EFFECTS OF EPOXYEICOSATRIENOIC ACIDS ON KATP CHANNEL

描述(改编自申请者摘要)：本提案旨在探索 EET对心脏KATP通道的作用机制。据推测，外星人 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亚基抑制的影响。 分子研究表明，KATP通道至少由两种类型组成 亚基：K通道亚基称为KIR6.2，另一个亚基 是磺酰脲受体亚单位(SUR)。KIR6.2是向内的一员 钾通道整流系列。SuR亚单位是ATP的成员之一 结合盒家族的蛋白质，并赋予通道敏感性 磺脲类药物。功能通道被假定为八聚体 由四个KIR6.2亚基和四个SUR亚基组成。胰岛β细胞 细胞KATP通道由KIR6.2和SUR1组成。心经包括 而平滑肌通道由KIR6.2和SUR2A组成 SUR2B。EET是一种强大的内皮衍生血管扩张剂，可以调节血管 增强血管钙激活钾通道的紧张素 平滑的肌肉。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个羟基生态系统四酸(HETE)。研究表明， 显示大鼠心脏含有大量的内源性EET，11，12 EET已被证明能促进心脏功能的恢复 缺血症。在正常情况下，EET以NM浓度存在于 血浆。在缺血条件下，细胞内皮细胞的形成可能是 因此，EET可能在心脏的调节中发挥作用 缺血时的电生理和血管张力。 这些假设将通过测试KATP通道上的EET来解决 电生理学。通道激活的EC50及三磷酸腺苷的作用 将对依赖抑制进行评估。EETs的结构决定因素 将探索调制信道功能所需的。的立体异构体 EET以及碳链延长和缩短的变体将是 学习。EET的分子机制将利用突变体Kir6.2进行研究 和SUR2a来确定调制的子单元要求，并将 行动地点。 第一个具体目标是确定四种EET异构体对 膜片钳方法研究大鼠心室肌细胞KATP通道。这个 灯盏细辛对大鼠心脏药理和电生理特性的影响 将对心脏KATP通道进行研究。据推测，外星人是 内源性通道激活剂。虽然情况可能是这样，但这些 实验将无法确定EET是否是内源性的 通过研究大鼠心肌细胞的激活剂。然而，这些实验将 提供了本地频道的重要特征。 第二个目标是确定EETs的结构决定因素，这些因素对 调制KATP通道。PI将调查5，6-，8，9-，11，12-和14，15- EETS探索活动的化学要求。这些实验似乎 经过深思熟虑，应该为 激活性和特异性。 第三个目标将确定EET对KATP通道影响的分子机制 通过使用克隆的KIR6.2/SUR2A通道。假设是EET调节了 通过改变三磷酸腺苷的相互作用来调节通道。从初步情况来看， 数据表明，11，12 EET导致ATP结合率下降。这将是 通过对单个KATP通道上这些作用的分析，进一步探讨了这些作用。

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.