褪黑素激活大鼠大脑中动脉BKCa通道的受体依赖和非受体依赖途径

目的：研究褪黑素通过大电导Ca²⁺激活K⁺（BK_Ca）通道介导大脑中动脉张力变化的作用机制。方法：8周龄雄性Wistar大鼠,麻醉后取大脑中动脉,酶消化法急性分离脑中动脉平滑肌细胞,采用膜片钳技术全细胞记录模式检测细胞外液加入褪黑素前后BK_Ca通道和电压门控钾（K_V）通道的电流密度,褪黑素受体抑制剂2-苯基-N-乙酰色胺（luzindole）孵育后,全细胞记录模式记录加入褪黑素后BK_Ca通道电流幅值和贴附式单通道记录模式记录加入褪黑素后BK_Ca通道Po值,内面向外记录模式检测加入褪黑素后BK_Ca通道电导（G）,开放概率（Po）,平均开放时间（To）和关闭时间（Tc）。结果：①褪黑素（100 μmol/L）显著增加全细胞BK_Ca通道电流密度,但对K_V通道电流密度无显著影响;②luzindole （1 μmol/L）显著抑制褪黑素引起的BK_Ca通道电流密度增加;③贴附式单通道记录模式下,褪黑素（100 μmol/L）增加BK_Ca单通道Po值,luzindole （1 μmol/L）显著抑制褪黑素引起的Po增加;④内面向外单通道记录模式下,褪黑素（1 μmol/L,100 μmol/L）缩短BK_Ca单通道的To和Tc值,且Tc较To显著缩短;结论：褪黑素通过受体依赖和非受体依赖途径激活BK_Ca通道,介导大脑中动脉血管舒张。     

Flag和GFP双标记的BKCa通道α亚基表达质粒的构建、鉴定和序列分析

目的：采用重叠PCR法构建表达质粒,为下一步研究通道功能奠定基础。方法：在已有编码大电导钙激活钾通道（BK_Ca）通道α亚单位的表达质粒pcDNA3.1-hSlo的基础上,采用重叠PCR法构建Flag和GFP双标签标记的表达质粒pcDNA3.1-Flag-hSlo-GFP （Flag-hSlo-GFP）。结果：构建的表达质粒Flag标签插入BK_Ca通道的S1-S2胞外环,GFP标签连接BK_Ca通道的胞内C末端,测序结果证实质粒构建成功。结论：成功构建BK通道基因表达质粒Flag-hSlo-GFP,重叠PCR能够很好的用于长片段基因扩增和插入片段的实验。     

钾通道活化剂对豚鼠气道平滑肌电压依赖性钾通道特性的影响

钾通道活化剂可激活钾离子通道并松驰支气管平滑肌,在急性分离的豚鼠支气管平滑肌细胞上,用膜片钳技术的细胞贴附式和内面向外式研究了其对电压依赖性钾通道的直接作用。结果证实：在全细胞记录条件下,卡吗克林和拉吗克林不影响静息膜电位。但在去极化时可使通道电导从７５．２±５．１ｐＳ分别增大到８５．９±１１．８ｐＳ和８２．１±５．５ｐＳ。通道动力学特性也发生了改变,通道平均开放时间的τｏ２值延长和开放概率显著增加,其中拉吗克林的作用更为强。两者均可诱发通道出现多级开放。表明这两类活化剂可使去极化时钾离子外流增加。     

过氧化氢诱发的小脑皮质凋亡神经元膜K+通道电流的变化

用新生ＳＤ大鼠小脑皮质细胞进行培养,用Ａｒａ－Ｃ抑制非神经元生长,以Ｈ２Ｏ２诱发神经元凋亡。用膜片细胞贴附式观察了凋亡神经元膜钾离子通道电流的变化,结果表明,凋亡小脑皮质神经元膜Ｋ通道在不同箍位电压下,通道电流（ＩＫ）幅度小于正常神经元的,单位电导小于正常神经元的,通道的平均开放时间,开放概率、短开放及长开放时间常数亦均小于正常神经元的。说明小脑皮质凋亡神经元Ｋ通道活动减弱     

心肌钠,钾离子通道的分子生物学进展

分子生物学和电生理学（膜片钳技术）的联合应用研究对于阐明心肌钠、钾离子通道的分子结构与功能表达已取得突破性进展。现已证明,心肌钠离子通道主要由基β－亚单位作为功能性单位以表达出钠通道竭尽成分,其α－亚单位的存在可能改变钠电流的动力学性质。多种钾离子通道及其电流成分亦已在心肌细胞上鉴定出来。分子生物学研究已揭示出与心肌钠、钾通道功能表达有关的基因结构。在生理（例如心肌发生学过程中）情况下或病变心肌时     

膜脂物理属性及化学成分对BKCa钾离子通道的调节作用

大电导钙激活电压门控型钾离子通道(BKCa通道)广泛分布于各种组织中,主要由胞内钙离子浓度增加和细胞膜去极化而激活.此外多种膜脂,如脂肪酸、胆固醇、鞘糖脂等可修饰该通道的功能.该通道参与细胞内信号转导、细胞的兴奋及代谢调节等多种生理过程,其功能异常牵涉到特发性癫痫、高血压等疾病的发生.因而对BKCa通道功能的调节作用的研究具有重要的生理学及病理学意义.文章将主要介绍膜脂对BKCa通道功能的调节作用.     

中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道

我们用电压箝方法研究了中华大蟾蜍卵母细胞的膜生理特性。发现卵母细胞膜去极化至-30mV及更偏正时,有一持续的外向电流出现,该电流与去极化程度约呈正比增加,当膜电位箝在20mV时其峰值达3.7±1.4μA。该电流被钾离子通道拮抗剂TEA和4-AP抑制,TEA半抑制浓度为2.6mmol/L。氯通道拮抗剂9-AC(2.5mmol/L)无抑制作用。无钙的或钙离子浓度增加三倍的胞外灌流液均对该电流无影响、该外向电流的逆转电位随胞外钾离子浓度的改变而变化。胞外钾离子浓度增加十倍,逆转电位约增加47.3mV,而胞外钠、钙或氯离子浓度的改变对逆转电位基本上无影响,因此该电流可被认为主要是电压依赖性钾离子流。取自冬眠蟾蜍的卵母细胞经孕酮诱发成熟后,电压依赖性钾离子流减小,仅为原来的1/20-1/30,而取自全年在高温饲养的蟾蜍的卵母细胞经孕酮处理后未见成熟,其电压依赖性钾离子流仅减小至原来的三分之一。     

BK通道功能性复合体的研究进展

离子通道可以与其他蛋白质耦合形成稳定的大分子复合物,以确保信号转导的效率和准确性。大电导、钙离子激活的钾离子通道(BK通道)的核心是由形成孔区的α亚基组成的四聚体,它具有BK通道的基本生理功能。在不同的组织内,BKα可以与不同的辅助性亚基结合,使通道功能变得复杂多样。BK通道可以将细胞兴奋性与细胞内的钙离子信号联接在一起,在血流、泌尿、免疫、神经递质释放等许多生命过程中发挥着重要的调节作用。近年来,大量的研究工作表明,BK通道可以与钙离子通道、细胞骨架蛋白、蛋白激酶等生物大分子形成功能性复合物,这对通道功能调控和信号转导等生命活动具有重要的生理意义。本文综述了这些BK通道功能复合体的主要分类、功能特性以及生理学意义,并对其未来的研究前景进行展望。     

BK通道功能性复合体的研究进展

离子通道可以与其他蛋白质耦合形成稳定的大分子复合物,以确保信号转导的效率和准确性。大电导、钙离子激活的钾离子通道（BK通道）的核心是由形成孔区的d亚基组成的四聚体,它具有BK通道的基本生理功能。在不同的组织内,BKα可以与不同的辅助性亚基结合,使通道功能变得复杂多样。BK通道可以将细胞兴奋性与细胞内的钙离子信号联接在一起,在血流、泌尿、免疫、神经递质释放等许多生命过程中发挥着重要的调节作用。近年来,大量的研究工作表明。BK通道可以与钙离子通道、细胞骨架蛋白、蛋白激酶等生物大分子形成功能性复合物,这对通道功能调控和信号转导等生命活动具有重要的生理意义。本文综述了这些BK通道功能复合体的主要分类、功能特性以及生理学意义,并对其未来的研究前景进行展望。     

双孔钾通道TREK-1结构、分布、调控因素及其抗癫痫作用的研究进展

钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70余种钾离子通道亚型,其中双孔钾离子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统的单孔钾离子通道差异很大。双孔钾离子通道,具有4个跨膜片段,形成独特的2个孔道结构域,主要介导背景钾电流。由于其介导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1几乎表达于机体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

Bupivacaine inhibits large conductance,voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K⁺ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca²⁺-activated K⁺ channels (BK_Ca). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K⁺ currents carried by BK_Ca channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC₅₀ 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K⁺ currents (~45% blockage) in which, under our working conditions, BK_Ca is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK_Ca channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Ginsenoside Rg<Subscript>3</Subscript> enhances large conductance Ca<Superscript>2+</Superscript>-activated potassium channel currents: A role of Tyr360 residue

Ginsenosides, active ingredients of Panax ginseng, are known to exhibit neuroprotective effects. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are key modulators of cellular excitability of neurons and vascular smooth muscle cells. In the present study, we examined the effects of ginsenosides on rat brain BK_Ca (rSlo) channel activity heterologously expressed in Xenopus oocytes to elucidate the molecular mechanisms how ginsenoside regulates the BK_Ca channel activity. Ginsenoside Rg₃ (Rg₃) enhanced outward BK_Ca channel currents. The Rg₃-enhancement of outward BK_Ca channel currents was concentration-dependent, voltage-dependent, and reversible. The EC₅₀ was 15.1 ± 3.1 μM. Rg₃ actions were not desensitized by repeated treatment. Tetraetylammonium (TEA), a K⁺ channel blocker, inhibited BK_Ca channel currents. We examined whether extracellular TEA treatment could alter the Rg₃ action and vice versa. TEA caused a rightward shift of the Rg₃ concentration-response curve (i.e., much higher concentration of Rg₃ is required for the activation of BK_Ca channel compared to the absence of TEA), while Rg₃ caused a rightward shift of the TEA concentration-response curve in wild-type channels. Mutation of the extracellular TEA binding site Y360 to Y360I caused a rightward shift of the TEA concentration-response curve and almost abolished both the Rg₃ action and Rg₃-induced rightward shift of TEA concentration-response curve. These results indicate that Tyr360 residue of BK_Ca channel plays an important role in the Rg₃-enhancement of BK_Ca channel currents.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Cloning of large-conductance Ca-activated K channel α-subunits in mouse cardiomyocytes

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BK_Ca channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BK_Ca channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BK_Ca channel subfamily M alpha member 1. These findings suggest that a unique BK_Ca channel α-subunit is expressed in mouse cardiomyocytes.     

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK_Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK_Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK_Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca²⁺ and voltage without altering the unitary conductance and the K⁺ selectivity. Our results suggest a novel mechanism by which exercise training increases the K⁺ currents by changing the BK_Ca channel activities and kinetics.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: Evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK_Ca channels opener NS1619 reversed thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; BK_Ca channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca²⁺ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK_Ca channels may play a regulatory role in it. (Mol Cell Biochem 269: 37–47, 2005)     

Role of Voltage-Gated K+ (KV) Channels in Vascular Function

Voltage-dependent K⁺ (K_V) channels represent the most diverse group of K⁺ channels ubiquitously expressed in vascular smooth muscles. The K_V channels, together with other types of K⁺ conductances, such as Ca²⁺-activated (BK_Ca), ATP-sensitive (K_ATP), and inward rectifier, play an important role in the control of the cell membrane potential and regulation of the vascular contractility. Comparison of the expression of different K_V channel isoforms obtained from RT-PCR studies showed that virtually all K_V genes could be detected in vascular smooth muscle cells (VSMC). Based on the analysis of both mRNA and protein expressions, it is likely that K_V1.1, K_V1.2, K_V1.3, K_V1.5, K_V1.6, K_V2.1, and K_V3.1b channel isoforms are mainly responsible for the delayed rectifier current characterized electrophysiologically in most VSMC types studied to date. It has been recently demonstrated by our research group and by others that functional expression of multiple K_V channel α-subunits is not homogeneous and varies in different vascular beds of small and large arteries. Growing evidence suggests that in some small arteries, e.g., cerebral arteries and arterioles, the K_V channels are activated at more negative membrane voltages than BK_Ca, thus making a greater contribution to the control of vascular tone. Our data also suggest that in some blood vessels, such as the rat aorta and mouse small mesenteric arteries, the K_V channel current (identified mainly as passed through K_V2.1 channels), but not BK_Ca, is the predominant conductance activated even under conditions where intracellular Ca₂₊ concentration is increased up to 200 nM. In addition, our data indicate that the K_V2.1 channel current could also contribute to the regulation of the induced rhythmic activity in the rat aorta in vitro acting as a negative feedback mechanism for membrane depolarization. We and other experimenters also demonstrated that functional expression of K_V channels is a dynamic process, which is altered under normal physiological conditions (e.g., during the development of the vessels), and in various pathological states (e.g., pulmonary hypertension developing during chronic hypoxia). Recent findings also suggest that activation of K_V channels can also play a role in vascular apoptosis (causing loss of intracellular K⁺ and subsequent cell shrinking, one of the essential prerequisites of cellular apoptosis). To summarize, the K^V channels are essential for normal vascular function, and their expression and properties are altered under abnormal conditions. Therefore, understanding of the molecular identity of native K_V channels and their functional significance and elucidation of the mechanisms, which govern and control the expression of the K_V channels in the vasculature, represent an important and challenging task and could also lead to the development of useful therapeutic strategies for the treatment of cardiovascular diseases.     

Biphasic effects of H2O2 on BKCa channels

Abstract

The inhibitory or activating effect of H₂O₂ on large conductance calcium and voltage-dependent potassium (BK_Ca) channels has been reported. However, the mechanism by which this occurs is unclear. In this paper, BK_Ca channels encoded by mouse Slo were expressed in HEK 293 cells and BK_Ca channel activity was measured by electrophysiology. The results showed that H₂O₂ inhibited BK_Ca channel activity in inside-out patches but enhanced BK_Ca channel activity in cell-attached patches. The inhibition by H₂O₂ in inside-out patches may be due to oxidative modification of cysteine residues in BK_Ca channels or other membrane proteins that regulate BK_Ca channel function. PI3K/AKT signaling modulates the H₂O₂-induced BK_Ca channel activation in cell-attached patches. BK_Ca channels and PI3K signaling pathway were involved in H₂O₂-induced vasodilation and H₂O₂-induced vasodilation by PI3K pathway was mainly due to modulation of BK_Ca channel activity.