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.     

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.     

Dihydropyridine Ca2+ channel antagonists and agonists block Kv4.2, Kv4.3 and Kv1.4 K+ channels expressed in HEK293 cells

(1) We have determined the molecular basis of nicardipine-induced block of cardiac transient outward K(+) currents (I(to)). Inhibition of I(to) was studied using cloned voltage-dependent K(+) channels (Kv) channels, rat Kv4.3L, Kv4.2, and Kv1.4, expressed in human embryonic kidney cell line 293 (HEK293) cells. (2) Application of the dihydropyridine Ca(2+) channel antagonist, nicardipine, accelerated the inactivation rate and reduced the peak amplitude of Kv4.3L currents in a concentration-dependent manner (IC(50): 0.42 micro M). The dihydropyridine (DHP) Ca(2+) channel agonist, Bay K 8644, also blocked this K(+) current (IC(50): 1.74 micro M). (3) Nicardipine (1 micro M) slightly, but significantly, shifted the voltage dependence of activation and steady-state inactivation to more negative potentials, and also slowed markedly the recovery from inactivation of Kv4.3L currents. (4) Coexpression of K(+) channel-interacting protein 2 (KChIP2) significantly slowed the inactivation of Kv4.3L currents as expected. However, the features of DHP-induced block of K(+) current were not substantially altered. (5) Nicardipine exhibited similar block of Kv1.4 and Kv4.2 channels stably expressed in HEK293 cells; IC(50)'s were 0.80 and 0.62 micro M, respectively. (6) Thus, at submicromolar concentrations, DHP Ca(2+) antagonist and agonist inhibit Kv4.3L and have similar inhibiting effects on other components of cardiac I(to), Kv4.2 and Kv1.4.     

Effects of norfluoxetine, the major metabolite of fluoxetine, on the cloned neuronal potassium channel Kv3.1.

The effects of fluoxetine and its major metabolite, norfluoxetine, were studied using the patch-clamp technique on the cloned neuronal rat K(+) channel Kv3.1, expressed in Chinese hamster ovary cells. In whole-cell recordings, fluoxetine and norfluoxetine inhibited Kv3.1 currents in a reversible concentration-dependent manner, with an IC(50) value and a Hill coefficient of 13.11+/-0.91 microM and 1.33+/-0.08 for fluoxetine and 0.80+/-0.06 microM and 1.65+/-0.08 for norfluoxetine at +40 mV, respectively. In inside-out patches, norfluoxetine applied to the cytoplasmic surface inhibited Kv3.1 with an IC(50) value of 0.19+/-0.01 microM. The inhibition of Kv3.1 currents by both drugs was characterized by an acceleration in the apparent rate of current decay, without modification of the activation time course and with relatively fewer effects on peak amplitude. The degree of inhibition of Kv3.1 by norfluoxetine was voltage-dependent. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.31+/-0.05. The association (k(+1)) and dissociation (k(-1)) rate constants for norfluoxetine-induced inhibition of Kv3.1 were 21.70+/-3.39 microM(-1) s(-1) and 14.68+/-3.94 s(-1), respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 0.68 microM. Norfluoxetine did not affect the ion selectivity of Kv3.1. The reversal potential under control conditions was about -85 mV and was not affected by norfluoxetine. Norfluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of norfluoxetine, were superimposed. The voltage dependence of steady-state inactivation was not changed by the drug. Norfluoxetine produced use-dependent inhibition of Kv3.1 at a frequency of 1 Hz and slowed the recovery from inactivation. It is concluded that at clinically relevant concentrations, both fluoxetine and its major metabolite norfluoxetine inhibit Kv3.1, and that norfluoxetine directly inhibits Kv3.1 as an open channel blocker.     

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 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.     

Basic arrhythmogenic mechanisms in both inherited and acquired long QT syndrome

The heterologous expression system will provide clues for understanding the basic mechanism of arrhythmogenicity in both inherited and acquired long QT syndrome, which are reviewed here, with emphasis on the K+ channels. Endothelin is implicated in the morphological and electrical remodeling of cardiac muscles in heart failure. The effects of endothelin on the transient outward K+ currents (Ito) were compared between Kv1.4 (rich in endocardial muscle) and Kv4.3 (rich in epicardial muscle) channels in the Xenopus oocytes expression system. Both Itos were decreased by stimulation of endothelin receptor ETA coexpressed with the K+ channels. Ito of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, whereas that of Kv4.3 was decreased by about 60%. By mutagenesis experiments, we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in Ito by ET-1. In Kv4.3 we identified a PKC phosphorylation site that is partly responsible for the decrease. Differences in the suppression of Ito could be due to the differences in intracellular signaling including the number of phosphorylation sites. These findings show some of the molecular mechanisms of ventricular arrhythmias in heart failure, resulting in dispersion and prolongation of action potential which elicit reentry and after depolarization.     

Effects of BRL 38227 on potassium currents in smooth muscle cells isolated from rabbit portal vein and human mesenteric artery.

1. Single smooth muscle cells were isolated from the rabbit portal vein and the human mesenteric artery and whole cell currents recorded at room temperature from either cell type by the whole cell voltage clamp technique. 2. In the rabbit portal vein cells addition of 10 microM BRL 38227 induced a quasi-instantaneous, voltage-insensitive and time-independent current which had a reversal potential of -75 mV under experimental conditions where the calculated EK was -83 mV. 3. Cells were held at 0 mV and BRL 38227 was added cumulatively to construct a dose-response relationship. BRL 38227 (0.03-10 microM) caused a dose-dependent outward shift in the holding current with an EC50 of 1.3 microM. 4. BRL 38227 (10 microM) had no effect on the delayed rectifier K+ current measured in the presence of 5 mM tetraethylammonium and no effect on the Ca(2+)-activated K+ current measured in the presence of 5 mM 4-aminopyridine. Similarly BRL 38227 had no effect on the Ca2+ current. 5. The BRL 38227-induced current was blocked by glibenclamide (10 microM) and phentolamine (100 microM), specific blockers of the ATP-sensitive K+ current in single cells. 6. In human isolated mesenteric artery cells, BRL 38227 (10 microM) induced a glibenclamide-sensitive current similar to, but smaller than, that observed in the rabbit portal vein. 7. We conclude that in these cells, BRL 38227 activates a potassium conductance which has the electrophysiological and pharmacological characteristics of ATP-sensitive K+ channels.     

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.     

Permeation properties and modulation of volume-activated Cl(-)-currents in human endothelial cells.

1. We have studied the permeation and pharmacological properties of a recently described volume-activated, calcium-insensitive, small-conductance Cl(-)-channel in endothelial cells from human umbilical vein. 2. The relative permeability for various anions was I- > Cl- approximately Br- > F- > gluconate- (1.63 +/- 0.36: 1:0.95 +/- 0.16:0.46 +/- 0.04:0.19 +/- 0.07, n = 10). 3. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) induced a fast and reversible block of the current (Ki = 29 mumol l-1). 4. Extracellular ATP induced a low-affinity block of the current, that showed a small voltage-dependence (K1 = 4.9 mmol l-1 at +80 mV and K1 = 8.2 mmol l-1 at -80 mV). 5. Extracellularly applied arachidonic acid (10 mumol l-1) irreversibly blocked the current in 5 out of 9 cells. This block seems to be non-specific, because other ionic currents, e.g. inwardly rectifying K+ currents, were blocked as well. 6. Tamoxifen induced a high affinity block of the current (K1 = 2.9 mumol l-1). Block and reversal of block were however much slower than with NPPB. 7. Cytotoxic compounds, which are substrates of the P-glycoprotein multidrug transporter, loaded into endothelial cells via the patch pipette, exerted only minor effects on the volume-activated current. Vinblastine and colcemid did not affect the volume-activated current, whereas daunomycin and vincristine induced a slow 'run-down' of the current.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle

BACKGROUND AND PURPOSE: Although azelnidipine is used clinically to treat hypertension its effects on its target cells, Ca2+ channels, in smooth muscle have not been elucidated. Therefore, its effects on spontaneous contractions and voltage-dependent L-type Ca2+ channels were investigated in guinea-pig portal vein. EXPERIMENTAL APPROACH: The inhibitory potency of azelnidipine on spontaneous contractions in guinea-pig portal vein was compared with those of other dihydropyridine (DHP)-derived Ca antagonists (amlodipine and nifedipine) by recording tension. Also its effects on voltage-dependent nifedipine-sensitive inward Ba2+ currents (IBa) in smooth muscle cells dispersed from guinea-pig portal vein were investigated by use of a conventional whole-cell patch-clamp technique. KEY RESULTS: Spontaneous contractions in guinea-pig portal vein were reduced by all of the Ca antagonists (azelnidipine, Ki = 153 nM; amlodipine, Ki = 16 nM; nifedipine, Ki = 7 nM). In the whole-cell experiments, azelnidipine inhibited the peak amplitude of IBa in a concentration- and voltage-dependent manner (-60 mV, Ki = 282 nM; -90 mV, Ki = 2 microM) and shifted the steady-state inactivation curve of IBa to the left at -90 mV by 16 mV. The inhibitory effects of azelnidipine on IBa persisted after 7 min washout at -60 mV. In contrast, IBa gradually recovered after being inhibited by amlodipine, but did not return to control levels. Both azelnidipine and amlodipine caused a resting block of IBa at -90 mV. Only nifedipine appeared to interact competitively with S(-)-Bay K 8644. CONCLUSIONS AND IMPLICATIONS: These results suggest that azelnidipine induces long-lasting vascular relaxation by inhibiting voltage-dependent L-type Ca2+ channels in vascular smooth muscle.     

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).     

Ion channel modulation by NS 1619, the putative BKCa channel opener, in vascular smooth muscle.

1. The effects of NS 1619, the putative BKCa channel opener, were investigated on rat intact portal veins and on single smooth muscle cells enzymatically separated from the same tissue. 2. Under whole-cell patch clamp conditions with K-rich pipettes, exposure of single cells held at -10 mV to NS 1619 (10-33 microM) induced a noisy, outward current which reached a maximum (33 microM NS 1619; mean 35.8 +/- 17 pA, n = 8) within about 6 min. 3. On stepping to test potentials (range -50 to +50 mV) from a holding potential of -10 mV, the NS 1619-induced noisy current exhibited time-dependent activation and marked outward rectification. 4. The stimulation of outward currents by NS 1619 at -10 mV was independent of the presence of Ca2+ in the bath or pipette solutions but was antagonized by either charybdotoxin (250 nM) or penitrem A (100 nM) in the bath solution. 5. Stationary fluctuation analysis of the noisy current induced by NS 1619 at -10 mV yielded a value of 70 +/- 8 pS (n = 4) (under the quasi-physiological conditions of the experiment) for the unitary conductance of the channel involved. 6. At -10 mV, NS 1619 (10-33 microM) rapidly inhibited spontaneous transient outward currents. 7. With a holding potential of -90 mV, NS 1619 (10-33 microM) produced a reduction of outward currents evoked by depolarizing steps to +50 mV, an effect associated with marked inhibition of the delayed rectifier current, IK(V). 8. NS 1619 (3-100 microM) produced a concentration-dependent inhibition of spontaneous activity in rat portal vein characterized by a reduction in the amplitude and duration of the tension waves. This inhibition was slightly potentiated in the presence of either charybdotoxin (250 nM) or penitrem A (1 microM). NS 1619 also totally inhibited contractions of rat aorta induced by KCl (both 20 mM and 80 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition of the activity of T lymphocyte Kv1.3 channels by extracellular zinc

The inhibitory effect of zinc on voltage-gated Kv1.3 channels in human T lymphocytes was investigated using the "whole-cell" patch-clamp technique. Application of 10 and 20 microM Zn caused a concentration-dependent shift of activation midpoint of the whole-cell currents from -19.65+/-1.03 mV (mean+/-SE) under control conditions to 9.84+/-0.66 mV upon application of 20 microM Zn. This effect was saturated at zinc concentrations higher than 20 microM. The activation rate was considerably slower, whereas the deactivation rate was not significantly affected by Zn. Inactivation midpoint was shifted from -53.06+/-0.44 mV under control conditions to -36.05+/-0.48 mV in the presence of 100 microM Zn. Inactivation rate was not significantly affected upon Zn treatment. Whole-cell potassium currents were reduced to about 70% of their control values with no clear concentration dependence in the zinc concentration range from 10 to 100 microM. When raising the zinc concentration to levels above 100 microM, a concentration-dependent inhibition of the whole-cell currents appeared additionally to the changes in channel gating. The channels were half-blocked at the zinc concentration of 346+/-40 microM and the Hill slope coefficient was 1.89+/-0.21. The inhibitory effect of zinc was not complete at micromolar concentrations and was saturated at concentrations higher than 1mM. This inhibitory effect was not accompanied by any further modification in the shift of the activation and inactivation midpoints nor by a slowing of the channel activation rate. The inhibitory effect of zinc was significantly diminished in the presence of 150 mM K(+) in the extracellular solution, whereas the zinc-induced shift of the activation threshold and slowing of the activation kinetics remained unchanged when raising extracellular potassium concentration. It is suggested that zinc acts on two independent binding sites on the channels. Binding to one site that is saturated at concentrations higher than 20 microM affects the channel gating. Binding to another site at concentrations higher than 100 microM inhibits the currents without affecting the channel gating.     

Open channel block of Kv3.1 currents by fluoxetine

The action of fluoxetine, a serotonin reuptake inhibitor, on the cloned neuronal rat Kv3.1 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Fluoxetine reduced Kv3.1 whole-cell currents in a reversible, concentration-dependent manner, with an IC(50) value and a Hill coefficient of 13.4 muM and 1.4, respectively. Fluoxetine accelerated the decay rate of inactivation of Kv3.1 currents without modifying the kinetics of current activation. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.38. The binding (k(+1)) and dissociation (k(-1)) rate constants for fluoxetine-induced block of Kv3.1 were 5.7 microM(-1)s(-1) and 53.5 s(-1), respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 9.3 microM. Fluoxetine did not affect the ion selectivity of Kv3.1. Fluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of fluoxetine, were superimposed. Inhibition of Kv3.1 by fluoxetine was use-dependent. The present results suggest that fluoxetine acts on Kv3.1 currents as an open-channel blocker.