乌头碱阻断表达在卵母细胞上的HERG通道的电药理特性

目的观察乌头碱对表达在卵母细胞上的HERG电流的影响。方法HERG通道表达在非洲爪蟾卵母细胞上,利用双电极电压钳技术测量其电流。结果①HERG通道可以稳定表达在卵母细胞上;②乌头碱以浓度和电压依赖性方式阻断表达在卵母细胞上的野生型HERG通道,阻断的半数抑制浓度值是1.801±0.332μmol/L;③乌头碱对HERG电流的阻断呈时间依赖性;④峰电流幅值被1μmol/L乌头碱显著降低,而稳态失活的半数失活电压(-39.10±1.04 mV vs-41.61±2.66 mV,P>0.05,n=6)没有被乌头碱的阻断显著改变,而斜率k(32.37±1.04 mV vs 41.05±4.19 mV,P<0.05,n=6)出现正向移动。结论乌头碱对HERG电流呈浓度、电压和时间依赖性阻断,而其对失活状态下的HERG通道无明显阻断作用,HERG通道可能是乌头碱致心律失常的关键离子靶点之一。     

二十碳四烯酸对转基因中国仓鼠卵母细胞hKv1.5钾通道电流的作用

目的观察二十碳四烯酸对转基因中国仓鼠卵母细胞(CHO细胞)hKv1.5钾通道电流的影响。方法把hKv1.5钾通道基因转入CHO细胞膜中表达,给予不同浓度的ETYA,用全细胞膜片钳技术引出电流并观察不同电压条件下hKv1.5钾通道电流的变化情况。结果ETYA对hKv1.5稳态电流有明显的可逆性抑制作用,且具有浓度依赖性和电压依赖性。ETYA对hkv1.5通道电流的抑制系直接作用于通道而产生,并非影响脂氧合酶的活性而起作用。结论ETYA对hKv1.5钾通道电流有抑制作用。     

厄贝沙坦对电压依赖性的Kv1.3和Kv1.5通道电流的阻断作用

目的通过观察厄贝沙坦对电压依赖性的Kv1.3和Kv1.5的阻断作用,探讨厄贝沙坦对此类通道的阻断可能具有的临床作用。方法使用双电极电压钳技术记录表达于非洲爪蟾卵母细胞的Kv1.3和Kv1.5钾通道电流,不同浓度厄贝沙坦灌流对其电流的影响。结果①厄贝沙坦浓度依赖性的阻断Kv1.3通道,阻断的IC50是2.46μmol/L,且阻断具有电压依赖性。②厄贝沙坦浓度依赖性的阻断Kv1.5通道,阻断的IC50是0.47μmol/L,且阻断具有显著的电压依赖性。结论厄贝沙坦阻断开放状态的Kv1.3可能是其发挥免疫调节和抗动脉粥样硬化作用的机制之一;而对开放状态的Kv1.5的阻断可能是其具备减少心房颤动发生率的作用机制之一。     

替米沙坦对电压依赖性的Kv1.3和Kv1.5通道电流阻断作用的实验研究

目的 通过观察替米沙坦对电压依赖性的Kv1.3和Kv1.5的阻断作用,探讨替米沙坦对此类通道的阻断可能具有的临床作用.方法 使用双电极电压钳技术记录表达于非洲爪蟾卵母细胞的Kv1.3和Kv1.5钾通道电流,不同浓度灌流观察其对电流影响.结果 (1)替米沙坦浓度依赖性的阻断Kv1.3通道,其阻断的IC50是2.05 μmol/L.替米沙坦对Kv1.3电流的阻断具有电压依赖性.(2)替米沙坦浓度依赖件的阻断Kv1.5通道,其阻断的IC50是2.37 μmol/L.替米沙坦对Kv1.5电流的阻断具有更显著的电压依赖性.结论 替米沙坦阻断开放状态的Kv1.3可能是其发挥免疫调节和抗动脉粥样硬化作用的机制之一.替米沙坦对开放状态的Kv1.5钾通道的阻断可能是其减少心房颤动发生率的作用机制之一.     

Kvβ1.3亚基显著影响DPO-1对表达在卵母细胞上的Kvl.5通道的阻断作用

目的:研究Kvβ1.3亚基和Kv1.5共表达时,对表达在非洲爪蟾卵母细胞的Kv1.5通道DPO-1的阻断作用的影响。方法:在非洲爪蟾卵母细胞上异源表达克隆Kv1.5及Kvβ1.3通道基因,用双电极电压钳技术记录全细胞电流,检测药物对Kv1.5通道及Kv1.5+Kvβ1.3共表达通道电流的影响。结果:DPO-1以电压、频率及浓度依赖方式抑制Kv1.5+Kvβ1.3共表达通道的电流。Kvβ1.3亚基存在时,DPO-1的阻断效应明显减弱,DPO-1阻断的IC50由(0.77士0.12)μmol/L显著增加至(47.21士5.18)μmol/L,增加了约60倍(P<0.01)。结论:Kvβ1.3亚基显著抑制DPO-1对表达在卵母细胞上的Kv1.5通道的阻断作用,但不改变其电压、频率及浓度依赖性,可能机制是Kvβ1.3亚基与DPO-1相互竞争Kv1.5孔区内部的某些结合位点。     

Kvβ1.3亚基显著影响DPO-1对表达在卵母细胞上的Kv1.5通道的阻断作用

目的:研究Kvβ1.3亚基和Kv1.5共表达时,对表达在非洲爪蟾卵母细胞的Kv1.5通道DPO-1的阻断作用的影响。方法:在非洲爪蟾卵母细胞上异源表达克隆Kv1.5及Kvβ1.3通道基因,用双电极电压钳技术记录全细胞电流,检测药物对Kv1.5通道及Kv1.5+Kvβ1.3共表达通道电流的影响。结果:DPO-1以电压、频率及浓度依赖方式抑制Kv1.5+Kvβ1.3共表达通道的电流。Kvβ1.3亚基存在时,DPO-1的阻断效应明显减弱,DPO-1阻断的IC50由(0.77士0.12)μmol/L显著增加至(47.21士5.18)μmol/L,增加了约60倍(P0.01)。结论:Kvβ1.3亚基显著抑制DPO-1对表达在卵母细胞上的Kv1.5通道的阻断作用,但不改变其电压、频率及浓度依赖性,可能机制是Kvβ1.3亚基与DPO-1相互竞争Kv1.5孔区内部的某些结合位点。     

阿司咪唑阻断HERG通道的生物学特性及分子机制

目的 观察阿司咪唑对野生型和Y652突变型HERG通道阻断的生物物理学特性,探讨HERG通道分子位点改变对阻断的影响.方法 将HERG通道表达于非洲爪蟾卵母细胞,利用双电极电压钳技术测量其电流,观察阿司咪唑不同浓度、不同电压、不同作用时间下,对野生型和Y652A、Y652R突变型HERG通道电流的阻断作用.结果 阿司咪唑以电压、浓度、时间依赖性阻断HERG通道电流;与野生型比较,Y652A和Y652R突变型可显著减弱阿司咪唑对HERG通道的阻断作用.结论 阿司咪唑优先阻断开放状态的HERG通道,Y652是阿司咪唑与通道结合的关键位点,其极性和侧链长度改变可影响阿司咪唑与通道结合.     

替米沙坦对表达在卵母细胞上Kv1.5通道的抑制效应

目的:研究替米沙坦对表达在卵母细胞上的克隆人类Kv1.5通道的作用,探讨其在心脏复极中的潜在效应。方法:在非洲爪蟾卵母细胞上异源表达克隆人类Kv1.5通道基因,使用双电极电压钳技术记录全细胞电流,检测药物对Ikur电流的影响。结果:替米沙坦以电压依赖性和浓度依赖性方式抑制Kv1.5通道电流,且对峰电流及1.5s末端电流的抑制效应不同,在1μmol/L浓度下,抑制效应分别达到(7.75±2.39)和(52.64±3.77),其半抑制浓度(IC50)分别为(2.25±0.97)μmol/L和(0.82±0.39)μmol/L。替米沙坦对通道的稳态失活没有显著改变,在对照条件下,V1/2的值为(14.47±3.71)mV,斜坡因子k为(23.24±3.86)mV;在1μmol/L替米沙坦作用下,V1/2和k的值分别为(14.38±4.62)mV和(26.26±5.04)mV(n=6,P>0.05)。同时,替米沙坦显著加速了Kv1.5通道的失活。在对照条件下,Kv1.5通道的失活慢时间常数是(693.74±23.16)ms,在应用1μmol/L替米沙坦后,其失活的慢时间常数下降为(523.85±10.28)ms(n=5,P<0.05)。结论:替米沙坦在临床有效浓度范围内能显著抑制表达在卵母细胞上的Ikur电流,提示它兼有选择性阻滞Kv1.5通道的作用。     

银杏叶提取物对超极化激活的环核苷酸门控基因编码的起搏通道电生理特性的影响

目的研究银杏叶提取物对异源表达在卵母细胞上的超极化激活的环核苷酸门控通道（HCN）2、4阻断的电药理学特性及分子机制。方法将人HCN2、4的mRNA显微注射到非洲爪蟾卵母细胞后,利用双电极电压钳技术记录通道电流。结果银杏叶提取物呈浓度依赖性阻滞表达在爪蟾卵母细胞上HCN2、4通道,药物半量抑制浓度IC50值分别为0.12±0.05 mg/ml和0.25±0.01 mg/ml。银杏叶提取物对HCN2、4通道阻断的阻断作用是不可逆的,经过15-20 min药物洗脱,阻滞的HCN电流部分并没有完全恢复到对照水平,只能恢复大约55%-75%。在-110 mV时银杏叶提取物对激活时间常数的值明显增大,对HCN2通道从504.6±39.8 ms（n=8）增为588.4±21.7 ms（0.03 mg/ml,n=8,P〈0.05）,1176.4±57.3 ms（0.3 mg/ml,n=8,P〈0.05）;对HCN4通道从1330.5±59.8 ms（n=8）增为1973.1±83.6 ms（0.03 mg/ml,n=8,P〈0.05）,而银杏叶提取物对HCN电流的去激活时间常数并未明显改变。结论银杏叶提取物以浓度依赖性方式不可逆的作用于HCN2、4通道,且对HCN4通道的抑制作用强于HCN2通道,减慢了通道的激活动力学,而对通道的去激活动力学无明显影响。     

地尔硫卓对Kv1.4钾通道动力学的影响

目的:研究地尔硫卓对异源表达在卵母细胞上的克隆fKv1.4钾通道电流的激活及失活动力学影响。方法: 在非洲爪蟾卵母细胞上异源表达雪貂心脏来源的去N端Kv1.4（fKv1.4ΔN）通道基因,采用双电极电压钳制技术记录电流、记录药物对fKv1.4ΔN通道电流的影响。结果: 地尔硫卓以频率依赖性、电压依赖性及浓度依赖性的方式抑制fKv1.4ΔN通道电流,其半抑制浓度（IC50）为(241.04±23.06) μmol/L(+50 mV)。对照条件下,fKv1.4ΔN通道电流失活的表现为单指数方程拟合,在应用地尔硫卓后,fKv1.4ΔN通道电流失活变为双指数方程拟合,即药物诱导的快速失活成分及较慢的C型失活成分。地尔硫卓可加快C型失活,但其不影响fKv1.4ΔN通道电流的激活过程。结论: 地尔硫卓为fKv1.4ΔN通道的开放状态阻滞剂,可加快Kv1.4ΔN通道的失活过程。     

Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X)

OBJECTIVE: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study. METHODS: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. RESULTS: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation. CONCLUSION: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.     

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.     

The acid-sensitive potassium channel TASK-1 in rat cardiac muscle

OBJECTIVE: The outward current flowing through the two-pore domain acid-sensitive potassium channel TASK-1 (I(TASK)) and its inhibition via alpha1-adrenergic receptors was studied in rat ventricular cardiomyocytes. METHODS: Quantitative RT-PCR experiments were carried out with mRNA from rat heart. Patch-clamp recordings were performed in isolated rat cardiomyocytes. TASK-1 and other K+ channels were expressed in Xenopus oocytes to study the pharmacological properties of a new TASK-1 channel blocker, A293. RESULTS: TASK-1 channels were found to be strongly expressed in rat heart. Analysis of the sensitivity of various K+ channels to A293 in Xenopus oocytes showed that at low concentrations A293 was a selective blocker of TASK-1 channels. I(TASK) in rat cardiomyocytes was dissected by application of A293 and by extracellular acidification to pH 6.0; it had an amplitude of approximately 0.30 pA/pF at +30 mV. Application of 200 nM A293 increased action potential duration (APD(50)) by 31+/-3% at a stimulation rate of 4 Hz. The plausibility of the effects of A293 on APD50 was checked with a mathematical action potential model. Application of the alpha1-adrenergic agonist methoxamine inhibited I(TASK) in Xenopus oocytes co-injected with cRNA for TASK-1 and alpha1A-receptors. In cardiomyocytes, methoxamine inhibited an outward current with characteristics similar to I(TASK). This effect was abolished in the presence of the alpha1A-antagonist 5-methyl-urapidil. CONCLUSIONS: Our results suggest that in rat cardiomyocytes I(TASK) makes a substantial contribution to the outward current flowing in the plateau range of potentials and that this current component can be inhibited via alpha1A-adrenergic receptors.     

老年大鼠耳蜗核钾离子通道在爪蟾卵母细胞中的表达

目的观察爪蟾卵母细胞表达的老年大鼠耳蜗核电压依赖性离子通道电流的变化，为研究感音神经性聋听觉中枢离子通道的变化建立方法学基础。方法分别自青年和老年大鼠耳蜗核提取多聚腺嘌呤信使核糖核酸（ｍＲＮＡ），注入非洲爪蟾卵母细胞表达有功能的钾离子通道，并利用电压钳方法记录电压依赖性的离子通道电流。结果在注射老年大鼠耳蜗核ｍＲＮＡ的卵母细胞膜上记录到电压依赖性的钾离子通道电流最大幅度（２７８±３７ｎＡ，１６只），明显小于青年组（３６４±４２ｎＡ，１７只，Ｐ＜０００１）。结论移植的老年大鼠耳蜗核钾离子通道功能的下降可能与老年性聋的发病有关     

Expression of Caenorhabditis elegans neurotransmitter receptors and ion channels in Xenopus oocytes

Injection of Caenorhabditis elegans polyA RNA into Xenopus laevis oocytes led to the expression of neurotransmitter receptors that generated some unique responses, including ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors as well as receptors that coupled to G proteins, such as those to octopamine, norepinephrine, and angiotensin, which activated the oocyte's own phosphatidylinositol system and calcium-gated chloride channels. The oocytes also expressed chloride-conducting glutamate receptors, muscarinic acetylcholine receptors, and voltage-operated calcium channels. Unexpectedly, serotonin (5-hydroxytryptamine), dopamine, GABA, and kainate did not generate ionic currents, suggesting that the corresponding receptors were not expressed or were not functional in the oocytes. The use of X. laevis oocytes for expressing worm RNA demonstrates that there are many molecular components whose role remains to be clarified in the nematode. Among them are the nature of the endogenous agonists for the octopamine and angiotensin receptors and the subunits that compose the ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and the norepinephrine receptors that couple to the phosphoinositide cascade.     

普罗帕酮对Kv1.4通道内口突变前后的影响

目的探讨普罗帕酮对编码瞬时外向钾电流(Ito)慢通道Kv1.4通道(fKv1.4ΔN)内口突变(fKv1.4[V561A]ΔN)前后的影响。方法将fKv1.4ΔN和fKv1.4[V561A]ΔN的cRNA注射到非洲爪蟾卵母细胞,孵育24～72 h后使用不同刺激程序用双微电极法记录通道电流的表达。用Clampfit 9.0对数据进行分析。部分电流用相应的方程拟合。结果普罗帕酮对fKv1.4ΔN和fKv1.4[V561A]ΔN的阻滞效应都呈电压和频率依赖性。通道内口的V561A突变使fKv1.4ΔN通道与普罗帕酮的结合能力减小,50%抑制浓度(IC50)在突变前后分别为100μmol/L和380μmol/L(P<0.01)。普罗帕酮能改变fKv1.4ΔN和fKv1.4[V561A]ΔN的失活特性。尽管普罗帕酮对fKv1.4ΔN的失活后恢复没有影响,但是它能延长fKv1.4[V561A]ΔN的50%失活后恢复时间。结论普罗帕酮是fKv1.4ΔN通道的开放通道阻滞剂,fKv1.4ΔN通道内口突变(V561A)能改变普罗帕酮与通道之间的结合能力,从而影响通道的失活和失活后恢复。     

Does Acetaldehyde Mediate Ethanol Action in the Central Nervous System?

BACKGROUND: Some of the effects of ethanol in the central nervous system are due to changes in function of ligand-gated ion channels. Production of detectable amounts of acetaldehyde, a primary metabolite of ethanol, has been demonstrated in brain homogenates. The aim of this study was to determine whether central actions that are often attributed to ethanol may actually be mediated by acetaldehyde. METHODS: The effects of acetaldehyde (1-1000 microM) were tested by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing 10 different ligand-gated ion channel receptors [alpha1 glycine; alpha1beta2gamma2Sgamma-aminobutyric acid (GABA)A; rho1 GABAc; 5-hydroxytryptamine-3A; NR1a/NR2A NMDA; GluR1/GluR2 AMPA; GluR6/KA2 kainate; and alpha4beta2, alpha4beta4, and alpha2beta4 nicotinic-acetylcholine] and the G-protein-coupled inward rectifying potassium channel GIRK2. We also investigated the effect of acetaldehyde on the dopamine transporter (DAT), performing dopamine uptake assays in oocytes expressing DAT. RESULTS: Acetaldehyde (1 and 10 microM) significantly enhanced alpha1 glycine receptor-mediated currents. Acetaldehyde did not affect the function of any of the other receptors tested or the potassium currents measured in GIRK2 channels. Moreover, acetaldehyde did not alter the DAT-mediated dopamine uptake. CONCLUSIONS: Our results suggest a potential minor role for acetaldehyde in the glycine receptor-mediated effects of ethanol. Otherwise, acetaldehyde does not modulate function of the neuronal receptors tested in this study, in GIRK channels or DAT, when expressed recombinantly in Xenopus laevis oocytes.