Structural Basis of Katp Channel Gating

ATP敏感性钾通道在细胞代谢与细胞兴奋性的偶联中起着关键作用， 控制多种生理过程，包括激素分泌、控制血管张力， 在缺血期间调节心肌和神经元的活动。这个项目的长期目标是 是了解KATP通道门控的结构基础。为了实现这一目标，我们的研究集中在 KATP通道的胰腺亚型，其是异多聚体复合物，每个由四个 内向整流钾通道Kir6.2亚基和4个调节性磺酰脲受体1亚基。在 在胰腺β细胞中，KATP通道作为葡萄糖传感器来调节胰岛素分泌。中的突变 Kir6.2或SUR 1导致通道功能丧失是先天性高胰岛素血症的主要原因， 尽管血浆葡萄糖水平很低，但仍持续分泌胰岛素的疾病。另一方面，在一项研究中， Kir6.2突变导致通道活性增强，最近已被证明会导致新生儿 糖尿病几种生理分子，包括细胞内ATP，MgADP和膜 磷酸肌醇，特别是PI-4，5-P2（PIP 2），调节KATP通道的活性。但结构性 对于这些分子控制通道活性至关重要的通道蛋白的特征不 明白了本申请的目的是深入了解KATP的结构-功能关系 通过研究疾病中发现的突变如何影响通道， 功能在第一个目标中，我们将确定由九个新的Kir6.2突变引起的通道缺陷。 在使用COS细胞的先天性高胰岛素血症中，解决通道生物发生/表达中的缺陷 和门控。然后，我们将评估这些突变如何影响p细胞生理学以及它们如何响应 潜在的分子或药理学治疗，通过在大鼠胰腺p细胞系中表达突变Kir6.2 INS-1。在第二个目标中，我们将对最近在新生儿中发现的Kir6.2突变进行类似的研究。 糖尿病在第三个目标中，我们将鉴定Kir6.2胞质结构域中的亚基间相互作用， 根据我们之前的发现，对于Kir6.2亚基之间的门控和物理关联很重要 Kir6.2中亚基间离子对的破坏损害了正常的通道门控。我们将专注于潜力 由在先天性高胰岛素血症中发现突变的残基介导的相互作用，或 新生儿糖尿病这项研究不仅有助于我们更好地了解结构-功能 KATP通道的相互关系，也是KATP通道引起胰岛素分泌疾病的分子基础 突变。这些知识可能有助于确定药物开发的新结构位点， 为这些疾病设计有效的治疗策略。

ATP-sensitive potassium (KAip) channels play a key role in coupling cell metabolism to cell excitability and govern diverse physiological processes including hormone secretion, control of vascular tone, and modulation of the activity of cardiac muscle and neurons during ischemia. The long-term goal of this project is to understand the structural basis of KATP channel gating. Towards this goal, our research has focused on the pancreatic subtype of KATP channels, which are heteromultimeric complexes each composed of four inwardly rectifying potassium channel Kir6.2 subunits and four regulatory sulfonylurea receptor 1 subunits. In pancreatic p-cells, KATP channels serve as glucose sensors to regulate insulin secretion. Mutations in either Kir6.2 or SUR1 that lead to loss of channel function are the major cause of congenital hyperinsulinism, a disease characterized by persistent insulin secretion despite low plasma glucose level. On the other hand, mutations in Kir6.2 that lead to gain of channel activity have recently been shown to cause neonatal diabetes. Several physiological molecules, including intracellular ATP, MgADP, and membrane phosphoinositides, especially PI-4,5-P2 (PIP2), regulate the activity of KATP channels. However, structural features of the channel proteins that are critical for control of channel activity by these molecules are not clearly understood. The goal of this application is to gain insight to the structure-function relationship of KATP channels using a forward genetics approach by studying how mutations identified in disease affect channel function. In the first aim, we will determine channel defects caused by nine novel Kir6.2 mutations identified in congenital hyperinsulinism using COS cells, addressing both defects in channel biogenesis/expression and gating. We will then evaluate how these mutations impact on p-cell physiology and how they respond to potential molecular or pharmacological treatments, by expressing mutant Kir6.2 in a rat pancreatic p-cell line INS-1. In the second aim, we will perform similar studies on Kir6.2 mutations recently identified in neonatal diabetes. In the third aim, we will identify intersubunit interactions in the cytoplasmic domain of Kir6.2 that are important for gating and for physical association between Kir6.2 subunits, based on our previous finding that disruption of an intersubunit ion pair in Kir6.2 impairs normal channel gating. We will focus on potential interactions that are mediated by residues that have been found mutated in congenital hyperinsulinism or neonatal diabetes. The proposed study will better our understanding of not only the structure-function relationships of KATP channels but also the molecular basis of insulin secretion diseases caused by channel mutations. Such knowledge may help identify novel structural sites for drug development and is essential for designing effective therapeutic strategies for these diseases.