SUMO-regulation of ion channels via PIP2

项目摘要 心肌细胞中的离子通道决定心跳的形状、持续时间和频率。许多因素 改变通道的功能会导致人类心脏病，包括基因突变，药物不良反应， 副作用，或改变调节通道活性的细胞信号通路。该提案基于 对人类心脏功能至关重要的两种离子通道类型的调节的新见解：（1） Kir 2通道传递IK 1，IK 1是一种有助于心脏活动长期平台期的电流 （2）电压门控钠通道，介导INa的NaV1.5， 动作电位的快速上升。 Kir 2通道（Kir2.1至Kir2.4）的活性依赖于普遍存在的阴离子膜 磷脂，磷脂酰肌醇4，5-二磷酸（PIP 2）。PIP 2调节多种细胞过程， 蛋白质，包括TRP，Kir和至少一些电压门控钾通道。重要的研究工作 表征了PIP 2和Kir 2通道的结构-功能关系，包括PIP 2的晶体结构 与Kir2.2复合。许多疾病相关的突变和细胞信号通路改变Kir 2的功能 通过干扰通道和PIP 2之间的交互来干扰通道。关于频道的了解要少得多- 通过SUMO化调节，这是一种翻译后修饰（PTM）途径，最终导致共价结合。 通过~100个氨基酸的SUMO蛋白修饰通道。尽管SUMO化调控了许多 在一系列可兴奋组织中的离子通道蛋白，包括心肌细胞、神经元和胰腺β细胞， 关于影响SUMO化的细胞刺激物，或者关于SUMO化作用的分子基础， 通道蛋白的相扑。我们最近发现，神经元NaV1.2通道迅速SUMO化， 对急性缺氧的反应，增加通过通道的电流。我们很想知道 应用于心肌细胞中NaV1.5和PIP 2依赖性Kir 2通道的调节模式。信息的途径 在异源系统和人心室心肌细胞中的初步研究， 多能干细胞（iPS-CMs）的研究支持了五个新的发现：（1）Kir2.1和2.4通道是SUMO调控的; (2)SUMO对这些通道的作用是PIP 2依赖性的;（3）NaV1.5通道是SUMO调节的;（4） 心脏Nav1.5的SUMO化在急性缺氧反应中迅速增加;（5），NaV1.5通道活性在急性缺氧反应中增加。 PIP 2依赖性。基于这些初步的观察，我们提出假设，Kir 2和NaV1.5 通道以PIP 2依赖的方式被SUMO调节，并描述了两个实验目的， 描述这些发现并检验我们的假设通过研究Kir 2和NaV1.5通道，我们期望 了解SUMO在Kir 2通道调节中的作用和PIP 2在NaV1.5调节中的作用 渠道此外，我们希望证明SUMO介导的对通道的作用是通过PIP 2发生的， 所有SUMO和PIP 2依赖性离子通道的统一分子机制。

Project Summary Ion channels in cardiomyocytes determine the shape, duration, and frequency of the heartbeat. Many factors that alter the function of channels cause cardiac disease in humans, including genetic mutations, adverse drug side-effects, or changes to cell-signaling pathways that regulation channel activity. This proposal is based on new insights into the regulation of two ion channel types that are critical to the function of the human heart: (1) The Kir2 channels that pass IK1, a current that contributes to the long-lasting plateau phase of the cardiac action potential and (2), the voltage-gated sodium channel, NaV1.5 that mediates INa, the current that underlying the rapid upstroke of the action potential. The activity of Kir2 channels (Kir2.1 to Kir2.4) is dependent on the ubiquitous, anionic membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 regulates multiple cellular processes and proteins, including TRP, Kir and at least some voltage-gated potassium channels. Significant research effort has characterized the structure-function relationship of PIP2 and Kir2 channels, including a crystal structure of PIP2 in complex with Kir2.2. Many disease-associated mutations and cell signaling pathways alter the function of Kir2 channels by interfering with the interaction between the channel and PIP2. Much less is known about channel- regulation by SUMOylation, a post translational modification (PTM) pathway that culminated in the covalent modification of channels by the an ~100-amino acid SUMO protein. Although SUMOylation regulates numerous ion channel proteins in a range of excitable tissues, including cardiomyocytes, neurons, and pancreatic β-cells, little is known about the cellular stimuli that impact SUMOylation, or about the molecular basis for the action of SUMO on channel proteins. We recently showed that neuronal NaV1.2 channels are rapidly SUMOylated in response to acute hypoxia, increasing current through the channels. We were intrigued to understand if this mode of regulation applied to NaV1.5 and PIP2-dependent Kir2 channels in cardiomyocytes. Data from our preliminary studies in heterologous systems and human ventricular cardiomyocytes, derived from induced pluripotent stem cells (iPS-CMs), support five novel findings: (1) Kir2.1 and 2.4 channels are SUMO-regulated; (2) the effect of SUMO on these channels is PIP2-dependent; (3) NaV1.5 channels are SUMO-regulated; (4) SUMOylation of cardiac Nav1.5 increases rapidly in response to acute hypoxia; (5), NaV1.5 channel activity is PIP2-dependent. Based on these preliminary observations, we propose the hypothesis that Kir2 and NaV1.5 channels are SUMO-regulated in a PIP2-dependent manner and describe two experimental aims designed to characterize these findings and test our hypothesis. By studying Kir2 and NaV1.5 channels, we expect to understand the role of SUMO in the regulation of Kir2 channels and the role of PIP2 in the regulation of NaV1.5 channels. Further, we expect to demonstrate that SUMO-mediated effects on channels occur via PIP2, providing a unifying molecular mechanism for all SUMO- and PIP2-dependent ion channels.