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。很多因素 改变经络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，或调节通道活动的细胞信号通路的变化。这项建议是基于 对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) 通过IK1的Kir2通道，IK1是一种有助于心脏活动的长期平台期的电流 电位和(2)，电压门控钠通道，NaV1.5，它中介Ina，基础电流 动作电位迅速上升。 Kir2通道(Kir2.1到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(PIP2)。PIP2调节多种细胞过程和 蛋白质，包括色氨酸、KIR和至少一些电压门控钾通道。重大的研究工作已经完成 表征了PIP2和Kir2通道的结构-功能关系，包括PIP2的晶体结构 在与Kir2.2的复合体中。许多与疾病相关的突变和细胞信号通路改变了Kir2的功能 通过干扰通道和PIP2之间的相互作用。对渠道的了解要少得多- 糖基化的调节，翻译后修饰(PTM)途径，最终形成共价 An~100氨基酸相扑蛋白对通道的修饰。尽管SUMO化调节了许多 包括心肌细胞、神经元和胰腺β细胞在内的一系列可兴奋组织中的离子通道蛋白， 对影响SUMO化的细胞刺激或作用的分子基础知之甚少。 相扑对通道蛋白的影响。我们最近发现，神经元NAV1.2通道在 对急性缺氧作出反应，增加流经通道的电流。我们很想知道这是不是 心肌细胞中依赖NaV1.5和PIP2的Kir2通道的调节模式。数据来自我们的 诱导分化的异种系统和人心肌细胞的初步研究 多能干细胞(iPS-CMS)，支持5个新的发现：(1)Kir2.1和2.4通道受相扑调节； (2)相扑对这些通道的作用依赖于PIP2；(3)NaV1.5通道受相扑调节；(4) 心脏NaV1.5的苏莫化迅速增加，以响应急性缺氧；(5)，NaV1.5通道活性是 依赖于PIP2。基于这些初步观察，我们提出了Kir2和NaV1.5的假设 通道以PIP2依赖的方式被相扑调节，并描述了两个设计用于 描述这些发现，并检验我们的假设。通过对Kir2和NaV1.5渠道的研究，我们希望 了解相扑在Kir2通道调节中的作用和PIP2在NaV1.5调节中的作用 频道。此外，我们希望证明相扑对通道的影响是通过PIP2发生的，提供了 所有相扑和PIP2依赖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.