COUPLING OF K ATP CHANNELS WITH CARDIAC ENERGETICS

本提案的目的是定义atp敏感的K+ (KATP)通道与心肌能量学的耦合机制，并将这些信息转化为对内源性心脏保护原理的理解。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传递系统（由肌酸激酶、腺苷酸激酶和糖酵解酶组成）中核苷酸通量的重新分布。转基因肌肉缺乏磷转移酶，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，磷酸化通量的重新分布是由KATP通道感知的，这是心肌保护能量重塑的基础。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)磷酸化通量的代谢状态依赖性分布是否调节KATP通道亚基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同位素测量细胞磷酸化通量，并在正常、预处理和磷酸化转移酶缺陷转基因心脏中进行膜片钳通道记录。这一提议有可能建立一种新的原理，通过核苷酸交换的内在催化，通过磷酸化转移反应耦合到细胞能量学，来调节KATP通道。这一概念在通道生物学中具有重要意义，对于理解细胞在代谢应激下的调节和保护至关重要。

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.