大鼠kir2.1通道重组质粒的构建及其在HEK293细胞中的表达

<正>内向整流钾电流(I K1)是心肌最主要的背景外向电流,参与静息电位的维持和心肌动作电位3期终末的复极,进而调控心肌的兴奋性和传导性[1,2]。已知心肌细胞I K1通道主要由Kir2亚家族(Kir2.x)中的Kir2.1、Kir2.2和Kir2.3构成,它们的编码基因分别是KCNJ2、KCNJ12和KCNJ4。其中,Kir2.1是最重要的亚型[3],也是组成人类心肌细胞I K1电流的主要亚单位[4],大量研究表明I Kir2.1减弱或增强均可导致严重的心律失常[1,2]。基于此,国内外众多学者在抗心律失常药物筛选     

丙基丁基多巴胺对心肌内向整流钾电流的抑制作用

研究丙基丁基多巴胺对内向整流钾电流的影响。方法：应用缓慢斜坡去极化脉冲程序测定离体豚鼠心室肌细胞准稳态电流－电压关系曲线。结果：ＰＢＤＡ５,５０和１００μｍｏｌ．Ｌ＾－１浓度依赖性地降低了心肌内向整流钾电流,表明该药对ＩＫ１具有抑制作用。     

常咯啉对豚鼠和家兔单个心肌细胞钾电流的影响(英文)

目的:研究常咯啉是否对心肌细胞的钾电流有影响作用。方法:采用高阻抗密封膜片箝全细胞技术,记录分离的豚鼠和家兔心肌单个细胞的钾电流。结果:在临床用量常咯啉50μmol·L~(-1)抑制家兔心房肌细胞的瞬间外向钾电流(I_(TO))17.7％±2.4％(n=8)。但并不影响电压依赖性通道。同一剂量的常咯啉对单个家兔心室肌细胞的内向整流钾电流(I_(Kl))和单个豚鼠心室肌细胞的延迟整流钾电流(I_K)并不产生任何作用。结论:提示常咯啉具有阻制(I_(TO))的作用,而对(I_K)和(I_(Kl))无任何作用。     

苦参碱对豚鼠心肌细胞钾电流的抑制作用(英文)

目的研究苦参碱对豚鼠心肌细胞动作电位时程和钾电流的影响,探讨其抗心律失常作用的可能机制。方法应用全细胞膜片钳技术记录心室肌细胞的动作电位时程和钾电流。结果在频率0.1Hz时,苦参碱100μmol.L-1以非频率依赖方式延长动作电位复极90%的时程达40%,在-120mV抑制内向整流钾电流(IK1)接近47%,减少快激活延迟整流钾电流的尾电流达50%,对慢激活延迟整流钾电流的尾电流无影响。结论苦参碱抗心律失常的机制可能与其抑制多种钾电流和延长动作电位时程有关。     

延胡索乙素对豚鼠单个心室肌细胞钾离子通道的影响

目的研究延胡索乙素(dltetrahydropalmatine,THP)对正常豚鼠心室肌细胞钾电流的影响,旨在探讨延胡索乙素抗心律失常作用机制。方法酶解法分离豚鼠单个心室肌细胞,应用全细胞膜片钳技术记录延胡索乙素对豚鼠单个心室肌细胞钾电流的影响。结果延胡索乙素可明显抑制延迟整流钾电流(IK)和内向整流钾电流(IK1),并呈剂量依赖性。结论延胡索乙素抗心律失常作用机制可能与它对心肌细胞钾通道作用有关,THP可抑制IK和IK1,使动作电位时程(APD)和有效不应期(ERP)延长从而发挥其抗心律失常作用。     

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。     

风湿性心脏病房颤患者Ⅰk1重构的分子基础

目的研究心房颤动(AF)患者内向整流钾电流(Ⅰk1)重构的分子基础.方法将46例风湿性心瓣膜病接受换瓣手术患者分为三组,窦性心律组(SR组)18例,阵发性房颤组(PAF)7例,慢性房颤组(CAF)21例.应用半定量逆转录-聚合酶链反应(RT-PCR)技术,检测心房组织Kir2.1mRNA表达.结果和窦性心律组相比,Kir2.1mRNA在慢性房颤组中的表达明显增加(P＜0.01).结论风湿性心瓣膜病伴房颤患者Kir2.1mRNA表达水平的增加可能是Ⅰx1上调的分子基础.     

冬虫夏草水提液对单个心室肌细胞钾通道的影响

目的　观察冬虫夏草水提液对豚鼠及大鼠心室肌细胞钾通道的作用 ,探讨冬虫夏草的抗心律失常作用机制。方法　应用全细胞膜片钳技术记录冬虫夏草水提物对豚鼠单个心室肌细胞内向整流钾电流 (IK1)、延迟整流钾电流 (IK)及大鼠心室肌细胞瞬时外向钾电流 (Ito)的影响。结果　应用 0 1g·L-1(生药浓度 )冬虫夏草水提液使豚鼠单个心室肌细胞内向整流钾电流在实验电压 - 12 0mV时从给药前(- 36 37± 5 15 ) pA/pF减少到 (- 2 9 70± 5 90 ) pA/ pF(n=5 ,P <0 0 5 ) ;延迟整流钾电流在实验电压 +70mV时 ,从给药前 (9 2 1± 2 4 2 ) pA/ pF增加至 (11 5 4± 2 98)pA/ pF(n =6 ,P <0 0 1) ;使大鼠心室肌细胞瞬时外向钾电流 (Ito)在实验电压 +5 0mV时 ,从给药前 (13 36± 0 88) pA/ pF增加至 (16 4 8± 1 0 9) (n =4 ,P <0 0 1)。结论　冬虫夏草抗心律失常作用与它对心肌细胞钾通道的作用有关。它增加IK,Ito的同时抑制IK1,将会使动作电位时程缩短而不至于发生早后除极和迟后除极     

黄连素对豚鼠心肌钾离子通道的抑制作用

利用膜片钳全细胞记录的方法研究了黄连素对豚鼠心肌钾离子通道亚型的作用。结果表明,黄连素可延长心室肌细胞动作电位时程,抑制内向整流钾通道及延迟整流钾通道,并能对抗ATP敏感钾通道开放剂cromakalim(BRL-34915)引起的K^aTP电流升高及动作电位时程缩短。表明黄连素可抑制ATP敏感钾通道,提示其降血糖和抗心律失常作用与钾通道抑制有关。     

氯胺酮对豚鼠心室肌细胞中延迟整流钾电流的作用

目的验证并研究局麻药氯胺酮对豚鼠心肌延迟整流钾电流（IK）的作用,并探讨其对心脏功能的影响。方法采用II型胶原酶急性分离豚鼠心室肌细胞,利用全细胞膜片钳技术观察氯胺酮对豚鼠心室肌细胞中IK的作用。结果氯胺酮对心室肌IK有较强的抑制作用,但不影响该类型电流的整流特征。结论临床剂量的氯胺酮可以抑制心肌细胞中的IK,显示氯胺酮有致心律失常之作用,或者具有抗心律失常作用。     

Inhibition of cardiac potassium currents by pentobarbital

The inhibitory effects of the anesthetic barbiturate pentobarbital on the slow ( I(Ks)) and fast component ( I(Kr)) of cardiac delayed rectifier potassium currents ( I(K)) and on the inward rectifier potassium currents ( I(K1)) were examined in Xenopus oocytes expressing the human minK, human ether-á-go-go related gene (HERG) and guinea pig Kir2.2, respectively. Block of native I(K) ( I(Ks) and I(Kr)) and inward rectifier potassium current ( I(K1)) by pentobarbital was examined in guinea pig ventricular myocytes.In oocytes using the two electrode voltage clamp technique potassium currents of hminK-, HERG- and Kir2.2-expressing oocytes were inhibited by pentobarbital with IC50 values of 0.20, 1.58 and 0.54 mM, respectively. I(Ks) block was time- and voltage-independent and had no influence on activation at positive voltages although it shifted voltage-dependent activation to more positive voltages. Pentobarbital-induced HERG inhibition was not dependent on voltage but influenced the deactivation kinetics and shifted half-maximal activation to more negative voltages. In guinea pig cardiomyocytes, using the patch clamp technique, I(Ks) and I(Kr) were inhibited by pentobarbital with IC50 values of 0.18 mM and 2.75 mM, respectively. I(Kr) deactivation and I(Ks) activation kinetics were only slightly influenced by pentobarbital, if at all. Block of I(K1) was weakly voltage-dependent with IC(50) values of 0.26 mM (-40 mV) and 0.91 mM (-120 mV). The data show that pentobarbital suppresses both cloned ( I(K), I(Kir2.2)) and native ( I(K), I(K1)) cardiac potassium currents with the highest affinity for I(Ks).     

高胆固醇血症改变大鼠血管功能及动脉平滑肌细胞钾通道基因表达(英文)

目的:研究高胆固醇血症对大鼠主动脉功能及主动脉平滑肌上内向整流和ATP敏感钾通道基因表达的影响。方法:大鼠灌胃高胆固醇乳剂两周,制备带内皮和去内皮的主动脉血管环检测收缩和舒张反应,应用RT-PCR观察基因表达的变化。结果:高胆固醇血症明显损伤血管内皮功能,但不影响血管内皮非依赖性舒张反应;在高胆固醇血症血管平滑肌中Kir6.2mRNA表达明显升高(P<0.05),而Kir3.1mRNA表达却显著降低(P<0.05)。结论:高胆固醇血症可改变血管功能,并影响某些内向整流和ATP敏感钾通道亚型的基因表达。     

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine.

1. We have investigated the inhibition of inwardly rectifying potassium channels by the alpha-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1). 2. In skeletal muscle and at a membrane potential of -50 mV, chloroethylclonidine (CEC), an agonist at alpha2-adrenergic receptors and an antagonist at alpha1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 microM. 3. The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of alpha1-(prazosin) or alpha2-(rauwolscine) antagonists. 4. The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at -50 mV c.f. approximately 10% block at -190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be approximately 8 s at 0 mV, and the rate of unblock was described by the relationship tau=exp((Vm+149)/22) s. 5. This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine. 6. The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of alpha-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.     

Direct block of inward rectifier potassium channels by nicotine

Nicotine has been shown to depolarize membrane potential and to lengthen action potential duration in isolated cardiac preparations. To investigate whether this is a consequence of direct interaction of nicotine with inward rectifier K(+) channels which are a key determinant of membrane potentials, we assessed the effects of nicotine on two cloned human inward rectifier K(+) channels, Kir2.1 and Kir2.2, expressed in Xenopus oocytes and the native inward rectifier K(+) current I(K1) in canine ventricular myocytes. Nicotine suppressed Kir2.1-expressed currents at varying potentials negative to -20 mV, with more pronounced effects on the outward current between -70 and -20 mV relative to the inward current at hyperpolarized potentials (below -70 mV). The inhibition was concentration dependent. For the outward currents recorded at -50 mV, the IC50 was 165 +/- 18 microM. Similar effects of nicotine were observed for Kir2.2. A more potent effect was seen with I(K1) in canine myocytes. Significant blockade ( approximately 60%) was found at a concentration as low as 0.5 microM and the IC50 was 4.0 +/- 0.4 microM. The effects in both oocytes and myocytes were partially reversible upon washout of nicotine. Antagonists of nicotinic receptors (mecamylamine, 100 microM), muscarinic receptors (atropine, 1 microM), and beta-adrenergic receptors (propranolol, 1 microM) all failed to restore the depressed currents, suggesting that nicotine acted directly on Kir channels, independent of catecholamine release. This property of nicotine may explain its membrane-depolarizing and action potential duration-prolonging effects in cardiac cells and may contribute in part to its ability to promote propensity for cardiac arrhythmias.     

苄普地尔对豚鼠甲亢性肥厚心肌中延迟整流钾电流的抑制作用

目的　研究苄普地尔(bepridil)对肥厚心肌细胞延迟整流钾电流(I_K)中快激活成份(I_Kr)和慢激活成份(I_Ks)及内向整流钾电流(I_K1)的作用。方法　全细胞膜片钳技术。结果　在肥厚心肌细胞中,Bepridil 30 μmol·L- 1 对I_Kr及I_Ks有阻断作用,抑制率分别为20.9% (0 mV)和27.2 % (+50 mV)。“Envelopeoftail”显示bepridil对I_Ks的阻断作用大于I_Kr。Bepridil(1 - 100 μmol·L^-1 )浓度依赖性的阻断I_Ks及I_Kr,其IC₅₀ 分别为23.8μmol·L^-1 和46.7μmol·L^-1 。Bepridil 30 μmol·L^-1 也能阻断I_K1 ,抑制率为15.1% (- 100 mV) ,但不影响其反转电位。结论　Bepridil对甲亢性豚鼠肥厚心肌中I_Ks,I_Kr及I_K1有阻断作用     

Block of heart potassium channels by clofilium and its tertiary analogs: relationship between drug structure and type of channel blocked

The whole-cell arrangement of the patch clamp was used to study delayed rectifier and inward rectifier K channels in isolated guinea pig ventricular cells. Block of these channels by an externally applied quaternary nitrogen compound, clofilium, and two of its tertiary nitrogen structural analogs (LY97241 and LY97119) were investigated. Clofilium reduced delayed rectifier current but had little effect on inward rectifier currents in concentrations as high as 100 microM. The block of delayed rectifier did not reverse upon washout. In contrast, lower concentrations of the tertiary analogs blocked both delayed rectifier and inward rectifier K currents. Onset of block of delayed rectifier was fast and block was reversible. The onset of block of inward rectifier by the tertiary compounds was slower than for delayed rectifier current and more difficult to reverse. We conclude from this work that tertiary, but not quaternary, clofilium blocks inward as well as delayed rectifier channels in these cells. Block of inward rectifier current is presumably caused by access to a receptor for the molecule that is gained by the tertiary, but not the quaternary, forms of the drug.     

Endothelin-1 inhibits inward rectifier K+ channels in rabbit coronary arterial smooth muscle cells through protein kinase C

We studied inward rectifier K+ (Kir) channels in smooth muscle cells isolated from rabbit coronary arteries. In cells from small- (<100 microm, SCASMC) and medium-diameter (100 approximately 200 microm, MCASMC) coronary arteries, Kir currents were clearly identified (11.2 +/- 0.6 and 4.2 +/- 0.6 pA pF at -140 mV in SCASMC and MCASMC, respectively) that were inhibited by Ba(2+) (50 microm). By contrast, a very low Kir current density (1.6 +/- 0.4 pA pF) was detected in cells from large-diameter coronary arteries (>200 microm, LCASMC). The presence of Kir2.1 protein was confirmed in SCASMC in a Western blot assay. Endothelin-1 (ET-1) inhibited Kir currents in a dose-dependent manner. The inhibition of Kir currents by ET-1 was abolished by pretreatment with the protein kinase C (PKC) inhibitor staurosporine (100 nM) or GF 109203X (1 microm). The PKC activators phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced Kir currents. The ETA-receptor inhibitor BQ-123 prevented the ET-1-induced inhibition of Kir currents. The amplitudes of the ATP-dependent K+ (KATP), Ca(2+)-activated K+ (BKCa), and voltage-dependent K+ (KV) currents, and effects of ET-1 on these channels did not differ between SCASMC and LCASMC. From these results, we conclude that Kir channels are expressed at a higher density in SCASMC than in larger arteries and that the Kir channel activity is negatively regulated by the stimulation of ETA-receptors via the PKC pathway.     

Inward rectifier potassium currents as a target for atrial fibrillation therapy

Subunits of inwardly rectifying potassium channels (Kir) are expressed in many different tissues of the human body. Inward rectifier currents expressed in the heart are constituted by pore-forming alpha-subunits of Kir2, Kir3, and Kir6 subfamilies. Characteristic properties of inward rectifiers comprise small outward conductances that nevertheless are important to terminal repolarization of cardiac action potentials. There is considerable difference in the regional expression of cardiac Kir channels, and subunits are additionally regulated by specific disease conditions. Resulting changes facilitate occurrence and persistence of atrial fibrillation (AF). For instance, upregulation of Kir2.1 protein and resultant current I K1 is a hallmark of AF-related ionic remodeling. Increased I K1 helps to stabilize atrial rotors, and current inhibition has accordingly been suggested as an antiarrhythmic approach for AF therapy. But there are caveats to I K1 inhibition per se, and there is no specific inhibitor of Kir2 channels. Modulation of I K1 rectification properties seems theoretically interesting for manipulation of Kir2 currents as an antiarrhythmic approach. Kir3-based muscarinic currents (I KACh) are functionally upregulated during AF through increased constitutive activity (passing current in the absence of an agonist). Upregulated I KACh supports sustenance of the arrhythmia. There is considerable intraatrial diversity in the expression of underlying Kir3.1/Kir3.4 subunits, but atrial-specific localization makes inhibition of this current a potentially interesting antiarrhythmic target devoid of ventricular side effects. Experimental studies of specific inhibitors indicate efficacy in various disease models. The role of I KATP remodeling under AF conditions has not been extensively studied, but present evidence indicates current downregulation and modulation of IKATP seems less promising than that of other inward rectifiers.