The regulation of ion channel activity by intracellular Ca2+ dynamics and survey of candidate molecules available for therapy of related diseases

细胞内Ca2动力学对离子通道活性的调节及可用于治疗相关疾病的候选分子的调查

• 批准号: 14370786
• 负责人: IMAIZUMI Yuji
• 金额: $ 8.7万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370786/
• 关键词: potassium channel; intracellular calcium dynamics; drug development; channel opener; calcium activated potassium channel; pimaric acid; BK channel; ピマル酸; K^+チャネル開口薬; イオンチャネル; 細胞内カルシウム; カルシウム依存性カリウムチャネル; カリウムチャネル開口薬; 虚血性細胞障害; 気管支喘息; カリウムチャネル

Although the increase in intracellular Ca^<2+> concentration ([Ca^<2+>]i) is commonly observed in responses to various types of stimuli, the excess increase in [Ca^<2+>]i, or in other words, overload of cells with calcium is one of the key steps and very popular in the process of accumulation in cellular damages under pathophysiological settings. To minimize calcium overload, cells have various systems to extrude Ca^<2+> to and/or prevent Ca^<2+> entry from outside. The Ca^<2+> entry is usually due to opening of two separate types of Ca^<2+> entry channels; voltage-dependent Ca^<2+> channels (VDCCs) and non selective cation channels. Ion channels, whose activities are directly modulated by [Ca^<2+>]i, strongly contributes to the regulation of Ca^<2+> entry via the changes in membrane potential. Large conductance Ca^<2+> activated K^+ (BK) channels are ubiquitously expressed in excitable cells except cardiac myocytes and also expressed in some non-excitable cells. The activation of BK c … More hannel induces membrane hyperpolarization, reduces VDCC activity and minimizes Ca^<2+> overload in excitable cells. We surveyed low molecular natural products mainly from plants to find out new prototype of BK channel opener, since this type of agents may reduce the hyper contractility of smooth muscle tissues or Ca^<2+> over load in neurons under pathophysiological conditions. Among over 60 natural products and their synthesized derivatives, we found pimaric acid (PiMA)and related compounds as potent openers of BK channel. Effects of PiMA and other compounds on BK channels were examined using HEK293 cells, in which either the a-subunit of BK channel (HEKBKα) or both α and 01 (HEKBKαβ1) subunits was heterologously expressed. Effects of these compounds (10μM) on the membrane potential of HEKBKαβ1 were monitored by use of DiBAC_4(3), a voltage-sensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BKαβ1 opening under whole cell voltage-clamp showed that these six compounds activated BKαβ1 in a very similar concentration range (1-10 μM), in contrast abietic acid, sclareol and methyl pimarate had no effect. PIMA did not affect the charybdotoxin-induced block of macroscopic BKaβ1 current. Single channel recordings of BKαβ1 in inside-out patches showed that 10 μM PiMA did not change channel conductance, but significantly increased its open probability due to increase in sensitivity to Ca^<2+> and voltage. Since co-expression of β1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BKα subunit. PiMA was selective to BK over cloned small and intermediate Ca^<2+> activated K^+ channels. It can be concluded that PiMA (>1μM) increases Ca^<2+> and voltage-sensitivity of BKα when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BKα activation. Moreover, we found that BK channel-like K^+ channels, which may be expressed in mitochondria of cardiac myocytes, are also activated PiMA. The protective effects of PiMA to reduced cell injury in ischemic conditions were also detected in rat cardiac myocytes. Taken together, we found a useful compound, PiMA, as a prototype of BK channel opener and obtained basic information about the activity-structure relationships for BK channel opener. Less

虽然细胞内Ca^<2+>浓度（[Ca^<2+>]i）的增加通常在对各种刺激的反应中观察到，但[Ca^<2+>]i的过度增加，或者换句话说，细胞钙超载是病理生理环境下细胞损伤积累过程中的关键步骤之一，也是非常普遍的。为了减少钙超载，细胞有不同的系统来挤压Ca^<2+>到和/或阻止Ca^<2+>从外部进入。Ca^<2+>进入通常是由于两种不同类型的Ca^<2+>进入通道的打开；电压依赖性Ca^<2+>通道（VDCCs）和非选择性阳离子通道。离子通道的活性由[Ca^<2+>]i直接调节，通过膜电位的变化对Ca^<2+>的进入有很大的调节作用。大电导Ca^<2+>激活的K^+ (BK)通道在除心肌细胞外的可兴奋细胞中普遍表达，也在一些不可兴奋细胞中表达。BK - c - More通道的激活可诱导细胞膜超极化，降低VDCC活性，使可兴奋细胞的Ca^<2+>过载最小化。我们主要从植物中寻找低分子天然产物，以寻找BK通道打开剂的新原型，因为这类药物可能在病理生理条件下降低平滑肌组织的超收缩性或神经元Ca^<2+>过载。在60多种天然产物及其合成衍生物中，我们发现海松酸（PiMA）及其相关化合物是有效的BK通道打开剂。以BK通道a亚基（HEKBKα）或α和01亚基（HEKBKαβ1）异种表达的HEK293细胞为实验对象，研究了PiMA等化合物对BK通道的影响。采用电压敏感染料DiBAC_4(3)监测了这些10μM化合物对HEKBKαβ1膜电位的影响。PiMA、异海松酸、檀香异海松酸、二氢海松酸、二氢异海松酸和二氢异海松醇诱导了大量的膜超极化。在全细胞电压箝位下直接测量BKαβ1的开度，结果表明，这6种化合物对BKαβ1的活化作用在非常相似的浓度范围（1 ~ 10 μM）内，而松木酸、香核醇和海海脂酸甲酯对BKαβ1没有作用。PIMA不影响炭黑毒素诱导的宏观BKaβ1电流阻滞。BKαβ1的单通道记录表明，10 μM PiMA没有改变通道电导，但由于对Ca^<2+>和电压的敏感性增加，通道打开概率显著增加。由于β1亚基的共表达不影响PiMA诱导的增强，因此PiMA的作用位点可能是BKα亚基。PiMA对克隆的Ca^<2+>激活的K^+小通道和中间通道具有选择性。结果表明，PiMA （> μ m）在细胞膜两侧均可增加Ca^<2+>和BKα的电压敏感性。PiMA和枞酸作为BK通道打开剂的效力存在显著差异，尽管它们的化学结构只有非常小的差异，这可能为了解控制BKα激活的基本结构-活性关系提供了见解。此外，我们发现心肌细胞线粒体中可能表达的BK通道样K^+通道也被PiMA激活。在大鼠心肌细胞中也检测到PiMA对缺血条件下减少细胞损伤的保护作用。综上所述，我们发现了一个有用的化合物PiMA作为BK通道打开剂的原型，并获得了BK通道打开剂的活性-结构关系的基本信息。少

项目成果

Purinergic modulation of pacemaker Ca^<2+> activity interstitial cells of Cajal

起搏器 Ca^<2> 活性 Cajal 间质细胞的嘌呤能调节

• 发表时间: 2005
• 期刊: Neuropharmacology 48
• 作者: Furuzono S.et al.

KB-R7943 reveals possible involvement of Na^+-Ca^<2+> exchanger in elevation of intracellular Ca^<2+> in rat carotide arterial myocytes

KB-R7943揭示Na^-Ca^2交换器可能参与大鼠颈动脉肌细胞细胞内Ca^2升高

• 发表时间: 2004
• 期刊: Journal of Smooth Muscle Research 40
• 作者: Liu H.-N.et al.;Liu H.-N.et al.;Furuzono S. et al.;Hatano N. et al.;Aoyama M. et al.;Takai N. et al.
• 通讯作者: Takai N. et al.

Hinata M, Yamamura H, Li L, Watano T, Imaizumi Y, Kimura J: "Stoichiometry of Na^+-Ca^<2+> exchange is 3 : 1 in guinea-pig ventricular myocytes"Journal of Physiology. 545・2. 453-461 (2002)

Hinata M、Yamamura H、Li L、Watano T、Imaizumi Y、Kimura J：“豚鼠心室肌​​细胞中 Na^+-Ca^<2+> 交换的化学计量为 3 : 1”生理学杂志 545・2。 .453-461 (2002)

Muraki K. et al.: "The TRV2 is a component of osmotically-sensitive cation channels in murine aortic myocytes"Circulation Research. 93(9). 829-838 (2003)

Muraki K. 等人：“TRV2 是小鼠主动脉肌细胞渗透敏感阳离子通道的一个组成部分”循环研究。

Hatano N. et al.: "Dihydropyridine Ca^<2+> channel antagonists and agonists block Kv4.2,Kv4.3,and Kv1.4K^+ channels in HEK293 cells"British Journal of Pharmacology. 139(3). 533-544 (2003)

Hatano N.等人：“二氢吡啶Ca 2+ 通道拮抗剂和激动剂阻断HEK293细胞中的Kv4.2、Kv4.3和K​​v1.4K 2 通道”英国药理学杂志。