Existence of a transient outward K(+) current in guinea pig cardiac myocytes

A novel transient outward K(+) current that exhibits inward-going rectification (I(to.ir)) was identified in guinea pig atrial and ventricular myocytes. I(to.ir) was insensitive to 4-aminopyridine (4-AP) but was blocked by 200 micromol/l Ba(2+) or removal of external K(+). The zero current potential shifted 51-53 mV/decade change in external K(+). I(to.ir) density was twofold greater in ventricular than in atrial myocytes, and biexponential inactivation occurs in both types of myocytes. At -20 mV, the fast inactivation time constants were 7.7 +/- 1.8 and 6.1 +/- 1.2 ms and the slow inactivation time constants were 85.1 +/- 14.8 and 77.3 +/- 10.4 ms in ventricular and atrial cells, respectively. The midpoints for steady-state inactivation were -36.4 +/- 0.3 and -51.6 +/- 0.4 mV, and recovery from inactivation was rapid near the resting potential (time constants = 7.9 +/- 1.9 and 8.8 +/- 2.1 ms, respectively). I(to.ir) was detected in Na(+)-containing and Na(+)-free solutions and was not blocked by 20 nmol/l saxitoxin. Action potential clamp revealed that I(to.ir) contributed an outward current that activated rapidly on depolarization and inactivated by early phase 2 in both tissues. Although it is well known that 4-AP-sensitive transient outward current is absent in guinea pig, this Ba(2+)-sensitive and 4-AP-insensitive K(+) current has been overlooked.     

Extracellular Ba(2+) blocks the cardiac transient outward K(+) current

Ba(2+) is widely used as a tool in patch-clamp studies because of its ability to block a variety of K(+) channels and to pass Ca(2+) channels. Its potential ability to block the cardiac transient outward K(+) current (I(to)) has not been clearly documented. We performed whole cell patch-clamp studies in canine ventricular and atrial myocytes. Extracellular application of Ba(2+) produced potent inhibition of I(to) with an IC(50) of approximately 40 microM. The effects were voltage independent, and the inactivation kinetics were not altered by Ba(2+). The potency of Ba(2+) was approximately 10 times higher than that of 4-aminopyridine (a selective I(to) blocker with an IC(50) of 430 microM) under identical conditions. By comparison, Ba(2+) blockade of the inward rectifier K(+) current was voltage dependent; the IC(50) was approximately 20 times lower (2.5 microM) than that for I(to) when determined at -100 mV and was comparable to I(to) as determined at -60 mV (IC(50) = 26 microM). Ba(2+) concentrations of 相似文献   

Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C

Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K(+) (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K(+) currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K(+) current (I(to)) that activated at potentials positive to -40 mV and partially inactivated during depolarizing voltage steps. I(to) was inhibited by the antiarrhythmic agent flecainide (100 microM) and BaCl(2) (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K(+) currents [I(K) fast (I(Kf)) and I(K) slow (I(Ks))] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited I(to) but had no effect on I(Kf) and I(Ks). Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 alpha-subunits but not Kv4.2 or Kv1.6 alpha-subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca(2+) mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.     

Ca modulates outward current throughI K1 channels

Summary Inward-rectifier channels in cardiac cells (I _K1) stabilize the resting membrane potential near the K equilibrium potential. Here we investigate the role ofI _K1 in the regulation of action potentials and link this to the influx of Ca during beating. Inward Ca current alters the open-channel probability of outwardI _K1 current. Thus Ca ions depolarize cells not only by carrying an inward current but also by blocking an outward current.     

A method for estimation of drug affinity constants to the open conformational state of calcium channels.

The affinity of D600 to calcium channels in the open state has been examined in isolated smooth muscle cells of the rabbit ear artery. Calcium channel currents were measured in high external barium solution by means of the patch-clamp technique. The current inhibition in various D600 concentrations (3-100 microM) on application of trains of short test pulses (20-80 ms) has been studied in nonmodified calcium channels and in cells where the calcium channels were modified by the agonist dihydropyridine (+) 202,791 (100 nM). The kinetics of the peak current decay has been analyzed with a mathematical model which is based on the experimental finding that D600 interacts primarily with calcium channels in the open conformational state. The model approach allows the estimation of drug affinity constants of D600 to the calcium channel in the open conformation. An association rate constant to the open conformational state of D600 of 6.16 x 10(4) M-1 s-1 was estimated. The association rate of the drug was not significantly changed after the calcium channels have been modified with 100 nM (+) 202,791. A method for correction of rate constants for possible drug trapping is discussed.     

A calcium-dependent transient outward current in Xenopus laevis oocytes

Membrane currents were investigated in Xenopus laevis oocytes under voltage clamp. Depolarizing pulses, given from a holding potential of about-100 mV, elicited a transient outward current when the membrane potential was made more positive than about-20 mV. As the potential was made increasingly positive the transient outward current first increased and then decreased. The amplitude of the transient current increased when the external Ca2+ concentration was raised; and the current was abolished by Mn2+. It appears that when the membrane is depolarized Ca2+ ions enter the oocyte and trigger an outward current, possibly by opening C1- channels.     

A transient voltage-dependent outward current in cultured cerebellar granules

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of –80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was –77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of –78 mV and completely inactivated with –50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.     

Estimation of binding constants by capillary electrophoresis

Capillary electrophoresis (CE) has become a useful technique for measuring binding constants. This review is focused on recent trends in the estimation of binding constants by affinity CE. First, we introduce several mathematical equations in which it is assumed that the stoichiometry of the binding between drug and protein is 1:1 as a simple model. In order to calculate accurate binding constants by affinity CE, several experimental considerations are described in this review. In addition, some recent methodologies, such as partial filling technique and multiple-step ligand injection method, are introduced. Among research publications within 3 years, recent applications for determining binding constants are reviewed.     

Effect of ajmaline on transient outward current in rat ventricular myocytes

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.     

Depressed transient outward potassium current density in catecholamine-depleted rat ventricular myocytes

The effect of catecholamine depletion (induced by prior treatment with reserpine) was studied in Wistar rat ventricular myocytes using whole cell voltage-clamp methods. Two calcium-independent outward currents, the transient outward potassium current (I(to)) and the sustained outward potassium current (I(sus)), were measured. Reserpine treatment decreased tissue norepinephrine content by 97%. Action potential duration in the isolated perfused heart was significantly increased in reserpine-treated hearts. In isolated ventricular myocytes, I(to) density was decreased by 49% in reserpine-treated rats. This treatment had no effect on I(sus). The I(to) steady-state inactivation-voltage relationship and recovery from inactivation remained unchanged, whereas the conductance-voltage activation curve for reserpine-treated rats was significantly shifted (6.7 mV) toward negative potentials. The incubation of myocytes with 10 microM norepinephrine for 7-10 h restored I(to), an effect that was abolished by the presence of actinomycin D. Norepinephrine (0.5 microM) had no effect on I(to). However, in the presence of both 0.5 microM norepinephrine and neuropeptide Y (0.1 microM), I(to) density was restored to its control value. These results suggest that the sympathetic nervous system is involved in I(to) regulation. Sympathetic norepinephrine depletion decreased the number of functional channels via an effect on the alpha-adrenergic cascade and norepinephrine is able to restore expression of I(to) channels.     

Calcium-dependent transient potassium outward current in the marine ciliate Euplotes vannus

In the marine hypotrichous ciliate Euplotes vannus, the transient K+ outward current, IK fast, was studied by use of a single-microelectrode voltage-clamp equipment. Activation and inactivation kinetics, and steady-state inactivation are comparable to the properties of A-currents. Not typical for this type of current is its insensitivity to either 4-AP or 3,4-AP and its Ca2+ dependence which was derived from its inhibition by either extracellular Cd2+, La3+, D-600, or by intracellular BAPTA. Actual amplitudes of IK fast were obtained from a composite current, by subtraction of early parts of a slowly activating K+ current, IK slow, and of the early, transient Ca2+ inward current, ICa fast, that is typical for ciliates. IK fast counteracts ICa fast during the first milliseconds after onset of depolarization such that the composite current is purely outward directed.     

间歇性低氧对大鼠心室肌细胞短暂外向电流的影响

利用全细胞膜片箝方法研究间歇性低氧后左、右心室肌细胞短暂外向电流（Ｉｔｏ）的变化,以探讨间歇性低氧增强心肌电稳定性的离子机制。大鼠间歇性暴露于低氧环境２８ｄ（Ｈ２８,６ｈ／ｄ）后,右心室肌细胞的Ｉｔｏ密度较常氧对照组明显增加（１６１８±４６１比６３２±１３５ｐＡ／ｐＦ,Ｐ＜００５）,而左心室肌细胞Ｉｔｏ密度与对照组无明显差异。间歇性低氧暴露４２ｄ（Ｈ４２）动物,其左、右心室肌细胞Ｉｔｏ密度与对照组无明显差异。Ｉｔｏ激活、失活和恢复动力学变化主要表现为Ｈ４２组左、右心室肌细胞的稳态失活曲线明显向负电压方向移位。左心室细胞的半数失活电压（－３８９±２３）ｍＶ与对照组（－３２８±５９）ｍＶ比较,具有显著性差异（Ｐ＜００１）;右心室细胞的半数失活电压（－４１９±４５）ｍＶ与对照组（－３３５±３５）ｍＶ比较,具有显著性差异（Ｐ＜０００１）。据此可推断,Ｉｔｏ密度的改变可反映心室在低氧早期阶段的不同动力学反应。失活动力学改变参与间歇性低氧心脏保护机制     

西洛他唑对人心房肌细胞瞬间外向钾电流的影响

目的:观察西洛他唑对人心房肌细胞瞬间外向钾电流(Ito1)的影响,探讨该药抗心律失常作用的机制.方法:二步酶解法分离人单个右心房肌细胞,应用全细胞膜片钳技术记录人心房肌细胞Ito1.结果:在保持电位-50 mV和去极化脉冲为 50 mV条件下,30 μmol/L西洛他唑显著降低Ito1,使Ito1幅值由加药前(8.16±0.70)pA/pF降至(4.84±0.60)pA/pF(P<0.01).西洛他唑在1～50 μmol/L范围内呈浓度依赖性的抑制Ito1,1 μmol/L时即产生作用,50 μmol/L时达最大效应(降低51.09%±3.00%),IC50为(13.18±2.60)μmol/L.此外,该药对Ito1的电压依赖性激活和失活曲线以及恢复曲线均无显著影响.结论:本实验结果表明西洛他唑浓度依赖性地阻滞人心房肌细胞的Ito1.     

Effect of acidosis on transient outward potassium current in isolated rat ventricular myocytes

The effect of acidosis on the transient outward K(+) current (I(to)) of rat ventricular myocytes has been investigated using the perforated patch-clamp technique. When the holding potential was -80 mV, depolarizing pulses to potentials positive to -20 mV activated I(to) in subepicardial cells but activated little I(to) in subendocardial cells. Exposure to an acid solution (pH 6.5) had no significant effect on I(to) activated from this holding potential in either subepicardial or subendocardial cells. When the holding potential was -40 mV, acidosis significantly increased I(to) at potentials positive to -20 mV in subepicardial cells but had little effect on I(to) in subendocardial cells. The increase in I(to) in subepicardial cells was inhibited by 10 mM 4-aminopyridine. In subepicardial cells, acidosis caused a +8.57-mV shift in the steady-state inactivation curve. It is concluded that in subepicardial rat ventricular myocytes acidosis increases the amplitude of I(to) as a consequence of a depolarizing shift in the voltage dependence of inactivation.     

Zn(2+) modulation of neuronal transient K(+) current: fast and selective binding to the deactivated channels

Modulation of voltage-dependent transient K(+) currents (A type K(+) or K(A) current) by Zn(2+) was studied in rat hippocampal neurons by the whole-cell patch-clamp technique. It is found that Zn(2+) selectively binds to the resting (deactivated or closed) K(A) channels with a dissociation constant (K(d)) of approximately 3 &mgr;M, whereas the affinity between Zn(2+) and the inactivated K(A) channels is 1000-fold lower. Zn(2+) therefore produces a concentration-dependent shift of the K(A) channel inactivation curve and enhances the K(A) current elicited from relatively positive holding potentials. It is also found that the kinetics of Zn(2+) action are fast enough to compete with the transition rates between different gating states of the channel. The rapid and selective binding of Zn(2+) to the closed K(A) channels keeps the channel in the closed state and explains the ion's concentration-dependent slowing effect on the activation of K(A) current. This in turn accounts for the inhibitory effect of Zn(2+) on the K(A) current elicited from hyperpolarized holding potentials. Because the molecular mechanisms underlying these gating changes are kinetic interactions between the binding-unbinding of Zn(2+) and the intrinsic gating processes of the channel, the shift of the inactivation curve and slowing of K(A) channel activation are quantitatively correlated with ambient Zn(2+) over a wide concentration range without "saturation"; i.e., The effects are already manifest in micromolar Zn(2+), yet are not saturated even in millimolar Zn(2+). Because the physiological concentration of Zn(2+) could vary over a similarly wide range according to neural activities, Zn(2+) may be a faithful physiological "fine tuner," controlling and controlled by neural activities through its effect on the K(A) current.     

A transient outward current dependent on external calcium in rat cerebellar granule cells

Summary The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to anI _A-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca²⁺, but significantly inhibited by 3mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED₅₀=0.7mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca²⁺, Mn²⁺ and Mg²⁺. In 2.8mM Mn²⁺, the transient potassium conductance was comparable to that in 2.8mM Ca²⁺, while in 2.8mM Mg²⁺ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca²⁺ was present, Mg²⁺ up to 5mM had no effect. The transient current increased with increasing concentrations of external Ca²⁺, [Ca²⁺] _o , and the maximum conductancevs. [Ca²⁺] _o curve could be approximated by a one-site model. In addition, the current recorded with 5.5mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca²⁺ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.