COUPLING OF K ATP CHANNELS WITH CARDIAC ENERGETICS

本研究的目的是明确ATP敏感性钾(KATP)通道与心肌能量学的偶联机制，并将这一信息转化为了解内源性心脏保护的原理。KATP通道是细胞代谢状态的独特感受器，在缺血预适应中，通道功能与心肌保护有关。然而，KATP通道与细胞能量学耦合的机制尚不清楚。我们发现KATP通道具有ATPase和腺苷酸激酶样活性，并受细胞内磷酸转移反应的调节。缺血预适应诱导由肌酸激酶、腺苷酸激酶和糖酵解酶组成的磷酸转移系统之间的核苷酸流量的重新分配。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偶联。我们假设，在代谢应激中，KATP通道感受到了磷酸化通量的重新分布，这是心肌保护性能量重塑的基础。在这里，我们将定义：1)KATP通道亚基是否具有固有的ATPase和/或腺苷酸激酶样活性；2)依赖代谢状态的磷酸化通量分布是否调节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同步；以及3)磷酸转移反应与KATP通道催化活性是否共同参与缺血预适应。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，结合质谱学和31P核磁共振技术来使用18O同位素来测量细胞的磷酸化通量，并在正常、预适应和磷转移酶缺陷的转基因心脏中进行膜片钳通道记录。这一建议有可能建立一种新的原理，即通过磷酸转移反应将核苷酸交换耦合到细胞能量学，从而调节KATP通道。这一概念在通道生物学中具有重要意义，并对了解代谢应激下的细胞调控和保护至关重要。

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.