Potassium Channels as Macromolecular Complexes

描述(申请人提供)：心肌和冠状动脉肌细胞中的ATP敏感钾[KATP]通道将细胞代谢状态与膜兴奋性结合在一起，从而有助于调节组织对生理和病理生理刺激的反应。在心肌中，KATP通道的开放参与了应激反应并对缺血发作起到保护作用。在冠状动脉血管系统中，K(ATP/NDP)通道参与基础血流的调节以及对代谢损伤(缺氧性扩张和缺血性反应性充血)的反应。我们发现糖酵解酶与KATP通道亚单位相关，我们假设糖酵解酶是KATP通道大分子复合体的组成部分，糖酵解酶在生理和病理生理条件下调节KATP通道的活性，在心肌细胞以及冠脉平滑肌和内皮中都是如此。在第一个特定目标中，我们将调查糖酵解复合体的酶与KATP通道相关的假设。利用免疫共沉淀法，我们将研究糖酵解酶与单个KATP通道亚基(Kir6.1、Kir6.2、SUR1、SUR2A和SUR2B)相互作用的特异性。将进行天然蛋白质的免疫共沉淀分析，以研究生理相关条件下的相互作用。还将使用先进的蛋白质组学方法(ICAT和iTRAQ)研究蛋白质相互作用，这有可能发现更多新的KATP通道相互作用蛋白质。在第二个目标中，我们将检验如下假设，即糖酵解酶与KATP通道亚单位的物理相互作用是必需的，并且通道调制是由于通道复合体微环境中核苷酸水平的改变而发生的。这将使用突变的KATP通道亚基(缺乏与糖酵解酶的相互作用或改变的核苷酸敏感性)来实现。在最后一个目标中，我们将研究糖酵解和KATP通道在心肌细胞和冠脉血管缺血保护方面的相互作用。为此，我们将利用我们的遗传小鼠模型，该模型专门在心肌细胞、平滑肌或内皮细胞中表达显性-负性K(ATP)通道亚单位。我们的发现可能对理解生理和非病理生理条件下KATP通道在心脏和冠状动脉血管系统中的作用有重要意义。

DESCRIPTION (provided by applicant): ATP-sensitive potassium [KATP] channels in the heart muscle and coronary myocytes couple cellular metabolic status to membrane excitability, thereby contributing to the regulation of tissue responses to physiological and pathophysiological stimuli. In the heart muscle, opening of KATP channels participate in the stress response and protect against ischemic episodes. In the coronary vasculature, K(ATP/NDP) channels contribute to the regulation of basal flow as well as responses to metabolic impairment (hypoxic dilatation and ischemic reactive hyperemia). We found glycolytic enzymes to associate with KATP channel subunits, We hypothesize that glycolytic enzymes are integral components of the KATP channel macromolecular complex and that glycolytic enzymes regulate KATP channel activity under physiological and pathophysiological conditions, both in the cardiac myocyte as well as in the coronary smooth muscle and endothelium. In a first Specific Aim, we will investigate the hypothesis that enzymes of the glycolytic complex are associated with the KATP channel. Using co-immunoprecipitation assays we will investigate the specificity of interaction of glycolytic enzymes with individual KATP channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B). Co-immunoprecipitation assays of native proteins will be performed to investigate interactions under physiologically relevant conditions. Protein interactions will also be investigated using advanced proteomic approaches (ICAT & ITRAQ), which has the potential to uncover additional novel KATP channel interacting proteins. In a second Aim, we will examine the hypothesis that physical interaction of glycolytic enzymes with KATP channel subunits is required and that channel modulation occurs because of altered nucleotide levels in the microenvironment of the channel complex. This will be accomplished using mutant KATP channel subunits (lacking interaction with glycolytic enzymes or altered nucleotide sensitivity). In a final Aim, we will investigate the interaction of glycolysis and KATP channels in the context of ischemic protection in cardiac myocytes and the coronary vasculature. To this end, we will utilize our genetic mouse models that express dominant-negative K(ATP) channel subunits specifically in the cardiac myocyte, smooth muscle or endothelium. Our findings may have important implications for understanding the role of KATP channels in the heart and coronary vasculature under physiological and non-pathophysiological conditions.