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.     

Mechanism of transient outward K(+) channel block by disopyramide.

The block of the transient outward K(+) current (I(to)) by disopyramide was studied in isolated rat right ventricular myocytes using whole cell patch-clamp techniques. Disopyramide at a concentration of 10 to 1000 microM reduced peak I(to) and accelerated the apparent rate of current inactivation. The onset of block was assessed using a double pulse protocol with steps from -70 to +50 mV. As the duration of the first (conditioning) pulse was increased from 1 to 50 ms, block was increased. Further prolongation of the conditioning pulse resulted in relief of block, which was nearly complete with a 1-s conditioning pulse. In the absence of drug, the recovery from inactivation of I(to) at -70 mV was fast and best fit with a single exponential function having a time constant of 33 +/- 13 ms. In contrast, in the presence of 100 microM disopyramide, recovery from apparent inactivation was biexponential with time constants of 35 +/- 13 ms and 7.16 +/- 1.5 s. The time course of the slow component was used to estimate recovery of channels from block by disopyramide. Recovery from block was voltage-dependent, suggesting that disopyramide was trapped by the open channel. Taken together, these results suggest that disopyramide rapidly blocks channels in the open state and that unblock occurs from the inactivated state.     

Amiodarone-induced block of sodium current in isolated cardiac cells

Sodium current (INa) block by amiodarone (AMI) was investigated in isolated single Purkinje and ventricular myocardial cells using the single suction-pipette voltage-clamp technique. AMI produced marked resting block that was enhanced at low holding potentials, findings consistent with a shift in the steady-state INa availability curve to more negative potentials (-16 +/- 3 mV). Resting block was not associated with any change in the time course of INa decay during a depolarizing clamp step. AMI also produced use-dependent block in conjunction with increases in rate (0.5-5.0 Hz) and pulse duration (2-200 msec). These changes are consistent with a slowing of the recovery from inactivation of the sodium channel. Brief depolarizing pulses produced little use-dependent block, suggesting that the onset of drug-induced block is slow. Thus, AMI blocks INa and shifts the availability curve in isolated myocytes, both of which contribute to the net tonic block. The results suggest that both rested state and inactivated state sodium channel block are factors in AMI's antiarrhythmic efficacy.     

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)     

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.     

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 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.     

Quantitative structure activity studies of antiarrhythmic properties in a series of lidocaine and procainamide derivatives

The use- and voltage-dependent depression of the maximum upstroke velocity of the cardiac action potential by a series of lidocaine and procainamide derivatives was studied in guinea pig papillary muscles. The derivatives were chosen to test the effects of the structural and physicochemical differences between lidocaine and procainamide on the kinetics of sodium channel block. Three derivatives were similar to lidocaine with a rapid onset of use-dependent block at fast stimulation rates and short time constants of recovery at normal resting potentials. Seven derivatives were similar to procainamide having slower rates of block development and longer recovery time constants. In order to quantify the differences in sodium channel block the data were analyzed by a model based on the modulated receptor hypothesis. This hypothesis proposes that each of the sodium channel states (rested, open and inactivated) has characteristic association and dissociation rate constants for each sodium channel blocker, drug bound channels do not conduct sodium and have altered inactivation kinetics. This model was solved for the dissociation constants of the drug for the rested and open states, the association and dissociation rate constants for the inactivated channels and the voltage shift of the inactivation kinetics for drug-bound channels. Quantitative structure-activity analysis on the derived parameters revealed that the affinity of the drugs for the open channel state is related to the compounds lipid solubility, the degree of voltage shift was proportional to molecular weight and the dissociation from the inactivated channels was correlated with both the molecular weight and charge.     

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.     

Verapamil-induced blockade of voltage-activated K+ current in small-cell lung cancer cells.

Verapamil, Ca++ channel antagonist, has proven clinically useful in the reversal of multiple drug resistance, which is a major detriment to chemotherapy. Recently, verapamil alone has been shown to diminish proliferation in a variety of neoplastic cell lines. Using the patch-clamp technique, the action of verapamil on voltage-gated K+ channels in two cell lines of human small-cell carcinoma of the lung, NCI-H146 and NCI-H82, was investigated. With inward Na+ current suppressed, virtually all control cells exhibited a slowly inactivating outward current that was insensitive to alterations in the external Ca++ concentration. Externally applied verapamil enhanced the rate and extent of outward K+ current (IK) inactivation. Verapamil at a concentration of 20 microM diminished peak IK, evoked by a test pulse to +60 mV from a holding potential of -80 mV, from 1.38 +/- 0.11 nA (mean +/- S.E.M., n = 29 cells) to 0.56 +/- 0.13 nA (n = 11) and caused IK to decay to less than 20% of the peak current within 60 msec. After blocking IK and Na+ current, Ca++ current (ICa) was measured in the presence of 10 mM Ca++. The addition of 100 microM verapamil to the external bath resulted in a 53% reduction of H146 ICa. Peak ICa fell from 81 +/- 9 pA (n = 22) to 38 +/- 8 pA (n = 12). Examination of the whole-cell K+ current on single cells before and immediately after the addition of 100 microM verapamil clearly revealed that the drug had no effect on the initial activation phase of IK, suggesting that K+ channels first open before interacting with the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blocking action of terodiline on calcium channels in single smooth muscle cells of the guinea pig urinary bladder.

The blocking action of terodiline, a nonspecific organic Ca++ antagonist, on smooth muscle Ca++ channels of the guinea pig urinary bladder was investigated. Inward Ca++ currents were recorded from smooth muscle cells isolated from the urinary bladder using the whole-cell patch-clamp technique. In the absence of terodiline, a use-dependent reduction in the amplitude of inward Ca++ current was observed at a stimulus frequency of 0.2 Hz. When terodiline (1-10 microM) was applied, the use-dependent reduction was accelerated markedly, depending on the stimulus frequency. The blocking action of terodiline was also dose-dependent; the Kd value as measured at the end of 20 times repetitive stimulation at 0.2 Hz was 1.7 microM. In addition to such a use-dependent block, terodiline produced a hyperpolarizing shift in the steady-state inactivation curve. The results suggest that terodiline preferentially binds to the Ca++ channel in the open state and also in the inactivated state.     

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.     

Kinetics of chlorpromazine block of sodium channels in single guinea pig cardiac myocytes

Block of sodium current by chlorpromazine in single ventricular myocytes isolated from guinea pigs was studied using the whole cell patch clamp technique. Chlorpromazine in micromolar concentrations reduced the amplitude of peak sodium current associated with step depolarizations from a holding potential of -140 mV. Concentration-response curves obtained with a holding potential of -140 mV were best fit by a 2:1 stoichiometry, and were shifted in the direction of lower concentrations when a holding potential of -100 mV was used. In agreement with this observation, the steady-state inactivation curve was shifted to more negative potentials by chlorpromazine. The block was not associated with any change in the time course of sodium current activation or inactivation during a depolarizing step. Chlorpromazine also produced marked use-dependent block as demonstrated by a cumulative increase in the block during a train of depolarizing pulses. This use dependence was due to a higher affinity of chlorpromazine for the inactivated state of sodium channels than for the resting state and to a very slow repriming of the drug-bound sodium channels from inactivation. These blocking actions could contribute to the antiarrhythmic effects of chlorpromazine at low concentrations and to the cardiotoxic effects at high concentrations.     

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)     

Actions of flunarizine, a Ca++ antagonist, on ionic currents in fragmented smooth muscle cells of the rabbit small intestine

Actions of flunarizine on the Ca++ inward and K+ outward currents were investigated using fragmented smooth muscle cells (smooth muscle ball) prepared from the longitudinal muscle layer of the rabbit ileum. Flunarizine dose dependently inhibited the Ca++ inward current (ID50 = 1.4 microM). The decay of the inward current consisted of two exponentials and flunarizine had no effect on these time constants. When command pulses (100 msec; stepped up to 0 mV from -60 mV) were applied every 20 sec, the peak amplitude of the inward current remained unchanged. Flunarizine above 0.3 microM slowly inhibited the peak amplitude of inward current, in a voltage- and use-dependent manner. Intracellular perfusion of flunarizine, up to 100 microM, did not modify the peak amplitude of the inward current. This Ca++ antagonist also inhibited the K+ outward current, in a dose-dependent manner (ID50 = 5.8 microM) and accelerated inactivations of this current. When the command pulses (300 msec; stepped up to +20 mV from -60 mV) were applied repetitively every 20 sec, amplitudes of the K+ outward current were not affected. However, flunarizine, above 1 microM, reduced the peak amplitude of the K+ outward current slowly. These results indicate that although flunarizine possesses the property of a Ca++ antagonist, it also inhibits the K+ outward current, in a manner different from that observed on the Ca++ inward current.     

Block of human heart hH1 sodium channels by amitriptyline

Amitriptyline is a tricyclic antidepressant used to treat major depression and various neuropathic pain syndromes. This drug also causes cardiac toxicity in patients with overdose. We characterized the tonic and use-dependent amitriptyline block of human cardiac (hH1) Na(+) channels expressed in human embryonic kidney cells under voltage-clamp conditions. Our results show that, near the therapeutic plasma concentration of 1 microM, amitriptyline is an effective use-dependent blocker of hH1 Na(+) channels during repetitive pulses (approximately 55% block at 5 Hz). The tonic block for resting and for inactivated hH1 channels by amitriptyline (0.1-100 microM) yielded IC(50) values (50% inhibitory concentration) of 24.8 +/- 2.0 (n = 9) and 0.58 +/- 0.03 microM (n = 7), respectively. Substitution of phenylalanine with lysine at the hH1-F1760 position, a putative binding site for local anesthetics, eliminates the use-dependent block by amitriptyline at 1 microM. The time constants of recovery from the inactivated-state amitriptyline block in hH1 wild-type and hH1-F1760K mutant channels are 8.0 +/- 0. 5 (n = 6) and 0.45 +/- 0.07 s (n = 6), respectively. A substitution at either hH1-F1760K or hH1-Y1767K significantly increases the IC(50) values for resting and inactivated states of amitriptyline, but the increase is much more pronounced with the hH1-F1760K mutation. Because these two residues were proposed to form a part of the local anesthetic binding site, we conclude that amitriptyline and local anesthetics interact with a common binding site. Furthermore, at therapeutic concentrations, the ability of amitriptyline to act as a potent use-dependent blocker of Na(+) channels may, in part, explain its analgesic actions.     

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.     

Methylmercury blocks N- and L-type Ca++ channels in nerve growth factor-differentiated pheochromocytoma (PC12) cells.

Effects of methylmercury (MeHg) on whole-cell Ba++ currents in rat pheochromocytoma (PC12) cells were examined. Based on biophysical characteristics and sensitivity to omega-conotoxin GVIA and dihydropyridine agonists and antagonists, voltage-activated Ba++ currents (IBa) in PC12 cells were mediated by N- and L-type Ca++ channels. Addition of MeHg (10 microM) to the extracellular solution caused a rapid and complete block of current carried by 20 mM Ba++. The rate of block of IBa by MeHg increased in a concentration-dependent manner between 1 and 20 microM. Increasing the frequency of stimulation from 0.1 to 0.4 Hz facilitated block of IBa by MeHg. A 2-min application of 10 microM MeHg in the absence of stimulation also reduced IBa by approximately 80%. Thus, block of IBa by MeHg is not state-dependent. Additionally, MeHg blocked IBa when the membrane holding potential was -40, -70 and -90 mV, indicating that both N- and L-type Ca++ channels are blocked by MeHg. Block of IBa by MeHg was voltage-dependent at a membrane holding potential of -40 mV, but not at holding potentials of -70 and -90 mV. Decreasing the extracellular concentration of Ba++ ([Ba++]e) from 20 mM to 10 mM increased the magnitude of block by MeHg from 45.6 to 77.3%. Increasing [Ba++]e to 30 mM caused no further antagonism of block. Block of IBa by MeHg was not reversed by washing with MeHg-free solution. The ionic permeability of PC12 cell Ca++ channels was Ca++ = Sr++ greater than Ba++. In the presence of MeHg, all three divalent cations were equally permeant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of the 5-hydroxytryptamine 3 antagonist class of antiemetic drugs with human cardiac ion channels

Administration of the 5-hydroxytryptamine 3 receptor class of antiemetic agents has been associated with prolongation in the QRS, JT, and QT intervals of the ECG. To explore the mechanisms underlying these findings, we examined the effects of granisetron, ondansetron, dolasetron, and the active metabolite of dolasetron MDL 74,156 on the cloned human cardiac Na(+) channel hH1 and the human cardiac K(+) channel HERG and the slow delayed rectifier K(+) channel KvLQT1/minK. Using patch-clamp electrophysiology we found that all of the drugs blocked Na(+) channels in a frequency-dependent manner. At a frequency of 3 Hz, the IC(50) values for block of Na(+) current measured 2.6, 88.5, 38.0, and 8.5 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Block was relieved by strong hyperpolarizing potentials, suggesting a possible interaction with an inactivated channel state. Recovery from inactivation was impaired at -80 mV compared with -100 mV, and the fractional recovery was impaired by drug in a concentration-dependent manner. IC(50) values for block of the HERG cardiac K(+) channel measured 3.73, 0.81, 5.95, and 12.1 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Ondansetron (3 microM) also slowed decay of HERG tail currents. In contrast, none of these drugs (10 microM) produced greater than 30% block of the slow delayed rectifier K(+) channel KvLQT1/minK. We concluded that the antiemetic agents tested in this study block human cardiac Na(+) channels probably by interacting with the inactivated state. This may lead to clinically relevant Na(+) channel blockade, especially when high heart rates or depolarized/ischemic tissue is present. The submicromolar affinity of ondansetron for the HERG K(+) channel likely underlies the prolongation of cardiac repolarization reported for this drug.     

ELECTROPHYSIOLOGICAL ANALYSIS OF THE ACTION OF NIFEDIPINE AND NICARDIPINE ON MYOCARDIAL FIBERS

The effects of nifedipine and nicardipine, 2 dihydropyridines (DHP) used in the treatment of cardiovascular disorders, were compared in frog atrial fibers. Rapid photolysis of nifedipine with a single UV flash (1-ms duration) reversed the block, allowing comparison of effects of both drugs on the same preparation, and manipulation of the calcium channel on a millisecond timescale. The results show that inhibition of the action potential (AP) and slow inward current (Isi) is more pronounced with nifedipine than with nicardipine. Concentration-effect relationships confirm that nicardipine (IC50 = 1 microM) is less potent than nifedipine (IC50 = 0.2 microM) in blocking cardiac calcium channels. Both DHP block calcium channels in the closed state at the resting potential, inducing a large tonic block (in the absence of stimulation). An additional phasic block can be observed with nifedipine and nicardipine. A slight voltage dependence to the block is observed for both DHP, their effects being enhanced depolarization holding potentials. Rapid unblocking of calcium channels by a single light flash, presented during the decay phase of Isi, reveals a higher affinity of DHP for inactivated channels. This effect is most pronounced when inactivation is slowed by using Ba++, Sr++, or Na+ ions as the current carriers. Open channel block is also suggested. The mechanism of DHP action on calcium channels can be described according to the "modulated receptor hypothesis". These DHP exhibit an additional nonspecific effect on potassium channels. It is concluded that nicardipine is a less potent Ca++ antagonist than nifedipine in atrial fibers and that the reduction of delayed potassium current, which occurs in a similar range of concentrations to the blockade of Isi, could also be involved in its therapeutic effects.