充血性心衰病人心房肌细胞瞬间外向钾电流和超快速延迟整流钾电流的特征

AIM: To study the properties of transient outward K+ current (Ito) and ultra-rapid delayed rectifier K+ current (IKur) in isolated human atrial myocytes from patients with congestive heart failure (CHF). METHODS: Single cells were isolated from CHF patients with collagenase and protease. Ito and IKur were recorded using whole cell patch-clamp technique. RESULTS: The activation and inactivation of I(to) were voltage-dependent and time-dependent. The half-activation and half-inactivation voltage were (15 +/- 12) mV and (-45 +/- 4) mV respectively. When membrane potential went up from -40 mV to +60 mV, the activation time constant means decreased from (6.9 +/- 2.3) ms to (1.40 +/- 0.20) ms, while the inactivation time constant means decreased from (69 +/- 17) ms to (21 +/- 14) ms. Otherwise, the mean reactivation time constants was (125 +/- 65) ms when the membrane potential was held at -80 mV, but the recovery was not complete during the interval observed. Ito showed less frequency-dependent reduction at test frequency between 0.2-2 Hz. Compared with Ito, the activation of IKur only showed voltage-dependence, without time-dependence. Its mean current densities was (3.4 +/- 0.7) pA/pF when test potential was +60 mV. The half activation voltage of IKur was (23 +/- 14) mV. No clear frequency-dependence was observed at the same frequency range of Ito either. CONCLUSION: I(to) and IKur are important outward potassium channel currents in isolated human atrial myocytes from CHF patients and they have different kinetic properties.     

未成年人心房肌细胞瞬间外向钾电流和内向整流钾电流的特性

AIM: To study the properties of transient outward K+ current (Ito) and inward rectifier K+ current (IKl) in immature human heart. METHODS: Ito and IKl were recorded using whole-cell patch-clamp technique in atrial myocytes isolated from 12 immature (aged from 6 months to 5 a) human hearts. RESULTS: Ito was voltage-dependent, activated and inactivated rapidly. The IC50 (95% confidence limits) of 4-AP on Ito was 0.64 (0.48-0.87) mmol.L-1. 4-AP 1 mmol.L-1 shifted V1/2 of activation from (6.6 +/- 2.0) mV to (19.8 +/- 3.0) mV (n = 4-10, P < 0.01). 4-AP 0.3 mmol.L-1 changed V1/2 of inactivation from (-49 +/- 4) mV to (-61.4 +/- 2.1) mV (n = 3, P < 0.01), but there were no obvious influence on voltage-dependent activation of Ito (P > 0.05). At the same concentration, the recovery time constant (tau value) was prolonged from (108 +/- 16) ms to (220 +/- 67) ms (n = 3-12, P < 0.01). IKl was also voltage-dependent. Its reverse potential was -40 mV. CONCLUSION: Both Ito and IKl are important K+ channel currents in immature human atrial myocytes. 4-AP can affect the inactivation and recovery of Ito at low concentration (0.3 mmol.L-1) and affect its activation at high concentration (1 mmol.L-1).     

The sulphonylurea glibenclamide inhibits voltage dependent potassium currents in human atrial and ventricular myocytes

1 It was the aim of our study to investigate the effects of the sulphonylurea glibenclamide on voltage dependent potassium currents in human atrial myocytes. 2 The drug blocked a fraction of the quasi steady state current (ramp response) which was activated positive to -20 mV, was sensitive to 4-aminopyridine (500 microM) and was different from the ATP dependent potassium current IK(ATP). 3 Glibenclamide dose dependently inhibited both, the peak as well as the late current elicited by step depolarization positive to -20 mV. The IC50 for reduction in charge area of total outward current was 76 microM. 4 The double-exponential inactivation time-course of the total outward current was accelerated in the presence of glibenclamide with a tau(fast) of 12.7+/-1.5 ms and a tau(slow) of 213+/-25 ms in control and 5.8+/-1.9 ms (P<0.001) and 101+/-20 ms (P<0.05) under glibenclamide (100 microM). 5 Our data suggest, that both repolarizing currents in human atrial myocytes, the transient outward current (Ito1) and the ultrarapid delayed rectifier current (IKur) were inhibited by glibenclamide. 6 In human ventricular myocytes glibenclamide inhibited Ito1 without affecting the late current. 7 Our data suggest that glibenclamide inhibits human voltage dependent cardiac potassium currents at concentrations above 10 microM.     

Effects of MiRP1 and DPP6 beta-subunits on the blockade induced by flecainide of Kv4.3/KChIP2 channels

BACKGROUND AND PURPOSE: The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore-forming Kv4.3 alpha-subunit, coassembled with modulatory beta-subunits as KChIP2, MiRP1 and DPP6 proteins. beta-Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on Kv4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 beta-subunits.Experimental approach:Currents were recorded in Chinese hamster ovary cells stably expressing K(V)4.3/KChIP2 channels, and transiently transfected with either MiRP1, DPP6 or both, using the whole-cell patch-clamp technique. KEY RESULTS: In control conditions, Kv4.3/KChIP2/MiRP1 channels exhibited the slowest activation and inactivation kinetics and showed an 'overshoot' in the time course of recovery from inactivation. The midpoint values (Vh) of the activation and inactivation curves for Kv4.3/KChIP2/DPP6 and Kv4.3/KChIP2/MiRP1/DPP6 channels were approximately 10 mV more negative than Vh values for Kv4.3/KChIP2 and Kv4.3/KChIP2/MiRP1 channels. Flecainide (0.1-100 microM) produced a similar concentration-dependent blockade of total integrated current flow (IC50 approximately 10 microM) in all the channel complexes. However, the IC50 values for peak current amplitude and inactivated channel block were significantly different. Flecainide shifted the Vh values of both the activation and inactivation curves to more negative potentials and apparently accelerated inactivation kinetics in all channels. Moreover, flecainide slowed recovery from inactivation in all the channel complexes and suppressed the 'overshoot' in Kv4.3/KChIP2/MiRP1 channels.Conclusions and implications:Flecainide directly binds to the Kv4.3 alpha-subunit when the channels are in the open and inactivated state and the presence of the beta-subunits modulates the blockade by altering the gating function.     

Direct effects of candesartan and eprosartan on human cloned potassium channels involved in cardiac repolarization

In the present study, we analyzed the effects of two angiotensin II type 1 receptor antagonists, candesartan (0.1 microM) and eprosartan (1 microM), on hKv1.5, HERG, KvLQT1+minK, and Kv4.3 channels expressed on Ltk(-) or Chinese hamster ovary cells using the patch-clamp technique. Candesartan and eprosartan produced a voltage-dependent block of hKv1.5 channels decreasing the current at +60 mV by 20.9 +/- 2.3% and 14.3 +/- 1.5%, respectively. The blockade was frequency-dependent, suggesting an open-channel interaction. Eprosartan inhibited the tail amplitude of HERG currents elicited on repolarization after pulses to +60 mV from 239 +/- 78 to 179 +/- 72 pA. Candesartan shifted the activation curve of HERG channels in the hyperpolarizing direction, thus increasing the current amplitude elicited by depolarizations to potentials between -50 and 0 mV. Candesartan reduced the KvLQT1+minK currents elicited by 2-s pulses to +60 mV (38.7 +/- 6.3%). In contrast, eprosartan transiently increased (8.8 +/- 2.7%) and thereafter reduced the KvLQT1+minK current amplitude by 17.7 +/- 3.0%. Eprosartan, but not candesartan, blocked Kv4.3 channels in a voltage-dependent manner (22.2 +/- 3.5% at +50 mV) without modifying the voltage-dependence of Kv4.3 channel inactivation. Candesartan slightly prolonged the action potential duration recorded in guinea pig papillary muscles at all driving rates. Eprosartan prolonged the action potential duration in muscles driven at 0.1 to 1 Hz, but it shortened this parameter at faster rates (2--3 Hz). All these results demonstrated that candesartan and eprosartan exert direct effects on Kv1.5, HERG, KvLQT1+minK, and Kv4.3 currents involved in human cardiac repolarization.     

普罗帕酮对钾通道亚型Kv4.2和Kv4.3电流的影响

目的　研究普罗帕酮对钾通道亚型Kv4 2和Kv4 3电流的影响。方法　采用全细胞膜片钳技术记录稳定表达Kv4 2和Kv4 3电流的人胚胎肾细胞株 (HEK2 93细胞 )电流的变化。结果　①普罗帕酮明显抑制Kv4 2和Kv4 3电流 ,呈浓度依赖性 ,IC50 分别为 1 0 3 μmol·L- 1 和 71 μmol·L- 1 ;②普罗帕酮明显加速Kv4 2和Kv4 3电流失活 ,1 0μmol·L- 1 的普罗帕酮可使Kv4 2电流衰减时间常数τ由(38 9± 2 1 )ms变为 (9 9± 1 8)ms ,半数最大失活膜电位V1 /2 由 (- 66 6± 0 8)mV左移至 (- 70 9± 1 1 )mV ;1 0 0μmol·L- 1 的普罗帕酮可使Kv4 3电流衰减时间常数τ(1 4 4 8± 2 0 8)ms变为 (1 8 5± 2 8)ms,半数最大失活膜电位V1 /2 由 (- 4 5 6± 1 9)mV左移至 (- 52 3± 2 1 )mV ;③普罗帕酮明显左移Kv4 2和Kv4 3电流的激活曲线 ,1 0μmol·L- 1 的普罗帕酮可使Kv4 2电流半数最大激活膜电位V1 /2 由 (- 4 1± 0 5)mV左移至 (- 1 6 1± 2 4)mV ;1 0 0μmol·L- 1 的普罗帕酮可使Kv4 3半数最大激活膜电位V1 /2由 (- 6 0± 1 1 )mV左移至 (- 1 6 5± 3 0 )mV。结论　普罗帕酮明显抑制Kv4 2 ,Kv4 3电流 ,该作用可能是其治疗心律失常的机制之一。     

丙咪嗪对大鼠心室细胞瞬间外向钾电流的抑制作用

目的：研究丙咪嗪大鼠心室细胞瞬间外向钾电流（Ｉｗ）的抑制作用,方法：膜片箝全细胞记录法。结果：丙咪嗪对Ｉｔｏ有浓度依赖性抑制作用,ＩＣ５０为６．０μｍｏｌ．Ｌ＾－１并明显加速该电流的灭活里程,在不同的测试电位下,丙咪嗪对该电流的抑制百分率没有差别,丙咪嗪对Ｉｔｏ的稳态激活和灭活苗曲线的半数膜电位都无明显影响,对Ｉｔｏ灭活后的再复活时程有延长趋势,但不显（τｃｏｎｔｒｏｌ＝３７±１１ｍｓ,τｄｒｕ     

Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis

1. In the present study, two new peptides, phrixotoxins PaTx1 and PaTx2 (29-31 amino acids), which potently block A-type potassium currents, have been purified from the venom of the tarantula Phrixotrichus auratus. 2. Phrixotoxins specifically block Kv4.3 and Kv4.2 currents that underlie I(to1), with an 5 < IC50 < 70 nM, by altering the gating properties of these channels. 3. Neither are the Shaker (Kv1), Shab (Kv2) and Shaw (Kv3) subfamilies of currents, nor HERG, KvLQT1/IsK, inhibited by phrixotoxins which appear specific of the Shal (Kv4) subfamily of currents and also block I(to1) in isolated murine cardiomyocytes. 4. In order to evaluate the physiological consequences of the Ito1 inhibition, mice were injected intravenously with PaTx1, which resulted in numerous transient cardiac adverse reactions including the occurrence of premature ventricular beats, ventricular tachycardia and different degrees of atrioventricular block. 5. The analysis of the mouse electrocardiogram showed a dose-dependent prolongation of the QT interval, chosen as a surrogate marker for their ventricular repolarization, from 249 +/- 11 to 265 +/- 8 ms (P < 0.05). 6. It was concluded that phrixotoxins, are new and specific blockers of Kv4.3 and Kv4.2 potassium currents, and hence of I(to1) that will enable further studies of Kv4.2 and Kv4.3 channel and/or I(to1) expression.     

Taurine inhibition of fast Na+ current in embryonic chick ventricular myocytes.

Effects of taurine on the fast Na+ current (INa) in 17-day-old embryonic chick ventricular myocytes were examined using the whole-cell voltage-clamp technique. The cells were spherical (10-15 microns diameter) and had a capacitance of 9.8 +/- 1.3 pF. The experiments were performed at room temperature (22 degrees C), and the holding potential was -90 mV. After the patch membrane was broken, peak INa initially increased, and then decreased and became stable within 3-5 min. The experiments on taurine were started after INa had stabilized. The characteristics of INa were as expected, including sensitivity to tetrodotoxin (10 microM). When added to the bath, taurine inhibited INa and shifted the reversal potential in the hyperpolarizing direction. At 10 mM, taurine inhibited INa by 38.2 +/- 4.3%, and shifted the reversal potential by 10.2 +/- 3.1 mV. The time to peak current was slowed: 0.83 +/- 0.20 ms (n = 11) in control, 1.03 +/- 0.18 ms (n = 9) in 10 mM taurine, and 1.10 +/- 0.19 ms (n = 10) in 20 mM taurine. These effects of taurine were not reversed by 30 min washout. At low concentrations, taurine actually enhanced INa in 3 of 8 cells at 1 mM, and in 4 of 10 cells at 5 mM; the reversal potential was still shifted in the hyperpolarizing direction by 5.7 +/- 1.6 mV. The time course of inactivation (fitted as a single exponential at test potential of -30 mV) was not affected: 1.1 +/- 0.5 ms in control 1.2 +/- 0.4 ms at 10 mM taurine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of propafenone on K currents in human atrial myocytes

1. The class Ic anti-arrhythmic agent, flecainide is known to inhibit the transient outward K current (Ito) selectively in human atrium. We studied the effects of propafenone, another class Ic antiarrhythmic agent, on K currents in human atrial myocytes using a whole-cell voltage-clamp method. 2. Propafenone inhibited both Ito and the sustained or ultra-rapid delayed rectifier K current (Isus or Ikur) evoked by depolarization pulses. The concentration for half-maximal inhibition (IC50) was 4.9 microM for Ito and 8.6 microM for Isus. Propafenone blocked Ito and Isus in a voltage- and use-independent fashion and accelerated the inactivation time constant of Ito [from 28.3 to 6.7 ms at 10 microM propafenone]. 3. The steady-state inactivation curve for Ito was unaffected by propafenone. Propafenone did not affect the initial current at depolarizing potentials, but it did produce a block that increased as a function of time after depolarization (time constant of 3.4 ms). This suggests that propafenone preferentially blocked Ito in the open state. 4. Propafenone had no significant effect on the rate at which Ito recovered from inactivation at -80 mV suggesting that propafenone dissociates rapidly from the channel. 5. The steady-state activation curve for Isus was not affected by propafenone. Propafenone slowed the time course of the onset of the Isus tail current. This suggests that propafenone blocked Isus in the open state. 6. The present results suggest that, unlike flecainide, propafenone blocks both Ito and Isus in human atrial myocytes in the open state at clinically relevant concentrations.     

Block of hERG channel by ziprasidone: biophysical properties and molecular determinants

Ziprasidone, an antipsychotic agent, delays cardiac repolarization and, thus, prolongs the QT interval of the cardiac ECG. In this study, we examined the biophysical properties and the molecular determinants of the ziprasidone block of wild-type hERG potassium channels stably expressed in HEK-293 cells or wild-type and mutant hERG channels expressed in Xenopus oocytes. In stably transfected HEK-293 cells, ziprasidone blocked wild-type hERG current in a voltage- and concentration-dependent manner (IC(50)=120nM, 0mV, 37 degrees C). Ziprasidone showed minimal tonic block of hERG current estimated during a depolarizing voltage (-20 or +30mV) or evaluated by the envelope of tails test (+30mV). Rate of the block onset was rapid, but not significantly affected by test potentials ranging from -20 to +30mV (time constant (tau)=114+/-14ms at +30mV). The time constant of the slow component of hERG current deactivation (at -50mV) was significantly increased by ziprasidone (tau=1776+/-90 versus 1008+/-71ms, P<0.01). Time course of channel inactivation was slowed by ziprasidone in a voltage-dependent manner. The V(1/2) values for steady-state activation and inactivation of hERG channel in HEK-293 cells were not significantly altered by ziprasidone. In Xenopus oocytes, ziprasidone exhibited less potent block of wild-type hERG current (IC(50)=2.8microM, 0mV, 23 degrees C). Mutation of the aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of hERG dramatically reduced the potency of channel block by ziprasidone (IC(50)>0.4 and 1mM at 0mV for Y652A and F656A, respectively). In conclusion, ziprasidone preferentially binds to and blocks open hERG channels. Tyr-652 and Phe-656 are two critical residues in the ziprasidone-binding site.     

Direct block by bisindolylmaleimide of the voltage-dependent K+ currents of rat mesenteric arterial smooth muscle

We investigated the effect of bisindolylmaleimide (I), a widely used protein kinase C (PKC) inhibitor, on the voltage-dependent K(+) (Kv) currents of rat mesenteric arterial smooth muscle cells using the whole-cell patch-clamp technique. Bisindolylmaleimide (I) reversibly and dose-dependently inhibited the Kv currents with an apparent K(d) value of 0.23+/-0.001 microM. The blockade was apparently through the acceleration of the decay rate of the Kv currents. The apparent rate constants of association and dissociation for bisindolylmaleimide (I) were 17.9+/-1.6 microM(-1) s(-1) and 4.1+/-1.5 s(-1), respectively. The inhibition of Kv current by bisindolylmaleimide (I) was steeply voltage-dependent between -30 and 0 mV (voltage range of channel activation). Bisindolylmaleimide (I) had no effect on the steady-state activation and inactivation of the Kv currents. Applications of trains of pulses at 1 or 2 Hz lead to a progressive increase in the bisindolylmaleimide (I)-blockade, and the recovery from bisindolylmaleimide (I)-block at -80 mV exhibited a time constant of 577.2+/-52.7 ms. Bisindolylmaleimide (V), an inactive analogue of bisindolylmaleimide (I), similarly inhibited the Kv currents with an apparent K(d) value of 1.48+/-0.004 microM, but other PKC inhibitor chelerythrine little affected the Kv currents. These results suggest that bisindolylmaleimide (I) directly inhibits the Kv currents of rat mesenteric arterial smooth muscle cells independently of PKC inhibition, in a state-, voltage-, time- and use-dependent manner.     

Spironolactone and its main metabolite canrenoic acid block hKv1.5, Kv4.3 and Kv7.1 + minK channels

Both spironolactone (SP) and its main metabolite, canrenoic acid (CA), prolong cardiac action potential duration and decrease the Kv11.1 (HERG) current. We examined the effects of SP and CA on cardiac hKv1.5, Kv4.3 and Kv7.1+minK channels that generate the human I(Kur), I(to1) and I(Ks), which contribute to the control of human cardiac action potential duration.hKv1.5 currents were recorded in stably transfected mouse fibroblasts and Kv4.3 and Kv7.1 + minK in transiently transfected Chinese hamster ovary cells using the whole-cell patch clamp. SP (1 microM) and CA (1 nM) inhibited hKv1.5 currents by 23.2 +/- 3.2 and 18.9 +/- 2.7%, respectively, shifted the midpoint of the activation curve to more negative potentials and delayed the time course of tail deactivation.SP (1 microM) and CA (1 nM) inhibited the total charge crossing the membrane through Kv4.3 channels at +50 mV by 27.1 +/- 6.4 and 27.4 +/- 5.7%, respectively, and accelerated the time course of current decay. CA, but not SP, shifted the inactivation curve to more hyperpolarised potentials (V(h)-37.0 +/- 1.8 vs -40.8 +/- 1.6 mV, n = 10, P < 0.05).SP (10 microM) and CA (1 nM) also inhibited Kv7.1 + minK currents by 38.6 +/- 2.3 and 22.1 +/- 1.4%, respectively, without modifying the voltage dependence of channel activation. SP, but not CA, slowed the time course of tail current decay.CA (1 nM) inhibited the I(Kur) (29.2 +/- 5.5%) and the I(to1) (16.1 +/- 3.9%) recorded in mouse ventricular myocytes and the I(K) (21.8 +/- 6.9%) recorded in guinea-pig ventricular myocytes.A mathematical model of human atrial action potentials demonstrated that K(+) blocking effects of CA resulted in a lengthening of action potential duration, both in normal and atrial fibrillation simulated conditions. The results demonstrated that both SP and CA directly block hKv1.5, Kv4.3 and Kv7.1 + minK channels, CA being more potent for these effects. Since peak free plasma concentrations of CA ranged between 3 and 16 nM, these results indicated that blockade of these human cardiac K(+) channels can be observed after administration of therapeutic doses of SP.Blockade of these cardiac K(+) currents, together with the antagonism of the aldosterone proarrhythmic effects produced by SP, might be highly desirable for the treatment of supraventricular arrhythmias.     

Effects of U50,488H on transient outward and ultra-rapid delayed rectifier K+ currents in young human atrial myocytes

The effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on transient outward K+ current (Ito1) and ultra-rapid delayed rectifier K+ current (IKur) in young human atrial myocytes were evaluated with a whole-cell patch-clamp technique. At +10 mV, U50,488H decreased Ito1 in a concentration-dependent manner (IC50=12.4+/-3.5 microM), while at +50 mV, U50,488H produced biphasic effects on Ito1-increasing and decreasing the current at 1-3 and 10-30 microM, respectively. U50,488H at 10 microM shifted the midpoint (V0.5) of Ito1 activation in a depolarizing direction by approximately 5 mV, accelerated the inactivation, and slowed the recovery from inactivation of Ito1. In addition, U50,488H inhibited IKur in a concentration-dependent manner (IC50=3.3+/-0.6 microM). The effects of U50,488H on the two types of K+ currents were not antagonized by either 5 microM nor-binaltorphimine or 300 nM naloxone. These results indicate that U50,488H affects both Ito1 and IKur in young human atrial myocytes in an opioid receptor-independent manner.     

Non-specific action of methoxamine on Ito, and the cloned channels hKv 1.5 and Kv 4.2

The alpha1-adrenoceptor agonist methoxamine acted independently of receptor activation to reduce Ito and the sustained outward current in rat ventricular myocytes, and hKv 1.5 and Kv 4.2 cloned K+ channel currents. Two hundred microM methoxamine reduced Ito by 36% in the presence of 2 microM prazosin, and by 37 and 38% after preincubation of myocytes with either N-ethylmaleimide or phenoxybenzamine (n=6). The EC50 values at +60 mV for direct reduction of Ito, hKv 1.5, and Kv 4.2 by methoxamine were 239, 276, and 363 microM, respectively, with Hill coefficients of 0.87-1.5. Methoxamine accelerated Ito and Kv 4.2 current inactivation in a concentration- and voltage-dependent manner. Apparent rate constants for methoxamine binding and unbinding gave Kd values in agreement with EC50 values measured from dose-response relations. The voltage-dependence of block supported charged methoxamine binding to a putative intracellular site that sensed approximately 20% of the transmembrane electrical field. In the presence of methoxamine, deactivating Kv 4.2 tail currents displayed a distinct rising phase, and were slowed relative to control, such that tail current crossover was observed. These observations support a dominant mechanism of open channel block, although closed channel block could not be ruled out. Single-channel data from hKv 1.5 patches revealed increased closed times with blank sweeps and decreased burst duration in the presence of drug, and a reduction of mean channel open time from 1.8 ms in control to 0.4 ms in 500 microM methoxamine. For this channel, therefore, both open and closed channel block appeared to be important mechanisms for the action of methoxamine.     

A quantitative and comparative study of the effects of a synthetic ciguatoxin CTX3C on the kinetic properties of voltage-dependent sodium channels

Ciguatoxins (CTXs) are known to bind to receptor site 5 of the voltage-dependent Na channel, but the toxin's physiological effects are poorly understood. In this study, we investigated the effects of a ciguatoxin congener (CTX3C) on three different Na-channel isoforms, rNa(v)1.2, rNa(v)1.4, and rNa(v)1.5, which were transiently expressed in HEK293 cells. The toxin (1.0 micromol l(-1)) shifted the activation potential (V(1/2) of activation curve) in the negative direction by 4-9 mV and increased the slope factor (k) from 8 mV to between 9 and 12 mV (indicative of decreased steepness of the activation curve), thereby resulting in a hyperpolarizing shift of the threshold potential by 30 mV for all Na channel isoforms. The toxin (1.0 micromol l(-1)) significantly accelerated the time-to-peak current from 0.62 to 0.52 ms in isoform rNa(v)1.2. Higher doses of the toxin (3-10 micromol l(-1)) additionally decreased time-to-peak current in rNa(v)1.4 and rNa(v)1.5. A toxin effect on decay of I(Na) at -20 mV was either absent or marginal even at relatively high doses of CTX3C. The toxin (1 micromol l(-1)) shifted the inactivation potential (V(1/2) of inactivation curve) in the negative direction by 15-18 mV in all isoforms. I(Na) maxima of the I-V curve (at -20 mV) were suppressed by application of 1.0 micromol l(-1) CTX3C to a similar extent (80-85% of the control) in all the three isoforms. Higher doses of CTX3C up to 10 micromol l(-1) further suppressed I(Na) to 61-72% of the control. Recovery from slow inactivation induced by a depolarizing prepulse of intermediate duration (500 ms) was dramatically delayed in the presence of 1.0 micromol l(-1) CTX3C, as time constants describing the monoexponential recovery were increased from 38+/-8 to 588+/-151 ms (n=5), 53+/-6 to 338+/-85 ms (n=4), and 23+/-3 to 232+/-117 ms (n=3) in rNa(v)1.2, rNa(v)1.4, and rNa(v)1.5, respectively. CTX3C exerted multimodal effects on sodium channels, with simultaneous stimulatory and inhibitory aspects, probably due to the large molecular size (3 nm in length) and lipophilicity of this membrane-spanning toxin.     

A novel enhancing effect of clofilium on transient outward-type cloned cardiac K+ channel currents.

1. The antiarrythmic drug, clofilium, has been shown to block several types of K+ channel currents. To investigate the effects of clofilium on the transient outward K+ current (Ito), a cloned Ito-type cardiac K+ channel (RHK1) was expressed in Xenopus oocytes and the drug effects were examined on whole cell currents. 2. Extracellular application of clofilium slightly inhibited the current at +60 mV from a holding potential of -80 mV. However, it unexpectedly enhanced the current from a holding potential of -60 mV in a dose-dependent manner (219 +/- 39% of control at 100 microM). 3. This enhancement is probably due to an increase in the ratio of channels in the resting state during steady depolarization, since clofilium shifted the inactivation curve in the depolarizing direction. 4. LY97119, a tertiary ammonium analogue of clofilium, did not exhibit this enhancing effect but only inhibited the current. 5. Clofilium may be useful for the study of channel inactivation because this type of phenomenon has not been reported for any other drug.     

Mechanism of inhibition of the sodium current by bepridil in guinea-pig isolated ventricular cells.

1. Effects of bepridil, a sodium-, calcium-, and potassium-antagonistic agent, on the Na+ current were studied by the whole cell voltage clamp technique (tip resistance = 0.5 MOhm, [Na]i and [Na]o 10 mmol l-1 at 20 degrees C). 2. Bepridil produced tonic block (Kdrest = 295.44 mumol l-1, Kdi = 1.41 mumol l-1; n = 4). 3. Bepridil (100 mumol l-1) shifted the inactivation curve in the hyperpolarization direction by 13.4 +/- 2.7 mV (n = 4) without change in the slope factor. 4. In the presence of 50 mumol l-1 bepridil, bepridil showed use-dependent block at 2 Hz, whereas changes in pulse duration did not significantly effect this use-dependent block (81% +/- 2% at 10 ms, 84% +/- 3% at 30 ms, 86% +/- 3% at 100 ms; n = 4). 5. After removal of fast inactivation of the Na+ current by 3 mmol l-1 tosylchloramide sodium, bepridil (50 mumol l-1) still showed use-dependent block which was independent of the holding potential. 6. The recovery time constant from the bepridil-induced use-dependent block was 0.48 s at holding potential of -100 mV and 0.51 s at holding potential of -140 mV. 7. These results indicate that bepridil could bind to the receptor in the sodium channel through the hydrophobic and the hydrophilic pathway and leave the receptor through the hydrophobic pathway in the lipid bilayer. The binding and dissociation kinetics of this drug were shown to be fast, and the accumulation of the drug in the sodium channel appeared to be small.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by fendiline of the transient outward current in rat ventricular cardiomyocytes.

The effects of fendiline on the transient outward current (Ito) were investigated in rat ventricular cardiomyocytes. Extracellularly applied fendiline reduced peak and steady-state current amplitude of Ito; the inactivation of Ito was accelerated by the drug, which reflects onset of block. The described effects were concentration dependent: half-maximal effects were achieved at approximately 3 microM fendiline. Intracellularly applied fendiline (3 microM) did not affect Ito within 5 min. The steady-state current amplitude of Ito was more efficiently suppressed by the drug at 22 +/- 1 degrees C than at 36 +/- 1 degrees C. The recovery of Ito was analyzed by the application of twin depolarizing voltage pulses, interrupted by variable pulse intervals. In the presence of fendiline, recovery of Ito was about twofold slower than that under control conditions, independent of the drug concentration used, which reflects offset from block. Concentration-dependent onset but concentration-independent offset of block suggest that the described time constants correspond to voltage-dependent net binding and unbinding, respectively, of fendiline at its receptor sites. It is proposed that fendiline binds extracellularly at positive potentials to Ito channels in their open state and dissociates from the channels at rest.