COUPLING OF K ATP CHANNELS WITH CARDIAC ENERGETICS

K ATP 通道与心脏能量的耦合

• 批准号: 6091474
• 负责人: ANDRE TERZIC
• 金额: $ 28.22万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6091474
• 关键词: adenosinetriphosphatase; adenylate kinase; bioenergetics; creatine kinase; enzyme activity; genetically modified animals; glycolysis; guinea pigs; high performance liquid chromatography; laboratory mouse; mass spectrometry; myocardial ischemia /hypoxia; myocardium; nuclear magnetic resonance spectroscopy; phosphoproteins; phosphorus metabolism; potassium channel; recombinant proteins; voltage /patch clamp

The objective of this proposal is to define mechanisms coupling ATP-sensitive K+ (KATP) channels with myocardial energetics, and translate this information into understanding principles of endogenous cardio-protection. KATP channels are unique sensors of the cellular metabolic state, and channel function has been associated with cardioprotection in ischemic preconditioning. However, the mechanisms that couple KATP channels with cellular energetics are unknown. We have discovered that KATP channels possess ATPase and adenylate kinase-like activities, and are regulated by cellular phosphotransfer reactions. Ischemic preconditioning induces re-distribution of nucleotide fluxes among phosphotransfer systems, comprised of creatine kinase, adenylate kinase and glycolytic enzymes. Transgenic muscles, lacking phosphotransfer enzymes, are more vulnerable to metabolic stress. Based on these findings, we put forward a new concept that KATP channel regulation is accomplished by intrinsic catalysis of nucleotide exchange, and coupled to cellular energetics through phosphotransfer reactions. We hypothesize that, in metabolic stress, re-distribution of phosphoryl flux is sensed by KATP channels, and underlies cardioprotective energetic remodeling of the myocardium. Here, we will define: 1) whether KATP channel subunits possess intrinsic ATPase and/or adenylate kinase-like activities; 2) whether metabolic state-dependent distribution of phosphoryl flux regulates nucleotide exchange and catalytic activities in KATP channel subunits and synchronizes channel function with cellular energetics; and 3) whether phosphotransfer reactions, coupled with KATP channel catalytic activities, contribute to ischemic preconditioning. We will use advanced molecular biology, biochemical and electrophysiological techniques to characterize nucleotide exchange, protein-protein interaction, and catalytic activity in channel subunits, in conjunction with mass spectrometric and 31P NMR techniques to measure cellular phosphoryl fluxes using 18O isotopes, and patch-clamp channel recording in normal, pre-conditioned and phosphotransfer enzyme-deficient transgenic hearts. This proposal has the potential to establish a novel principle of KATP channel regulation by intrinsic catalysis of nucleotide exchange coupled through phosphotransfer reactions to cellular energetics. Such concept is of fundamental importance in channel biology, and essential to understand cellular regulation and protection under metabolic stress.

该提案的目的是定义 ATP 敏感 K+ (KATP) 通道与心肌能量耦合的机制，并将这些信息转化为理解内源性心脏保护的原理。 KATP 通道是细胞代谢状态的独特传感器，通道功能与缺血预处理中的心脏保护有关。然而，KATP 通道与细胞能量耦合的机制尚不清楚。 我们发现KATP通道具有ATP酶和腺苷酸激酶样活性，并受到细胞磷酸转移反应的调节。 缺血预处理诱导磷酸转移系统​​中核苷酸通量的重新分配，该系统由肌酸激酶、腺苷酸激酶和糖酵解酶组成。 缺乏磷酸转移酶的转基因肌肉更容易受到代谢压力的影响。 基于这些发现，我们提出了一个新概念：KATP 通道调节是通过核苷酸交换的内在催化来完成的，并通过磷酸转移反应与细胞能量耦合。 我们假设，在代谢应激中，磷酰基通量的重新分布被 KATP 通道感知，并且是心肌的心脏保护性能量重塑的基础。在这里，我们将定义：1）KATP通道亚基是否具有内在的ATP酶和/或腺苷酸激酶样活性； 2）磷酰基通量的代谢状态依赖性分布是否调节KATP通道亚基中的核苷酸交换和催化活性并使通道功能与细胞能量学同步； 3) 磷酸转移反应与 KATP 通道催化活性相结合是否有助于缺血预处理。 我们将使用先进的分子生物学、生化和电生理学技术来表征通道亚基中的核苷酸交换、蛋白质-蛋白质相互作用和催化活性，并结合质谱和 31P NMR 技术，使用 18O 同位素测量细胞磷酰基通量，并在正常、预调节和磷酸转移中进行膜片钳通道记录 缺乏酶的转基因心脏。 该提议有可能通过磷酸转移反应与细胞能量耦合的核苷酸交换的内在催化作用，建立 KATP 通道调节的新原理。这个概念在通道生物学中具有根本重要性，对于理解代谢应激下的细胞调节和保护至关重要。