deactivation, but markedly slowed T channel recovery following an inactivating prepulse. Mibefradil inhibited adrenocorticotropin-stimulated cortisol secretion from AZF cells with an IC(50) value of 3.5 microM. These results show that mibefradil is a relatively potent antagonist of T-type Ca(2+) channels in cortisol-secreting cells. The enhanced potency of mibefradil with sustained or repetitive depolarizations, its shifting of the steady-state inactivation curve, and its slowing of recovery all indicate that this drug preferentially interacts with Ca(2+) channels in the open or inactivated state. The inhibition of cortisol secretion by mibefradil at concentrations similar to those that block I(T) is consistent with a requirement for these channels in corticosteroidogenesis.     

Block of Na+ channels by imipramine in guinea-pig cardiac ventricular cells.

The effects of imipramine on the Na+ current of guinea-pig ventricular myocytes were examined by the whole-cell clamp method. Imipramine inhibited the Na+ current with a dissociation constant value of 25 microM at a -130 mV holding potential. At 1 microM, imipramine caused a negative shift of the channel availability curve by 4.0 +/- 1.03 mV with its steepness unaffected. The inactivation time constants were not changed by 30 microM imipramine. Paired pulse experiments revealed that imipramine binds to the inactivated Na+ channels with time constants of 3.7 +/- 0.27 sec at -65 mV and 2.4 +/- 0.58 sec at -20 mV, and that it dissociates from the channels with time constants of 5.9 +/- 1.05 sec at -90 mV and 2.0 +/- 0.87 sec at -130 mV. From these paired pulse experiments, the dissociation constant for the interactions between imipramine and inactivated channels was calculated to be 0.67 microM, a value within its therapeutic plasma concentration. These slow interactions of imipramine with inactivated Na+ channels resulted in a slow onset of the frequency-dependent extrablock in the effects of imipramine on the Na+ current. Consequently, the imipramine-induced extrablock sufficient to terminate re-entrant tachyarrhythmias would not develop shortly after their initiation. Short depolarizations of 1- to 3-msec duration sustained appreciable extra blockage when a high concentration of 10 microM imipramine was used, or they were repeatedly applied at a high frequency. However, access of imipramine to the open channels seems to play a minor role in the drug-channel interactions.     

Direct block by bisindolylmaleimide of rat Kv1.5 expressed in Chinese hamster ovary cells

The interaction of bisindolylmaleimide (BIM), widely used as a specific protein kinase C (PKC) inhibitor, with rat brain Kv1.5 (rKv1.5) channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. BIM (I) and its inactive analog, BIM (V), inhibited rKv1.5 currents at +50 mV in a reversible concentration-dependent manner with an apparent K(d) value of 0.38 and 1.70 microM, respectively. BIM (I) accelerated the decay rate of inactivation of rKv1.5 currents but did not significantly modify the kinetics of current activation. Other specific PKC inhibitors, chelerythrine and PKC 19-36, had no effect on rKv1.5 and did not prevent the inhibitory effect of BIM (I). The inhibition of rKv1.5 by BIM (I) and BIM (V) was highly voltage-dependent between -30 and 0 mV (voltage range of channel opening), suggesting that both drugs interact preferentially with the open state of the channel. The additional inhibition by BIM (I) displayed a voltage dependence (delta = 0.19) in the full activation voltage range positive to 0 mV, but was not shown in BIM (V) (delta = 0). The rate constants of association and dissociation for BIM (I) were 9.63 microM(-1) s(-1) and 5.82 s(-1), respectively. BIM (I) increased the time constant of deactivation of tail currents from 26. 35 to 45.79 ms, resulting in tail crossover phenomenon. BIM (I) had no effect on the voltage dependence of steady-state inactivation. BIM (I) produced use-dependent inhibition of rKv1.5, which was consistent with the slow recovery from inactivation in the presence of drug. These results suggest that BIM (I) directly inhibits rKv1.5 channels in a phosphorylation-independent, and state-, voltage-, time-, and use-dependent manner.     

Effects of irbesartan on cloned potassium channels involved in human cardiac repolarization

We studied the effects of irbesartan, a selective angiotensin II type 1 receptor antagonist, on human ether-a-go-go-related gene (HERG), KvLQT1+minK, hKv1.5, and Kv4.3 channels using the patch-clamp technique. Irbesartan exhibited a low affinity for HERG and KvLQT1+minK channels (IC(50) = 193.0 +/- 49.8 and 314.6 +/- 85.4 microM, respectively). In hKv1.5 channels, irbesartan produced two types of block, depending on the concentration tested. At 0.1 microM, irbesartan inhibited the current in a time-dependent manner (22 +/- 3.9% at +60 mV). The blockade increased steeply with channel activation increasing at more positive potentials. However, at 10 microM, irbesartan induced a time-independent blockade that occurred in the range of potentials of channel opening, reaching its maximum at approximately 0 mV, and remaining unchanged at more positive potentials (24.0 +/- 1.0% at +60 mV). In Kv4.3 currents, irbesartan produced a concentration-dependent block, which resulted in two IC(50) values (1.0 +/- 0.1 nM and 7.2 +/- 0.6 microM). At 1 microM, it inhibited the peak current and accelerated the time course of inactivation, decreasing the total charge crossing the membrane (36.6 +/- 7.8% at +50 mV). Irbesartan shifted the inactivation curve of Kv4.3 channels, the blockade increasing as the amount of inactivated channels increased. Molecular modeling was used to define energy-minimized dockings of irbesartan to hKv1.5 and HERG channels. In conclusion, irbesartan blocks Kv4.3 and hKv1.5 channels at therapeutic concentrations, whereas the blockade of HERG and KvLQT1+minK channels occurred only at supratherapeutic levels. In hKv1.5, a receptor site is apparent on each alpha-subunit of the channel, whereas in HERG channels a common binding site is present at the pore.     

Characterization of mibefradil block of the human heart delayed rectifier hKv1.5

The goal of this study was to analyze the effects of mibefradil on a human cardiac K(+) channel (hKv1.5) stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Mibefradil inhibited in a concentration-dependent manner the hKv1.5 current with a K(D) value of 0.78 +/- 0.05 microM and a Hill coefficient of 0.97 +/- 0.06. Block induced by mibefradil was voltage dependent, consistent with a value of electrical distance of 0.13. The apparent association (k) and dissociation (l) rate constants measured at +50 mV were found to be 7.3 +/- 0.5 x 10(6) M(-1).s(-1) and 4.3 +/- 0.1 s(-1), respectively. Block increased rapidly between -20 and +10 mV, coincident with channel opening and suggested an open channel block mechanism, which was confirmed by a slower deactivation time course resulting in a "crossover" phenomenon when tail currents recorded under control conditions and in the presence of mibefradil were superimposed. Shifts toward negative potentials of the maximum conductance and the activation curve were observed, confirming the voltage dependence of block. Mibefradil induced a significant use-dependent block when trains of depolarization at frequencies between 0.02 and 2 Hz were applied. In the presence of mibefradil, recovery of inactivation was faster than under control conditions, suggesting that mibefradil might compete with the inactivation gate of hKv1.5. These results indicate that mibefradil blocks hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutic range.     

Recovery from use-dependent block of Vmax and restitution of action potential duration in canine cardiac Purkinje fibers

The recovery kinetics during diastole of various plateau currents are thought to control the restitution of action potential duration (APD). Based on the assumption that the recovery of the residual plateau Na current parallels that of Vmax, the hypothesis that Na current recovery kinetics influence the restitution of APD was tested. Drugs that reduced Vmax in a use-dependent manner (tetrodotoxin 3 microM, lidocaine 15 microM, mexiletine 20 microM) were compared with interventions that reduced Vmax in a simply tonic fashion [( Na]o 75 mM, [K]o 6.5 mM, disopyramide 30 microM). Microelectrode techniques and programmed stimulation were used to determine in vitro the kinetics of restitution of APD and of time-dependent recovery of Vmax during rest. Tetrodotoxin, lidocaine and mexiletine induced a blockade of Vmax that showed partial or full time-dependent unblocking in accordance with the known use dependence of their blocking action. Dissipation of the time-dependent component of the block in each case followed a single exponential time course, time constants being 163 +/- 12, 115 +/- 12 and 121 +/- 20 ms, respectively. Analysis of the kinetics of the APD restitution curves showed that the time constant of the fast decaying exponential component of restitution (T1) was prolonged by these drugs from 129 +/- 5 in control fibers to 295 +/- 17, 235 +/- 11 and 242 +/- 26 ms for tetrodotoxin, lidocaine and mexiletine, respectively (P less than .05). Low [Na]o and disopyramide reduced Vmax in a simply tonic fashion and did not significantly prolong the T1 component of APD restitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of L-type calcium channel blockade by nifedipine and/or cadmium in guinea pig ventricular myocytes

We tested the assumption that nifedipine blocks L-type calcium current [I(Ca(L))] at +10 mV and unmasks Na(+)/Ca(2+) exchange-triggered contractions in guinea pig isolated ventricular myocytes. Voltage-clamp pulses elicited I(Ca(L)) at +10 mV and evoked contractions in myocytes superfused with Tyrode's solution (35 degrees C). Nifedipine blocked I(Ca(L)) with an IC(50) of 0.3 microM; this decreased to 50 nM at a holding potential of -40 mV, indicating preferential block of inactivated L-type Ca(2+) channels. Use-independent block of I(Ca(L)) increased with concentration (10-100 microM) and application time when nifedipine was rapidly applied (t(1/2) = approximately 0.2 s) during rest intervals (5-30 s). The fraction of use-dependent block of I(Ca(L)) diminished with increasing drug concentration. Nifedipine also accelerated I(Ca(L)) inactivation on the first test pulse. The combination of 30 microM nifedipine/30 microM Cd(2+) (Nif 30/Cd 30) was as effective as 100 microM nifedipine to suppress I(Ca(L)) on the first test pulse at +10 mV. The incidence of complete block of contractions, as for complete block of I(Ca(L)), increased as a function of nifedipine concentration and application time. Neither nifedipine nor Nif 30/Cd 30 affected Na(+)/Ca(2+) exchange current at +10 to +100 mV. Contractions at +100 mV, although as large as those at +10 mV, were delayed in onset and resistant to nifedipine or Nif 30/Cd 30. We conclude that nifedipine-sensitive I(Ca(L)) triggers contractions at +10 mV, whereas nifedipine-resistant Na(+)/Ca(2+) exchange current initiates those at +100 mV.     

Neuroprotective agent riluzole dramatically slows inactivation of Kv1.4 potassium channels by a voltage-dependent oxidative mechanism

The modulation of Kv1.4 K+ channels by the neuroprotective agent riluzole was studied in bovine adrenal zona fasciculata cells by using whole-cell patch clamp. At concentrations ranging from 1 to 100 microM, riluzole reversibly inhibited Kv1.4 channels (IC50 = 70 microM) and irreversibly slowed Kv1.4 inactivation. Riluzole (100 microM) increased the inactivation time constant (tau(i)) from a control value of 28.9 +/- 3.9 to 623 +/- 47.6 ms (n = 13). The slowing of bKv1.4 inactivation was not affected by substituting poorly hydrolyzable nucleotides for ATP in the pipette solution, or by the addition of cAMP. Riluzole-induced slowing of bKv1.4 inactivation was nearly eliminated by the presence of the antioxidant reduced glutathione (3 mM) or dithiothreitol (3-5 mM) in the recording pipette, or when cells were superfused with riluzole at a holding potential of -40 mV rather than -80 mV. These results are consistent with a model in which riluzole inhibits bKv1.4 currents and slows inactivation by separate mechanisms. Slowing of inactivation is independent of protein kinases, but probably involves oxidation of a cysteine in the N-terminal inactivation domain. Failure of riluzole to slow inactivation when applied to a depolarized cell suggests that this cysteine is protected in an inactivated Kv1.4 channel. The neuroprotective action of riluzole involves inhibition of glutamate release from presynaptic terminals within the central nervous system. Kv1.4 K+ channels are distributed throughout the brain in axons and nerve terminals, including those from which glutamate is released. The pronounced slowing of Kv1.4 inactivation by riluzole in these neurons could be an important mechanism underlying the inhibition of glutamate release and the therapeutic actions of this drug.     

Disopyramide block of cardiac sodium current after removal of the fast inactivation process in guinea pig ventricular myocytes.

To determine the necessity of sodium channel fast inactivation for the block of sodium current (INa) by disopyramide, we studied the effects of disopyramide on INa in guinea pig ventricular myocytes treated with chloramine-T, which removes the fast component of INa inactivation. After exposure to chloramine-T (2 mM), INa amplitude was reduced at all voltages and INa decay was irreversibly prevented. Disopyramide (20 microM) produced both tonic block and use-dependent block of INa in chloramine-T-treated myocytes. Before treatment with chloramine-T, the time course of both the onset of and recovery from use-dependent block by disopyramide were best fit by the sum of double exponential functions, and the time constant of the slow phase of recovery increased as the membrane was hyperpolarized. After removal of the fast component of INa inactivation by chloramine-T, the fast phase of the onset block and the fast phase of recovery from block were abolished. However, the voltage dependency of the time course of recovery from block was unchanged. Thus, although the fast sodium inactivation process is not required for tonic and use-dependent block of INa by disopyramide, it contributes to the fast phase of block development and unbinding from use-dependent block.     

Mechanism of myricetin stimulation of vascular L-type Ca2+ current

An in-depth analysis of the mechanism of the L-type Ca(2+) current [I(Ca(L))] stimulation induced by myricetin was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Myricetin increased I(Ca(L)) in a frequency-, concentration-, and voltage-dependent manner. At holding potentials (V(h)) of -50 and -90 mV, the pEC(50) values were 4.9 +/- 0.1 and 4.2 +/- 0.1, respectively; the latter corresponded to the drug-apparent dissociation constant for resting channels, K(R), of 67.6 microM. Myricetin shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction but did not modify the threshold for I(Ca(L)) or the T-type Ca(2+) current. The Ca(2+) channel blockers nifedipine, verapamil, and diltiazem antagonized I(Ca(L)) in the presence of myricetin. Myricetin increased the time to peak of I(Ca(L)) in a voltage- and concentration-dependent manner. Washout reverted myricetin effect on both current kinetics and amplitude at V(h) of -90 mV while reverting only current kinetics at V(h) of -50 mV. At the latter V(h), myricetin shifted the voltage dependence of inactivation and activation curves to more negative potentials by 6.4 and 13.0 mV, respectively, in the mid-potential of the curves. At V(h) of -90 mV, myricetin shifted, in a concentration-dependent manner, the voltage dependence of the inactivation curve to more negative potentials with an apparent dissociation constant for inactivated channels (K(I)) of 13.8 muM. Myricetin induced a frequency- and V(h)-dependent block of I(Ca(L)). In conclusion, myricetin behaves as an L-type Ca(2+) channel agonist that stabilizes the channel in its inactivated state.     

Propafenone and its metabolites preferentially inhibit IKr in rabbit ventricular myocytes

Propafenone is an antiarrhythmic agent with recognized cardiac myocyte repolarizing K+ current inhibitory effects. It has two known electropharmacologically active metabolites, 5-hydroxy- and N-depropylpropafenone, whose K+ current inhibitory effects are less thoroughly elucidated than those of the parent compound. This study characterizes and directly compares the pharmacologic interaction of all three compounds with two key repolarizing K+ currents, the rapidly activating delayed rectifier IKr and the transient outward current Ito, using the whole-cell patch-clamp technique in isolated rabbit ventricular myocytes. All three agents potently inhibited IKr with IC50 values of 0.80 +/-0.14, 1.88 +/-0.21, and 5.78 +/-1.24 microM for propafenone, 5-hydroxypropafenone, and N-depropylpropafenone, respectively, based on reduction of peak tail current amplitude following repolarization from +50 mV to -30 mV. IKr inhibition was concentration- and weakly voltage-dependent, with a time course from channel activation that was well described by a single exponential model and consistent with open channel block. Propafenone and its 5-hydroxy and N-depropyl metabolites also blocked Ito with IC50 values of 7.27 +/-0.53, 40.29 +/-7.55, and 44.26 +/-5.73 microM, respectively, at +50 mV. No significant drug effects were observed with respect to Ito voltage dependence of steady-state inactivation or time course of recovery from inactivation. The preferential interaction of propafenone and its metabolites with IKr relative to Ito in ventricular myocytes sheds new light on the anti- and proarrhythmic activity of propafenone in vivo.     

Isoflurane inhibits NaChBac, a prokaryotic voltage-gated sodium channel

Volatile anesthetics inhibit mammalian voltage-gated Na(+) channels, an action that contributes to their presynaptic inhibition of neurotransmitter release. We measured the effects of isoflurane, a prototypical halogenated ether volatile anesthetic, on the prokaryotic voltage-gated Na(+) channel from Bacillus halodurans (NaChBac). Using whole-cell patch-clamp recording, human embryonic kidney 293 cells transfected with NaChBac displayed large inward currents (I(Na)) that activated at potentials of -60 mV or higher with a peak voltage of activation of 0 mV (from a holding potential of -80 mV) or -10 mV (from a holding potential of -100 mV). Isoflurane inhibited I(Na) in a concentration-dependent manner over a clinically relevant concentration range; inhibition was significantly more potent from a holding potential of -80 mV (IC(50) = 0.35 mM) than from -100 mV (IC(50) = 0.48 mM). Isoflurane positively shifted the voltage dependence of peak activation, and it negatively shifted the voltage dependence of end steady-state activation. The voltage dependence of inactivation was negatively shifted with no change in slope factor. Enhanced inactivation of I(Na) was 8-fold more sensitive to isoflurane than reduction of channel opening. In addition to tonic block of closed and/or open channels, isoflurane enhanced use-dependent block by delaying recovery from inactivation. These results indicate that a prokaryotic voltage-gated Na(+) channel, like mammalian voltage-gated Na(+) channels, is inhibited by clinical concentrations of isoflurane involving multiple state-dependent mechanisms. NaChBac should provide a useful model for structure-function studies of volatile anesthetic actions on voltage-gated ion channels.     

Preferential block of inactivation-deficient Na+ currents by capsaicin reveals a non-TRPV1 receptor within the Na+ channel

Capsaicin elicits burning pain via the activation of the vanilloid receptor (TRPV1). Intriguingly, several reports showed that capsaicin also inhibits Na+ currents but the mechanisms remain unclear. To explore this non-TRPV1 action we applied capsaicin to HEK293 cells stably expressing inactivation-deficient rat skeletal muscle Na+ mutant channels (rNav1.4-WCW). Capsaicin elicited a conspicuous time-dependent block of inactivation-deficient Na+ currents. The 50% inhibitory concentration (IC50) of capsaicin for open Na+ channels at +30 mV was measured 6.8+/-0.6 microM (n=5), a value that is 10-30 times lower than those for resting (218 microM) and inactivated (74 microM) wild-type Na+ channels. On-rate and off-rate constants for capsaicin open-channel block at +30 mV were estimated to be 6.37 microM(-1) s(-1) and 34.4 s(-1), respectively, with a calculated dissociation constant (KD) of 5.4 microM. Capsaicin at 30 microM produced approximately 70% additional use-dependent block of remaining rNav1.4-WCW Na+ currents during repetitive pulses at 1 Hz. Site-directed mutagenesis showed that the local anesthetic receptor was not responsible for the capsaicin block of the inactivation-deficient Na+ channel. Interestingly, capsaicin elicited little time-dependent block of batrachotoxin-modified rNav1.4-WCW Na+ currents, indicating that batrachotoxin prevents capsaicin binding. Finally, neuronal open Na+ channels endogenously expressed in GH3 cells were as sensitive to capsaicin block as rNav1.4 counterparts. We conclude that capsaicin preferentially blocks persistent late Na+ currents, probably via a receptor that overlaps the batrachotoxin receptor but not the local anesthetic receptor. Drugs that target such a non-TRPV1 receptor could be beneficial for patients with neuropathic pain.     

Developmental changes in the effects of lidocaine on sodium channels in rat cardiac myocytes.

Previous studies have suggested that there are developmental changes in the sodium channel blocking properties of class I antiarrhythmic drugs, yet this hypothesis has not been well tested using measurements of sodium current. In this study we defined the effects of lidocaine on the cardiac sodium current in neonatal (1-2-day old) and adult rat ventricular myocytes using the whole-cell variation of the patch-clamp technique (16 degrees C, [Na]i = 15 mM, [Na]o = 25 mM). Lidocaine (30 microM) produced significantly more tonic block at negative holding potentials (e.g., -140 mV) in neonatal myocytes (23.2 +/- 7.0%, mean +/- S.E.M., n = 9) compared to adult (6.5 +/- 1.1%, n = 12) (P less than .05). The percentage of use-dependent block obtained during trains of 10-msec pulses at a cycle length of 200 msec was also significantly greater in neonatal myocytes (22.5 +/- 5.6%, n = 9) compared to adult myocytes (6.9 +/- 2.2%, n = 7) (P less than .02). Analysis of the kinetics of block development at -20 mV indicated that neonatal cells have a lower dissociation constant for lidocaine interaction with inactivated channels (10.1 +/- 1.3 microM) compared to adult cells (16.5 +/- 1.9 microM)(P less than .02). A marked difference was found for the time constant of recovery from channel block, where neonates recovered from block approximately twice as slowly as adults (e.g., at -140 mV tau = 1.54 +/- 0.28 sec, n = 11 in neonates vs. tau = 0.64 +/- 0.07 sec, n = 13 in adults) (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of taurine with the fast Na-current in isolated rabbit myocytes.

We studied in whole cell configuration with the patch clamp method the effect of taurine on the macroscopic Na current in adult ventricular rabbit myocytes. Because these cells have a large surface [13,750 +/- 704 microns2 (19), mean +/- S.E.M. (n)], we reduced [Na]o to 45 mM and worked at room temperature to obtain acceptable voltage control. When the cells were held at -80 mV, taurine (20 mM) had the following effects: 1) The current voltage relationships crossed over so that taurine increased INa at potentials negative to -45 mV, and at more positive potentials it depressed the current; 2) taurine reduced the maximal Na conductance from 536.3 +/- 72.2 to 253.6 +/- 33.6 microS.cm-2; 3) the crossing over of the I/V curves was mainly caused by a hyperpolarizing shift of V1/2 of the steady-state activation by 6.3 mV; 4) the crossing over was independent of a -4.6 mV shift of V1/2 of the steady-state inactivation and 5) taurine increased significantly the time constant of reactivation between -90 and -70 mV, but we did not find evidence that taurine changed the time constant of inactivation between -40 and +20 mV. We conclude that positive to -45 mV taurine causes a block of INa channels that resembles that of local anesthetic antiarrhythmic drugs. Negative to -45 mV taurine counteracts the local anesthetic effect causing increased excitability and improved conduction in the range of the threshold potential and -45 mV.     

Block of sodium channel current by anticonvulsant U-54494A in mouse neuroblastoma cells.

(+/- ) -cis-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzamide (U-54494A), structurally related to a kappa opioid agonist U-50448H, is a potent anticonvulsant without analgesic or sedative effects of the opioid agonist in intact animal studies (VonVoigtlander et al., 1987). To explore the mechanism of its anticonvulsant action, we investigated the interaction of U-54494A with the voltage-gated sodium channel using the whole cell patch clamp technique in mouse neuroblastoma cells (NIE-115). The drug reversibly and dose-dependently reduced the tetrodotoxin-sensitive inward Na current without affecting its activation or inactivation kinetics or the reversal potential. Nearly half of this resting block by 50 microM U-54494A at a holding potential of -80 mV was reversed upon further hyperpolarization to -120 mV. We also observed a hyperpolarization shift (9.3 mV) of the steady-state slow inactivation curve in the presence of 50 microM drug with no shift in the steady-state activation or the fast inactivation curves. These results indicate that the drug interacts with the resting and the slowly inactivated channels. The drug appears not to interact with the open state, judging from the absence of a time-dependent block in chloramine-T-treated cells. The recovery rate of the inactivated channel was markedly delayed by the drug, and apparently is responsible for its use-dependent block upon repetitive depolarizations. Our results suggest that voltage- and use-dependent block of the Na channel by U-54494A may be an important pharmacological basis for its anticonvulsant action.