Structural Basis of Katp Channel Gating

描述(申请人提供)：ATP敏感钾(KATP)通道在细胞代谢和细胞兴奋性之间起关键作用，并调控多种生理过程，包括激素分泌，血管张力控制，以及在缺血期间心肌和神经元活动的调节。本项目的长期目标是了解KATP通道门控的结构基础。为此，我们的研究集中在KATP通道的胰腺亚型，它是由四个内向整流钾通道Kir6.2亚基和四个调节性磺酰脲受体1亚基组成的异构体复合体。在胰腺p细胞中，KATP通道作为葡萄糖感受器调节胰岛素的分泌。导致通道功能丧失的Kir6.2或SUR1突变是先天性高胰岛素血症的主要原因，这种疾病的特征是尽管血糖水平较低，但仍持续分泌胰岛素。另一方面，Kir6.2基因突变导致通道活性增加，最近被证明会导致新生儿糖尿病。几种生理分子，包括细胞内的ATP、MgADP和膜上的磷脂酰肌醇，特别是PI-4，5-P2(PIP2)，调节KATP通道的活性。然而，对这些分子控制通道活性至关重要的通道蛋白的结构特征尚不清楚。这项应用的目标是通过研究疾病中发现的突变如何影响通道功能，使用正向遗传学方法来洞察KATP通道的结构-功能关系。在第一个目标中，我们将使用COS细胞来确定由先天性高胰岛素血症中发现的九个新的Kir6.2突变引起的通道缺陷，解决通道生物发生/表达和门控方面的缺陷。然后，我们将通过在大鼠胰腺细胞系INS-1中表达突变Kir6.2来评估这些突变对细胞生理的影响以及它们对潜在的分子或药物治疗的反应。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，我们将识别Kir6.2细胞质区域中对门控和Kir6.2亚基之间的物理关联至关重要的亚基间相互作用，这是基于我们之前的发现，即Kir6.2亚基间离子对的破坏会损害正常的通道门控。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的突变残基所介导的潜在相互作用。该研究不仅有助于我们更好地了解KATP通道的结构与功能关系，也将有助于我们更好地了解通道突变引起的胰岛素分泌性疾病的分子基础。这些知识可能有助于确定药物开发的新结构部位，并对设计有效的治疗这些疾病的策略至关重要。

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.