Structural Basis of Katp Channel Gating

描述（由申请人提供）：ATP敏感性钾（KATP）通道在将细胞代谢与细胞兴奋性偶联中发挥关键作用，并控制多种生理过程，包括激素分泌、血管张力控制以及缺血期间心肌和神经元活性的调节。本项目的长期目标是了解KATP通道门控的结构基础。为了实现这一目标，我们的研究集中在胰腺亚型的KATP通道，这是异源多聚体复合物，每个由四个内向整流钾通道Kir6.2亚基和四个调节磺酰脲受体1亚基。在胰腺β细胞中，KATP通道作为葡萄糖传感器调节胰岛素分泌。导致通道功能丧失的Kir6.2或SUR 1突变是先天性高胰岛素血症的主要原因，先天性高胰岛素血症是一种以持续胰岛素分泌为特征的疾病，尽管血糖水平较低。另一方面，Kir6.2中导致通道活性增加的突变最近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。然而，通道蛋白的结构特征，这些分子的通道活性的控制是至关重要的是不清楚。本申请的目标是通过研究疾病中鉴定的突变如何影响通道功能，使用正向遗传学方法深入了解KATP通道的结构-功能关系。在第一个目标中，我们将使用COS细胞确定由先天性高胰岛素血症中鉴定的9种新型Kir6.2突变引起的通道缺陷，解决通道生物发生/表达和门控中的缺陷。然后，我们将通过在大鼠胰腺细胞系INS-1中表达突变Kir6.2来评估这些突变如何影响细胞生理学以及它们如何对潜在的分子或药物治疗作出反应。在第二个目标中，我们将对最近在新生儿糖尿病中发现的Kir6.2突变进行类似的研究。在第三个目标中，我们将确定Kir6.2的胞质结构域中的亚基间相互作用，这对于Kir6.2亚基之间的门控和物理关联非常重要，这是基于我们之前的发现，即Kir6.2中亚基间离子对的破坏损害了正常的通道门控。我们将重点关注在先天性高胰岛素血症或新生儿糖尿病中发现突变的残基介导的潜在相互作用。这项研究不仅有助于我们更好地了解KATP通道的结构-功能关系，而且有助于我们更好地了解由KATP通道突变引起的胰岛素分泌疾病的分子基础。这些知识可能有助于确定药物开发的新结构位点，并且对于设计这些疾病的有效治疗策略至关重要。

DESCRIPTION (provided by applicant): ATP-sensitive potassium (KATP) 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 ¿-cell physiology and how they respond to potential molecular or pharmacological treatments, by expressing mutant Kir6.2 in a rat pancreatic ¿-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.