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

虽然细胞内钙离子浓度([Ca^&lt；2&gt；]i)的增加通常见于对各种刺激的反应，但在病理生理环境下，细胞内钙超载是细胞损伤积累过程中的关键步骤之一。为了最大限度地减少钙超载，细胞有不同的系统来排出和/或阻止钙从外部进入。钙离子进入通常是由于两种不同类型的钙离子进入通道的开启：电压依赖性钙离子通道(VDCC)和非选择性阳离子通道。离子通道的活动直接受[Ca^&lt；2&gt；]i的调节，它通过改变膜电位对钙离子的进入起着重要的调节作用。除心肌细胞外，除心肌细胞外，大电导激活的K^(BK)通道广泛表达于非兴奋性细胞，也存在于一些非兴奋性细胞。BK-c-…的激活在可兴奋的细胞中，更多的HANNEL诱导膜超极化，降低VDCC活性，最大限度地减少钙超载。我们对主要来自植物的低分子天然产物进行了综述，以寻找BK通道开放剂的新原型，因为这类药物在病理生理条件下可以降低平滑肌组织的超收缩力或降低神经元的钙超载。在60多种天然产物及其合成的衍生物中，我们发现海马酸(PIMA)及其相关化合物是BK通道的强效开放剂。用异源表达BK通道α亚单位(HEKBKα)或同时表达α和01(HEKBKαβ1)亚基的HEK293细胞，观察PIMA等化合物对BK通道的影响。用电压敏感染料DiBaC_4(3)观察这些化合物(10μM)对HEKBKαβ-1膜电位的影响。PIMA、异海松酸、沙地松异海松酸、二氢海松酸、二氢异海松酸、二氢异海马酸和二氢异海马林醇均可引起膜的大量超极化。在全细胞电压钳下直接测定BKαβ1的开放程度表明，这6种化合物对BKαβ1的激活浓度范围非常接近(1~10μM)，而松香酸、紫杉醇和海马酸甲酯则无此作用。PIMA不影响轮藻毒素对BKAβ1宏观电流的阻断作用。BKαβ1内向外贴片的单通道记录显示，10μM PIMA没有改变通道电导，但由于对钙和电压的敏感性增加，显著增加了通道的开放概率。由于β1亚基的共表达不影响PIMA诱导的增强作用，因此PIMA的作用部位可能是BKα亚基。PIMA通过克隆的小、中钙激活的钾通道对BK具有选择性。结果表明，PIMA(&gt；1μM)可增加BK-α从细胞膜两侧施加时的钙离子浓度和电压敏感性。尽管它们的化学结构只有很小的差异，但作为BK通道开放剂的PIMA和松香酸在效力上的显著差异，可能有助于深入了解控制BKα激活的基本结构-活性关系。此外，我们还发现，可能表达于心肌细胞线粒体的BK通道样钾通道也被激活。在大鼠心肌细胞上检测了PIMA对减轻缺血条件下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通道开放剂的原型，并获得了BK通道开放剂的活性-结构关系的基本信息。较少

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