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Ca^2+浓度（[Ca^2+]i）的增加通常在对各种类型的刺激的响应中观察到，但[Ca^2+]i的过度增加，或者换句话说，细胞内钙超载是病理生理环境下细胞损伤的积累过程中的关键步骤之一并且非常普遍。为了使钙超载最小化，细胞有各种系统将Ca^2+挤出和/或阻止Ca^2+从外部进入。Ca^2+内流通常是由于两种不同类型的Ca^2+内流通道的开放所致：电压依赖性Ca^2+通道（VDCC）和非选择性阳离子通道。离子通道的活性直接受[Ca^2+]i的调节，通过膜电位的变化对Ca^2+内流的调节起重要作用。大电导Ca^2+激活的K^+（BK）通道广泛表达于除心肌细胞外的可兴奋细胞，也表达于一些非兴奋细胞。BK c的激活 ...更多信息 通道诱导膜超极化，降低VDCC活性并使可兴奋细胞中的Ca^2+过载最小化。我们对主要来自植物的低分子天然产物进行了调查，以寻找BK通道开放剂的新原型，因为这类药物可以在病理生理条件下减轻平滑肌组织的过度收缩或神经元中的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BK通道的有效开放剂。使用HEK 293细胞检查PiMA和其他化合物对BK通道的作用，其中BK通道的α-亚基（HEKBKα）或α和01（HEKBKαβ1）亚基均异源表达。这些化合物（10μM）对HEKBKαβ1膜电位的影响通过使用电压敏感染料DiBAC_4（3）来监测。PiMA、异海松酸、山达海松酸、二氢海松酸、二氢异海松酸和二氢异海松醇诱导大量的膜超极化。全细胞电压钳直接测量BKαβ1的开放表明，这6种化合物在1-10 μM浓度范围内激活BKαβ1，而松香酸、香紫苏醇和海松酸甲酯则无此作用。PIMA不影响Charybdotoxin对BKaβ1电流的阻断作用。在由内而外的膜片上记录BKαβ1的单通道结果显示，10 μM PiMA并不改变通道电导，但由于对Ca^2+和电压的敏感性增加，其开放概率显著增加。由于β1亚基的共表达不影响PiMA诱导的增强作用，因此推测PiMA的作用位点是BKα亚基。PiMA对BK的选择性高于克隆的小型和中型Ca^2+激活的K^+通道。可以得出结论，当从细胞膜的任一侧施加PiMA（>1μM）时，PiMA增加了Ca^<2+>和BKα的电压敏感性。PiMA和松香酸作为BK通道开放剂的效力存在显著差异，尽管它们的化学结构差异很小，但这可能有助于了解控制BKα激活的基本结构-活性关系。此外，我们还发现可能在心肌细胞线粒体中表达的BK通道样K^+通道也被激活。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。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