小电导钙激活钾通道磷酸化相关调节及其在心血管疾病中的作用

心血管疾病严重威胁人类健康,研究表明其发生发展多与离子通道活动相关。小电导钙激活钾通道（Small conductance calcium—activated potassium channels,SK channels）广泛存在于心血管系统的可兴奋细胞中,在调节心肌细胞复极化及维持心脏正常活动中起着重要作用,为心律失常的治疗提供了高度可能的靶点。SK通道的结构及Ca2＋门控特性已有相关综述描述,本综述主要讨论SK通道的磷酸化相关调节及其在心血管系统中的病理生理作用。     

BK通道及其β1亚单位在高血压发生发展中的作用

原发性高血压(hypertension,HT)已成为危害人们健康的心血管常见病、多发病,也是一系列心脑血管疾病(如中风、冠心病、糖尿病等)的主要危险因素[1].因此深入理解HT发生、发展中的分子机制,对于预防、治疗HT有重要意义.阻力动脉的血管张力持续增加是HT的基本病理生理机制,而血管平滑肌细胞离子通道的活动是血管张力调节的主要因素[2].近年研究发现,太电导钙激活K 通道(large conduction calcium-activated potassium channels,BK)是血管平滑肌分布最丰富、单通道电导最大(＞200 pS)的K 通道,在血管张力调节中可能起负反馈调节作用,它的功能或量的改变可能是高血压的发病机制之一.     

SK4通道和BK通道对心脏起搏功能的影响

钙离子激活的钾离子通道根据其电导的大小主要分为三种类型：小电导钙离子激活的钾离子通道SK（SK1-3）,中电导钙离子激活的钾离子通道(IK或SK4)和大电导钙离子激活的钾离子通道(BK)。既往研究已表明SK、SK4和BK在心律失常,高血压,心肌再灌注损伤等的发生发展中发挥重要作用。最近研究提示SK4和BK具有调节心脏起搏的功能。本文旨在综述这两种离子通道在心脏起搏中的作用和机制及构建生物起搏的潜在可能     

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.     

心房颤动患者小电导钙激活钾通道及蛋白激酶C对其调控的研究进展

心房颤动(atrial fibrillation,AF)是临床上最为常见而复杂的心律失常,容易引起心房内血栓、脑栓塞等严重并发症而导致脑卒中、痴呆和心力衰竭.引起房颤的原因有很多,小电导钙激活钾通道(small conductance calcium-activatedpotassium channels,SK channels) 是引起房颤的新的离子通道,由于它具有心房选择性特点,从而作为新的药物治疗靶点,对SK 通道的调控可能成为治疗某些疾病的新途径[ 1 ].人类SK 通道上存在蛋白激酶C(PKC)磷酸化氨基酸序列[ 2 ],同时PKC 参与了体内多种物质的生物合成,是重要的信号传导介质.本文对近年来心房颤动患者小电导钙激活钾通道与PKC 对其调控因素的研究进展作一综述.     

Melatonin ameliorates hippocampal nitric oxide production and large conductance calcium-activated potassium channel activity in chronic intermittent hypoxia

Melatonin protects against hippocampal injury induced by intermittent hypoxia (IH). IH-induced oxidative stress is associated with decreases in constitutive production of nitric oxide (NO) and in the activity of large conductance calcium-activated potassium (BK) channels in hippocampal neurons. We tested the hypothesis that administration of melatonin alleviates the NO deficit and impaired BK channel activity in the hippocampus of IH rats. Sprague-Dawley rats were injected with melatonin (10 mg/kg, i.p.) or vehicle before daily IH exposure for 8 hr for 7 days. The NO and intracellular calcium ([Ca2+]i) levels in the CA1 region of hippocampal slices were measured by electrochemical microsenor and spectrofluorometry, respectively. The activity of BK channels was recorded by patch-clamping electrophysiology in dissociated CA1 neurons. Malondialdehyde levels were increased in the hippocampus of hypoxic rats and were lowered by the melatonin treatment. Levels of NO under resting and hypoxic conditions, and the protein expression of neuronal NO synthase (nNOS) were significantly reduced in the CA1 neurons of hypoxic animals compared with the normoxic controls. These deficits were mitigated in the melatonin-treated hypoxic rats with an improved [Ca2+]i response to acute hypoxia. The open probability of BK channels was decreased in the hypoxic rats and was partially restored in the melatonin-treated animals, without alterations in the expression of channel subunits and unitary conductance. Acute treatment of melatonin had no significant effects on the BK channel activity or on the [Ca2+]i response to hypoxia. Collectively, these results suggest that melatonin ameliorates the constitutive NO production and BK channel activity via an antioxidant mechanism against an IH-induced down-regulation of nNOS expression in hippocampal neurons.     

Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes.

In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.     

多索茶碱对支气管哮喘患者外周血嗜酸粒细胞钙依赖性钾离子通道的影响

目的研究多索茶碱对支气管哮喘(简称哮喘)患者外周血嗜酸粒细胞(EOS)钙依赖性钾离子(KCa)通道的作用及机制。方法分离8例哮喘急性发作期患者外周血EOS,并均分为对照组和多索茶碱孵育组,采用细胞贴附式膜片钳技术封接成功后在浴槽液中加入0.2μm ol/L血小板活化因子(PAF)激活KCa通道,比较两组EOS KCa通道动力学的改变。结果细胞贴附式时浴槽液中加入0.2μm ol/L PAF时出现电流活动,对照组通道开放概率为0.135±0.021、开放时间为(5.75±0.40)m s、关闭时间为(2.17±0.50)m s,多索茶碱孵育组其相应值分别为0.044±0.018、(2.39±0.13)m s、(23.73±2.50)m s,两组比较差异均有统计学意义(P均<0.05)。结论PAF能开放EOSKCa通道,多索茶碱可通过缩短EOS KCa通道开放时间,延长关闭时间,降低哮喘患者EOS KCa通道开放概率。     

Unusual calcium-activated potassium channels of bovine parathyroid cells.

Parathyroid cells have unusual responses to an increase in the external calcium concentration--a reduction in secretion and a depolarization. In an attempt to explain this behavior, we have studied voltage-clamped inside-out patches from bovine parathyroid cells. We found a potassium-selective channel that requires internal calcium to open and has a 175-pS conductance. This channel differs from calcium-activated potassium channels that have been found in other cells in that it closes when the internal calcium concentration is increased above about 160 nM. This channel can account for the depolarization that occurs in parathyroid cells in high-calcium solutions. It may also account for the reduced secretion in high-calcium solutions.     

持续性心房颤动患者心房肌细胞小电导钙激活钾通道电流的增强

目的 比较风湿性心脏病窦性心律(SR)患者和持续性心房颤动(AF)患者心房肌细胞小电导钙激活钾通道(SK2)的电流变化和通道蛋白在细胞上的分布.方法 将临床行心脏体外循环手术的患者分为SR组(15例)和AF组(7例,AF持续时间≥6个月),应用常规膜片钳全细胞记录技术记录SK2通道电流的变化,应用免疫荧光染色检测SK2在心房肌细胞上的分布.结果 在SR组和AF组心房肌细胞上均记录到内向整流钾通道混合电流,AF组电流密度明显大于SR组.在-130mV,SR组的电流密度为(-6.59±2.24)pA/pF,AF组为(-16.42±5.32)pA/pF,P＜0.01;在此基础上加入SK2特异性阻断剂apamin 100 nmol/L后,使内向电流和外向电流均有所减小,内向电流减小较为明显,两者相减的为SK2电流.AF组的SK2电流明显高于SR组;在-130 mV测试电压下,其电流密度分别为(-9.81±2.54)pA/pF与(-3.67±0.37)pA/pF,P＜0.01.免疫荧光染色试验证明SR组与AF组中均存在SK2通道蛋白.结论 两组心房肌细胞上均有SK2通道蛋白的存在,AF时SK2通道电流明显增加,这表明心肌细胞上的一种新型通道SK2也参与和介导了AF的心房电重构过程.

Abstract：

Objective To compare the amplitude of the SK2 current (small conductance calciumactivated potassium channel ) in human atrial myicytes with or without persistent atrial fibrillation(AF).Methods Right atrial appendage was obtained from 15 patients with sinus rate (SR) and 7 patients with AF underwent surgical valve replacement. Single myocyte was isolated by enzymatic dissociation method and the SK2 channel current density was recorded using whole-cell patch clamp techniques to detect the changes.Immunofluorescence was used to observe SK2 channel protein distribution on right atrial appendage. Results Using the whole cell patch-clamp recording techniques, an inward rectifier K+ mix currents could be obtained from both SR (n = 15 ) and AF (n =7 ) samples, IK1 mix currents density in single myocyte of AF group was significantly increased than in SR group [( - 16. 42 ±5.32) pA/pF vs ( -6. 59 ±2. 24) pA/pF,P ＜0. 01], which could be partially inhibited by apamin (100 nmol/L). The apamin-sensitive current was obtained by subtraction of the currents before and after treatment with apamin. SK2 current density was significantly increased in AF group than that of SR group [( - 9. 81 ± 2. 54) pA/pF vs ( - 3.67 ± 0. 37)pA/pF,P ＜ 0. 01]. SK2 channel protein was evidenced with immunofluorescence method in right atrial appendage from AF group and SR group. Conclusion SK2 channel protein and current were present in atrial myocytes. The SK2 current density was significantly increased in AF group than in SR group suggesting that the increase of SK2 current might contribute to the electrical remodeling in AF patients.     

Role of vascular calcium-activated potassium channels in the regulation of human peripheral conduit artery diameter

The role of an endothelium-derived hyperpolarizing factor (EDHF), acting through the opening of vascular calcium-activated potassium (K(Ca)) channels, in the regulation of the basal diameter of human peripheral conduit arteries has never been investigated in vivo. We measured in 7 healthy subjects the effect of the local infusion of an inhibitor of K(Ca) channels, tetraethylammonium chloride (TEA, 9 micromol/min, 8 min), on radial artery diameter (echotracking) and flow (Doppler). Endothelium-independent dilatation was assessed before and after TEA using sodium nitroprusside (SNP: 5, 10 and 15 nmol/min, 3 min each). TEA induced a decrease in radial artery diameter (2.65 +/- 0.09 to 2.52 +/- 0.09 mm: p < 0.05) and flow (9.4 +/- 1.2 to 7.4 +/- 1.1 ml/min; p < 0.01) without modification in the radial artery dilatation in response to SNP (NS). The decrease in radial artery diameter was still significant even when the decrease in flow was taken as covariate into analysis (p < 0.05). These results demonstrate the role of vascular K(Ca) channels in the regulation of basal peripheral conduit artery diameter and arteriolar tone in human strongly suggesting the involvement of an EDHF a these two levels.     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

目的 探讨二十二碳六烯酸(DHA)增加冠状动脉平滑肌细胞(SMC)大电导钙离子激活钾离子流(BK)的机制.方法酶消化法分离正常大鼠冠状动脉SMC.采用不同钾通道阻滞剂,对冠状动脉SMC的钾离子通道进行鉴定.采用全细胞膜片钳实验技术研究DHA及其代谢产物16,17-环氧二十二碳五烯酸(16,17-EDP)对冠状动脉平滑肌细胞BK通道的影响,并观察加入细胞色素P450环氧化酶抑制剂SKF525A孵育SMC后BK通道电流的变化.结果正常冠状动脉平滑肌细胞BK通道电流约占总钾离子流的(64.2±2.7)%(n=20).DHA可激活BK通道,半效浓度为(0.23±0.03)μmoL/L,但当使用细胞色素P450环氧化酶抑制剂SKF525A预孵育SMC后,DHA对BK通道的激活作用消失,而DHA代谢产物16,17-EDP可产生与DHA相同的BK通道激活作用,半效浓度为(19.7±2.8)nmol/L.结论 DHA通过细胞色素P450环氧化酶代谢途径激活平滑肌细胞BK通道,从而扩张冠状动脉.

Abstract：

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.     

大鼠肝星状细胞大电导钙激活钾通道特性与细胞增殖的关系

目的探讨肝星状细胞（HSC）钙激活钾通道特性,以及钾通道对HSC增殖的影响。方法以胶原酶循环灌流法分离大鼠HSC,用结蛋白抗体及α-抗平滑肌抗体进行分离细胞鉴定,采用单通道细胞膜片钳技术检测传代HSC钾通道的特性,通过MTT法、流式细胞术分别检测钾离子通道阻滞剂四乙胺（TEA）对HSC的增殖、细胞周期的影响。结果所分离细胞为HSC。传代活化的HSC有单通道存在,其电导值为（210.86±5.49）pS（n=7）,属于大电导通道;其通道表现出明显的电压依赖性以及对钙敏感性,TEA可明显减少外向钾离子通道开放概率。TAE对HSC的增殖抑制效应具有时间依赖与剂量依赖的特点,可使细胞周期阻滞于G0/G1期。结论 HSC上大电导钙激活钾通道可能参与了HSC活化增殖。     

Functional regulation of large conductance Ca2+-activated K+ channels in vascular diseases

The large conductance Ca²⁺-activated potassium channels, the BK channels, is widely expressed in various tissues and activated in a Ca²⁺- and voltage-dependent manner. The activation of BK channels hyperpolarizes vascular smooth muscle cell membrane potential, resulting in vasodilation. Under pathophysiological conditions, such as diabetes mellitus and hypertension, impaired BK channel function exacerbates vascular vasodilation and leads to organ ischemia. The vascular BK channel is composed of 4 pore-forming subunits, BK-α together with 4 auxiliary subunits: β₁ subunits (BK-β₁) or γ₁ subunits (BK-γ₁). Recent studies have shown that down-regulation of the BK β₁ subunit in diabetes mellitus induced vascular dysfunction; however, the molecular mechanism of these vascular diseases is not well understood. In this review, we summarize the potential mechanisms regarding BK channelopathy and the potential therapeutic targets of BK channels for vascular diseases.