EFFECTS OF EPOXYEICOSATRIENOIC ACIDS ON KATP CHANNEL

描述（改编自申请人摘要）：本提案旨在探索 EET对心脏KATP通道的作用机制。据推测， 是KATP通道的内源性激活剂，这些作用是由 通过对ATP介导的Kir6.2亚基抑制的作用。 分子研究表明，KATP通道由至少两种类型的 亚基：K通道亚基被称为KIR6.2，另一个亚基被称为KIR6.2。 是磺酰脲受体亚单位（SUR）。KIR6.2是一个内向的成员 钾通道整流器家族。SUR亚基是ATP的一个成员， 结合盒家族的蛋白质，并赋予通道敏感性， 磺酰脲类药物。假设功能通道为八聚体 由四个KIR6.2亚基和四个SUR亚基组成。胰腺β 细胞KATP通道由KIR6.2和SUR 1组成。心脏通道由 平滑肌通道由KIR6.2和SUR 2A组成，而平滑肌通道由KIR6.2和SUR 2A组成。 SUR2B。内皮素是有效的内皮源性血管扩张剂，其调节血管紧张素转换酶的活性。 通过增强血管中钙激活钾通道来增强张力 平滑肌细胞色素P450单加氧酶将花生四烯酸转化为4 环氧二十碳三烯酸区域异构体，包括5，6-、8，9-、11，12-和14，15- EET，以及19和20羟基ecosatetronoic酸（HETE）。研究 显示大鼠心脏含有大量的内源性EET，11，12 EET已被证明可以促进全球心脏功能的恢复。 缺血在正常条件下，E12以nM浓度存在于 等离子体在局部缺血的条件下，细胞Ehrs的形成可能是 增强，因此EET可能在心脏的调节中起作用。 电生理学和血管张力。 这些假设将通过使用KATP通道测试Eclampin来解决。 电生理学通道激活的EC 50和ATP的作用 将评价依赖性抑制。Escherichia coli的结构决定因素 将探索调制通道功能所需的功能。的立体异构体 Escherichia coli和碳链延长和缩短的变体将是 研究了EET的分子机制将使用突变的Kir6.2进行检查。 和SUR 2A，以确定调制的亚基要求，并映射 行动的地点。 第一个具体目标是确定四种EET异构体对 大鼠心室肌细胞KATP通道的膜片钳研究。的 Escherichia coli的药理学和电生理学特性的影响 将研究心脏KATP通道。据推测， 通道的内源性激活剂。虽然情况可能如此，但这些 实验将无法确定Ehrs是否是内源性的 激活剂通过研究大鼠心肌细胞。然而，这些实验将 提供了原生通道的重要特征。 第二个目的是确定Ehrs重要的结构决定因素， 调节KATP通道。PI将调查5、6、8、9、11、12和14、15- 探索活动的化学要求。这些实验似乎 经过深思熟虑，应该提供新的见解的机制， 激活和特异性。 第三个目标是确定EET对KATP通道作用的分子机制 通过使用克隆的KIR6.2/SUR 2A通道。假设是，雌二醇调节 通过改变ATP的相互作用。初步认为， 数据表明，11，12 EET导致ATP结合率下降。这将是 通过分析这些对单个KATP通道的作用进一步探索。

DESCRIPTION (Adapted from Applicant's Abstract): This proposal aims to explore the mechanisms of EET on KATP channels in heart. It is hypothesized that EETs are endogenous activators of KATP channels, and these actions are mediated through effects on ATP mediated inhibition of the Kir6.2 subunit. Molecular studies indicate that KATP channels consist of at least two types of subunits: the K channel subunit is referred to as KIR6.2, and the other subunit is a sulfonylurea receptor subunit (SUR). KIR6.2 is a member of the inward rectifier family of potassium channels. The SUR subunit is a member of the ATP binding cassette family of proteins and confers channel sensitivity to the sulfonylurea drugs. The functional channel is assumed to be an octomer consisting of four KIR6.2 subunits and four SUR subunits. The pancreatic beta cell KATP channel is composed of KIR6.2 and SUR1. The cardiac channel consists of KIR6.2 and SUR2A whereas the smooth muscle channel consists of KIR6.2 and SUR2B. EETs are potent endothelium-derived vasodilators that modulate vascular tone by way of enhancement of calcium-activated potassium channels in vascular smooth muscle. Cytochrome P450 monooxygenases convert arachidonic acid to 4 epoxyeicosatrienoic acid regioisomers, including 5,6-, 8,9-, 11,12- and 14,15- EET, as well as the 19 and 20 hydroxyecosatetronoic acids (HETE). Studies have shown that rat heart contains substantial amounts of endogenous EET, and 11, 12 EET has been shown to enhance the recovery of cardiac function following global ischemia. Under normal conditions, EETs are present at nM concentrations in plasma. During conditions of ischemia, formation of cellular EETs may be enhanced, thus EET's may play a role in the modulation of cardiac electrophysiology and vascular tone during ischemia. These hypotheses will be addressed by testing EETs on KATP channels using electrophysiology. EC50s for channel activation and the effects of ATP dependent inhibition will be evaluated. The structural determinants of EETs required in modulating channel function will be explored. The stereoisomers of EETs and as well as carbon chain elongated and shortened variants will be studied. The molecular mechanisms of EET will be examined using mutant Kir6.2 and SUR2A to determine the subunit requirements for modulation and to map the sites of action. The first specific aim is to determine the effects of the four EET isomers on KATP channels in rat ventricular myocytes using patch clamp methods. The effects of EETs on the pharmacological and electrophysiological properties of cardiac KATP channels will be investigated. It is hypothesized that EETs are endogenous activators of the channel. Although this may be the case, these experiments will not be able to determine whether EETs are endogenous activators by studying rat myocytes. Nevertheless, these experiments will provide an important characterization of the native channels. The second aim is to identify the structural determinants of EETs important for modulating KATP channels. The PI will investigate 5,6-, 8,9-, 11,12- and 14,15- EETs to explore the chemical requirements for activity. These experiments seem well thought out and should provide novel insights into the mechanisms of activation and specificity. A third aim will determine molecular mechanisms of EET effects on KATP channels by using cloned KIR6.2/SUR2A channels. The hypothesis is that EETs modulate the channel through altering the ATP interaction. It is believed from preliminary data that 11,12 EET caused a decrease in the ATP binding rate. This will be further explored through analysis of these actions on single KATP channels.