Structural Basis of Katp Channel Gating

Katp 通道选通的结构基础

• 批准号: 7036279
• 负责人: Show-Ling Shyng
• 金额: $ 26.04万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-06 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036279
• 关键词: RNA interference; adenosine triphosphate; cell line; diabetes mellitus; disease /disorder onset; endocrine pharmacology; gene mutation; hyperinsulinism; insulin; molecular assembly /self assembly; pancreatic islet function; pancreatic islets; potassium channel; protein biosynthesis; protein protein interaction; protein structure; protein structure function; site directed mutagenesis; transfection

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.

描述（由应用程序提供）：ATP敏感的钾（KATP）通道在将细胞代谢与细胞的兴奋性耦合和控制多样的物理过程中起着关键作用，包括本人分泌，血管张力的控制以及缺血期间心肌和神经元的活性调节。该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KATP通道的胰腺亚型上，它们是异植物复合物，每个复合物由四个内部整流的钾通道KIR6.2亚基和四个调节性磺酰脲受体1个亚基组成。在胰腺P细胞中，KATP通道是调节胰岛素分泌的葡萄糖传感器。导致通道功能丧失的KIR6.2或SUR1的突变是先天性高胰岛素主义的主要原因，尽管血浆葡萄糖水平较低，但仍以持续的胰岛素分泌为特征。另一方面，最近已显示导致通道活性获得的突变导致新生儿糖尿病。几种物理分子，包括细胞内ATP，MGADP和膜磷酸肌醇，尤其是PI-4,5-P2（PIP2），可调节KATP通道的活性。但是，尚未清楚地了解这些分子控制通道活性至关重要的通道蛋白的结构特征。该应用的目的是通过研究疾病中鉴定的突变如何影响通道功能，从而深入了解KATP通道的结构功能关系。在第一个目标中，我们将确定由使用COS细胞在先天性高胰岛素中鉴定出的九种新型KIR6.2突变引起的通道缺陷，从而解决了通道生物发生/表达和门控中的两个缺陷。然后，我们将通过在大鼠胰腺胰岛线INS -1中表达突变体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，我们将确定Kir6.2细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至关重要，这是我们以前的发现，即在KIR6.2中破坏Kir6.2中的亚基离子对损害了正常的通道底盘。我们将专注于潜在的相互作用，这些相互作用是由在先天性高胰岛素或新生儿糖尿病中突变的残留物介导的。拟议的研究不仅将使我们对KATP通道的结构功能关系，而且还将更好地理解胰岛素分泌疾病的分子基础。这种知识可能有助于确定药物开发的新结构部位，对于为这些疾病设计有效的治疗策略至关重要。