鸡胚心肌细胞膜上外向钾电流的特征(英文)

目的:研究原代培养黄羽鸡胚心肌细胞上存在的外向钾电流特性,方法:膜片箝技术的“全细胞”记录,结果:此细胞模型上的钾电流动力学和药理学特征均不同于白羽鸡胚心肌细胞上的报道,钙通道阻断剂镉离子可消除此电流,异丙肾上腺素和cAMP通过磷酸化增加电流的峰值和缩短达到最大峰电流的时间,通道被磷酸化修饰后出现频率依赖性负性效应,结论:黄羽鸡胚心肌细胞上存在着钙激活的钾通道,磷酸化修饰不仅改变其开放速率还延长通道失活后再复活的时间,此结果也说明,同一种动物其细胞膜上离子通道的特性可因不同种系而各异。     

地高辛衍生物对人胚肾细胞293延迟整流钾电流的作用

目的 研究地高辛衍生物对人胚肾细胞(human embryonic kidney 293 cell,HEK-293)上延迟整流钾电流(delayed after potassium current,IK)的影响.方法 应用磷酸钙瞬时转染的方法,将人心肌细胞上的延迟整流钾电流通道蛋白基因(human Ether-a-go-go Related Gene,HERG)转染进人胚肾细胞,全细胞膜片钳技术记录药物浓度分别为1、10、100 nmol·L-1的地高辛甲、乙两种衍生物对人胚肾细胞上HERG钾通道时间依赖性电流(Istep)和尾电流(I tail)的影响.结果 地高辛衍生物甲和地高辛类似,对HERG钾通道时间依赖性电流和尾电流均呈现剂量依赖性抑制,其半数最大抑制浓度IC50分别为20.61、22.69 nmol·L-1;未见地高辛衍生物乙对HERG钾通道延迟整流钾电流的抑制作用.结论 地高辛衍生物甲对转染HERG钾通道的人胚肾细胞的延迟整流钾电流呈浓度依赖性抑制;而地高辛衍生物乙对转染HERG钾通道的人胚肾细胞的延迟整流钾电流无抑制作用.     

东亚钳蝎中新分离的毒素BmTx3B抑制大鼠海马神经元延迟整流性钾电流

目的：研究从东亚钳蝎毒素中新分离的短肽BmTx3B对电压门控性钾通道的作用．方法：在酶解打散的新生大鼠海马细胞,采用全细胞电压箝位方式记录,并根据动力学特性分离二种电压依赖性钾电流．结果：BraTx3B(10-100μmol／L)选择地抑制延迟整流性钾电流(IK),不影响瞬时性快钾电流(IA)．此抑制作用是可逆的,呈现浓度依赖性,但无电压依赖性．BmTx3B对延迟整流性钾电流的稳态激活和稳态失活的动力学特性无影响．结论：蝎毒短肽BmTx3B选择地抑制海马神经元延迟整流性钾通道。     

大鼠肺内动脉平滑肌细胞钾通道的研究

目的　研究大鼠肺内动脉平滑肌细胞钾电流。方法　用急性酶解分离法得到单个大鼠肺内动脉平滑肌细胞,采用膜片钳全细胞记录方式研究钾通道特性。结果　将平滑肌细胞钳制在-70 mV,给予-70～50 mV的斜坡刺激,时间为600 ms。可引出一随电压逐渐增大的电流,在+50 mV时其值为359±31 pA。细胞内用CsCl取代KCl后,该电流几乎完全消失;细胞外用无钙台氏液灌流,电极内液用高浓度的EGTA时,电流可被抑制50%±1%;细胞外给予特异性钙激活钾通道阻断剂TEA和延迟整流钾通道阻断剂4-AP均可使该电流明显下降。结论　大鼠肺内动脉平滑肌细胞的钾电流主要由钙激活钾电流和延迟整流钾电流组成。     

血管平滑肌钾通道及其调节因素

血管张力是决定血管阻力和血流量的重要因素 ,而改变钾通道活性能直接影响血管张力。钾通道开放引起钾外流 ,细胞膜超极化 ,关闭电压依赖性钙通道 ,钙内流减少 ,血管舒张 ;当钾通道受抑制时 ,可使细胞膜去极化 ,从而使电压依赖的钙通道开放 ,细胞外钙内流 ,钙离子使肌球蛋白轻链磷酸化 ,粗细肌丝发生相对运动 ,血管收缩。本文介绍血管平滑肌上 4种钾通道的基因结构、电生理学与药理学特性     

勾藤碱对大鼠和豚鼠心肌细胞钾离子通道的影响

用膜片钳全细胞记录的方法研究了勾藤碱对大鼠和豚鼠心肌细胞上电压依赖性钾通道的影响。CdC1₂0.1mmol/L存在时,勾藤碱30μmol/L减少大鼠心肌细胞一40至60mV去极化时产生的瞬时外向K⁺电流(I_A)30%,并减少一40至50mV去极化时,豚鼠心肌细胞的延迟整流K+电流(I_K)60%,同样剂量的勾藤碱对大鼠和豚鼠心肌细胞的内向整流K⁺通道(I_K1)无影响。提示勾藤碱的抗心律失常作用除了Ca通道阻断外,与其r通道抑制作用有关。     

人心肌细胞钾道通研究进展

钾通道是心肌细胞上一类具有重要功能的离子通道,是Ⅲ类抗心律失常药物作用的靶点。近年来,随着分子生物学技术的引入、钾通道克隆的出现及人心肌细胞成功分离和单通道的研究,对人心肌细胞上钾通道的激活和失活动力学特征,电压门控机制及离子的选择性等电生理学特性和药理学意义有了一个较全面深入的了解。这些对于抗心律失常药物的开发与研究具有重要的理论指导意义。     

激活视网膜水平细胞NMDA受体抑制内向整流钾通道活动

目的探讨视网膜水平细胞上激活NMDA受体对其内向整流钾通道的调控作用。方法采用酶解分离的视网膜水平细胞进行膜片钳全细胞记录,在给药激活NMDA受体前后,分别记录内向整流钾通道的电流大小;另外在无钙及螯合胞内钙条件下,观察NMDA受体对内向整流钾通道的作用。结果激活NMDA受体后,内向整流钾通道电流减小,灌流洗脱后电流恢复;在无钙和螯合胞内钙的条件下,激活NMDA受体不能改变内向整流钾通道活动。结论激活视网膜水平细胞NMDA受体可以通过胞内钙信号抑制内向整流钾通道电流。     

血管平滑肌ATP敏感性钾通道研究进展

ATP敏感性钾通道 (KATP)广泛存在于各类细胞和组织中 ,是药物作用的重要靶点。KATP是由内向整流钾通道Kir和磺酰脲类受体SUR亚基组成。与血管舒缩特性密切相关的是SUR2B/Kir6 1,电导值小 ,对ATP的抑制作用不敏感 ,需要有NDP才能被开放 ,故这类血管平滑肌KATP又被称为NDP依赖性钾通道。内源性和外源性的很多因子引起的血管舒缩反应与血管平滑肌上的KATP有关 ,此信号途径与PKA、PKC等磷酸化激酶有密切联系。不同血管对钾通道开放剂(potassiumchannelopeners,KCO)的反应有差异 ,KCO对血管的选择性作用机制仍不明确。本文就血管平滑肌KATP的分子结构、电生理、药理学特征、信号转导途径和KCO对血管的选择性作用进行综述     

CaMKⅡ 在心血管疾病中作用的研究进展

钙离子/钙调素依赖性蛋白激酶Ⅱ（CaMKⅡ）具有多重作用,并在心血管事件中有着举足轻重的地位。特别是其可以作用于多种分子信号通路中的下游靶点,促进血管疾病、心衰、心肌肥厚和心律失常发生发展。CaMKⅡ通过磷酸化L型钙通道,兰尼碱受体（RyR2）和受磷蛋白（PLN）等多种钙调蛋白能够影响心肌细胞钙平衡、增加钙渗漏,亦可影响钠通道及钾通道,调节晚钠电流及ATP敏感性钾电流IKATP。此外更可直接通过激活丝裂原活化蛋白激酶（MAPKs）及脱乙酰化酶（HDAC）影响心肌细胞转录调控。而这些机制在心肌肥厚、心力衰竭、心律失常的发生发展中都有着重要作用。因此深入了解CaMKⅡ的结构与作用机制将有助于制定新的心血管疾病治疗策略。     

Bay K 8644 enhances Ca2+ channel activities in embryonic chick heart cells without prolongation of open times.

The effects of the Ca2+ channel agonist, Bay K 8644, on the slow (L-type) Ca2+ channels was examined in young (3-day-old) embryonic chick heart cells, which naturally exhibit long-lasting openings. Bay K 8644 (5 microM) increased (a) the peak amplitude of the ensemble-averaged current by 3.9 +/- 0.9-fold (mean +/- S.E.) and (b) the maximal number of simultaneous opening from 2.6 +/- 0.4 to 4.4 +/- 0.9. Bay K 8644 had no effect on the unitary conductance (27 pS in control), and relatively little effect in the open-close kinetic analysis. The mean open times were 4.2 ms and 5.2 ms, in control and Bay K 8644, respectively. These results suggest that the agonistic effect of Bay K 8644 involves a mechanism other than open-time prolongation, such as activation of silent channels.     

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).     

Nitric oxide modulation of calcium-activated potassium channels in postganglionic neurones of avian cultured ciliary ganglia.

1. A study has been made of the modulation of calcium-activated potassium channels in cultured neurones of avian ciliary ganglia by sodium nitroprusside and L-arginine. 2. Sodium nitroprusside (100 microM) reduced the net outward current by 22 +/- 1% at 4.8 ms (mean +/- s.e. mean) and 25 +/- 1% at 350 ms during a test depolarization to +40 mV from a holding potential of -40 mV. The outward current remained reduced for the duration of the recording following a single application of sodium nitroprusside. These effects did not occur if the influx of calcium ions was first blocked with Cd2+ (500 microM). Application of ferrocyanide (100 microM) reduced the net outward current by only 6 +/- 3% at 350 ms during a test depolarization to +40 mV. 3. L-Arginine (270 microM) reduced the net outward current on average by 19 +/- 2% at 4.8 ms and 22 +/- 2% at 350 ms during a test depolarization to +40 mV. The current remained in this reduced state for the duration of the recording following a single application of L-arginine. These effects were reduced to 11 +/- 1% at 4.8 ms and 11 +/- 2% at 350 ms in the presence of N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4. In order to alleviate the dependence of calcium-activated potassium channels (Ik(Ca)) on the inward flux of calcium ions, the patch-clamp pipettes were filled with a solution containing 100 microM CaCl2, and the Ca2+ in the bathing solution was replaced with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different effects of endothelin-1 on calcium and potassium currents in canine ventricular cells

Effects of endothelin-1 (ET-1) on the L-type calcium current (ICa) and delayed rectifier potassium current (IK) were studied in isolated canine ventricular cardiomyocytes using the whole-cell configuration of the patch-clamp technique. ET-1 (8 nM) was applied in three experimental arrangements: untreated cells, in the presence of 50 nM isoproterenol, and in the presence of 250 microM 8-bromo-cAMP. In untreated cells, ET-1 significantly decreased the peak amplitude of ICa by 32.3+/-4.8% at +5 mV (P<0.05) without changing activation or inactivation characteristics of ICa. ET-1 had no effect on the amplitude of IK, Ito (transient outward current) or IK1 (inward rectifier K current) in untreated cells; however, the time course of recovery from inactivation of Ito was significantly increased by ET-1 (from 26.5+/-4.6 ms to 59.5+/- 1.8 ms, P < 0.05). Amplitude and time course of intracellular calcium transients, recorded in voltage-clamped cells previously loaded with the fluorescent calcium indicator dye Fura-2, were not affected by ET-1. ET-1 had no effect on force of contraction in canine ventricular trabeculae. Isoproterenol increased the amplitude of ICa to 263+/-29% of control. ET-1 reduced ICa also in isoproterenol-treated cells by 17.8+/-2% (P<0.05); this inhibition was significantly less than obtained in untreated cells. IK was increased by isoproterenol to 213+/-18% of control. This effect of isoproterenol on IK was reduced by 31.8+/-4.8% if the cells were pretreated with ET-1. Similarly, in isoproterenol-treated cells ET-1 decreased IK by 16.2+/-1.5% (P<0.05). Maximal activation of protein kinase A (PKA) was achieved by application of 8-bromo-cAMP in the pipette solution. In the presence of 8-bromo-cAMP ET-1 failed to alter ICa or IK It was concluded that differences in effects of ET-1 on ICa and IK may be related to differences in cAMP sensitivity of the currents.     

The influence of phenylephrine outward potassium currents in single smooth muscle cells from the rabbit mesenteric artery.

In mesenteric artery smooth muscle cells, depolarizing voltage steps activated outward K+ currents whose amplitude was decreased by about 20% with phenylephrine (1-10 microM: n = 14 cells). Attenuation of outward current was only partly dependent on [Ca2+]i, because it persisted, although reduced, with 10 mM BAPTA in the patch pipette and was abolished in the presence of 3 mM 3,4-diaminopyridine (n = 13). In outside-out patches, phenylephrine did not exert any direct effect on the unitary current amplitude or open probability of large conductance K+ channels. Outward current was significantly increased (>100% in both cases) by 10 mM caffeine, presumably owing to the release of internal Ca2+ stores. With 10 mM BAPTA in the pipette, the only response to caffeine was a small decrease (9 +/- 3.7%, n = 10) in the K+ current. These observations show that a minor effect of phenylephrine is to reduce outward K+ current (probably Kv) in mesenteric cells.     

Electrophysiological properties of cardiomyocytes isolated from CYP2J2 transgenic mice

CYP2J2 is abundant in cardiac tissue and active in the biosynthesis of eicosanoids such as epoxyeicosatrienoic acids (EETs). To determine the effects of CYP2J2 and its eicosanoid products in the heart, we characterized the electrophysiology of single cardiomyocytes isolated from adult transgenic (Tr) mice with cardiac-specific overexpression of CYP2J2. CYP2J2 Tr cardiomyocytes had a shortened action potential. At 90% repolarization, the action potential duration (APD) was 30.6 +/- 3.0 ms (n = 22) in wild-type (Wt) cells and 20.2 +/- 2.3 ms (n = 19) in CYP2J2 Tr cells (p < 0.005). This shortening was probably due to enhanced maximal peak transient outward K(+) currents (I(to,peak)), which were 38.6 +/- 2.8 and 54.4 +/- 4.9 pA/pF in Wt and CYP2J2 Tr cells, respectively (p < 0.05). In contrast, the late portion of the transient outward K(+) current (I(to,280ms)), the slowly inactivating outward K(+) current (I(K,slow)), and the voltage-gated Na(+) current (I(Na)) were not significantly altered in CYP2J2 Tr cells. N-Methylsulphonyl-6-(2-proparglyloxy-phenyl)hexanamide (MS-PPOH), a specific inhibitor of EET biosynthesis, significantly reduced I(to,peak) and increased APD in CYP2J2 Tr cardiomyocytes but not in Wt cells. Intracellular dialysis with a monoclonal antibody against CYP2J2 also significantly reduced I(to,peak) and increased APD in CYP2J2 Tr cardiomyocytes. Addition of 11,12-EET or 8-bromo-cAMP significantly reversed the MS-PPOH- or monoclonal antibody-induced changes in I(to,peak) and APD in CYP2J2 Tr cells. Together, our data demonstrate that shortening of the action potential in CYP2J2 Tr cardiomyocytes is associated with enhanced I(to,peak) via an EET-dependent, cAMP-mediated mechanism.     

Blockade by dendrotoxin homologues of voltage-dependent K+ currents in cultured sensory neurones from neonatal rats.

1. Homologues of dendrotoxin (Dtx) were isolated from the crude venom of Green and Black Mamba snakes and examined for K+ channel blocking activity in neonatal rat dorsal root ganglion cells (DRGs) by whole-cell patch clamp recording. 2. Outward potassium current activated by depolarization was composed of two major components: a slowly inactivating current (SIC, tau decay approximately 50 ms, 200 ms and 2s), and a non-inactivating current (NIC, tau decay > 2 min). Tail current analysis revealed two time constants of deactivation of total outward current, 3-12 ms and 50-150 ms (at -80 mV) which corresponded to SIC and NIC, respectively. 3. All the homologues (alpha-, beta-, gamma- and delta-Dtx and toxins I and K) blocked outward current activated by depolarization in a dose-dependent manner. The most potent in blocking total outward current was delta-Dtx (EC50 of 0.5 +/- 0.2 nM), although there were no statistically significant differences in potency between any of the homologues. 4. Qualitative differences in the nature of the block were noted between homologues. In particular, the block by delta-Dtx was time-dependent, whereas that by alpha-Dtx was not. 5. alpha-Dtx was a much better blocker of SIC (EC50 = 1.0 +/- 0.4 nM) than was delta-Dtx (EC50 = 17.6 +/- 5.8 nM). Furthermore, delta-Dtx was selective for NIC (EC50 +/- 0.24 +/- 0.03 nM) over SIC and reduced the slow component of tail currents (NIC), preferentially.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cocaine blocks HERG, but not KvLQT1+minK, potassium channels

Cocaine causes cardiac arrhythmias, sudden death, and occasionally long QT syndrome in humans. We investigated the effect of cocaine on the human K(+) channels HERG and KvLQT1+minK that encode native rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier K(+) channels in the heart. HERG and KvLQT1+minK channels were heterologously expressed in human embryonic kidney 293 cells, and whole-cell currents were recorded. Cocaine had no effect on KvLQT1+minK current in concentrations up to 200 microM. In contrast, cocaine reversibly blocked HERG current with half-maximal block of peak tail current of 7.2 microM. By using a protocol to quickly activate HERG channels, we found that cocaine block developed rapidly after channel activation. At 0 mV, the time constants for the development of block were 38.2 +/- 2.1, 15.2 +/- 0.8, and 6.9 +/- 1.1 ms in 10, 50 and 200 microM cocaine, respectively. Cocaine-blocked channels also recovered rapidly from block after repolarization. At -100 mV, recovery from block followed a biphasic time course with fast and slow time constants of 3.5 +/- 0.7 and 100.3 +/- 15.4 ms, respectively. Using N-methyl-cocaine, a permanently charged, membrane-impermeable cocaine analog, block of HERG channels rapidly developed when the drug was applied intracellularly through the patch pipette, suggesting that the cocaine binding site on the HERG protein is located on a cytoplasmic accessible domain. These results indicate that cocaine suppresses HERG, but not KvLQT1+minK, channels by preferentially blocking activated channels, that it unblocks upon repolarization, and does so with unique ultrarapid kinetics. Because the cocaine concentration range we studied is achieved in humans, HERG block may provide an additional mechanism for cocaine-induced arrhythmias and sudden death.     

Quinidine-induced open channel block of K+ current in rat ventricle.

1. The effects of quinidine on calcium-independent outward K+ currents in rat ventricular myocytes were studied using whole-cell patch clamp techniques. 2. Quinidine sulphate (6 microM) significantly prolonged repolarization of the ventricular action potential. This effect was larger during early repolarization (25% level) than at later times (90% level). 3. Quinidine reduced the amplitude of a transient outward current, and accelerated its rate of decay by approximately 4 fold at membrane potentials between 0 to +50 mV. Quinidine also reduced the amplitude of a slowly inactivating, tetraethylammonium-sensitive 'pedestal' component of the outward current. 4. The quinidine-induced block of the transient outward current was dependent on time and membrane potential. Maximal block occurred with depolarizations of about 100 ms duration, and longer depolarizations (up to 1.5 s) produced little additional block. The membrane potential dependence of quinidine-induced block was very similar to the membrane potential dependence of activation of the transient outward current. The membrane potential dependence of steady-state inactivation of the transient outward current was not significantly affected by quinidine. 5. These results show that quinidine blocks outward K+ currents in rat ventricular cells. The time and potential dependence of this block suggests that quinidine blocks the transient outward K+ current by acting primarily on the open state of these channels.