COUPLING OF K ATP CHANNELS WITH CARDIAC ENERGETICS

K ATP 通道与心脏能量的耦合

• 批准号: 6390722
• 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/6390722
• 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.

本研究的目的是阐明ATP敏感性钾通道（KATP）与心肌能量学的耦合机制，并将其转化为对内源性心脏保护机制的认识。 KATP通道是细胞代谢状态的独特传感器，其功能与缺血预处理的心脏保护作用有关。然而，耦合KATP通道与细胞能量学的机制是未知的。 我们已经发现KATP通道具有ATP酶和腺苷酸激酶样活性，并受细胞磷酸转移反应的调节。 缺血预处理诱导磷酸转移系统，包括肌酸激酶，腺苷酸激酶和糖酵解酶之间的核苷酸流量重新分配。 缺乏磷酸转移酶的转基因肌肉更容易受到代谢应激的影响。 基于这些发现，我们提出了一个新的概念，即KATP通道的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耦合到细胞能量学。 我们假设，在代谢应激，磷酰基流量的重新分配是由KATP通道，并根据心脏保护的心肌能量重塑。在这里，我们将定义：1）KATP通道亚基是否具有固有的ATP酶和/或腺苷酸激酶样活性; 2）磷酸流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以及细胞能量学的ATP通道功能; 3）磷酸转移反应与KATP通道催化活性偶联是否有助于缺血预处理。 我们将使用先进的分子生物学，生物化学和电生理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，结合质谱和31 P NMR技术，使用18 O同位素测量细胞磷酰基通量，并在正常，预处理和磷酸转移酶缺陷的转基因心脏中记录膜片钳通道。 这一建议有可能建立一个新的原则KATP通道调节的内在催化的核苷酸交换耦合通过磷酸转移反应的细胞能量。这一概念在通道生物学中具有根本的重要性，并且对于理解代谢应激下的细胞调节和保护至关重要。