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（由申请人提供）：ATP 敏感钾 (KATP) 通道在细胞代谢与细胞兴奋性的耦合中发挥着关键作用，并控制多种生理过程，包括激素分泌、血管张力控制以及心肌和神经元活动的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，我们的研究重点是 KATP 通道的胰腺亚型，它们是异多聚体。每个复合物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。在胰腺 p 细胞中，KATP 通道充当葡萄糖传感器来调节导致胰岛素分泌丧失的 Kir6.2 或 SUR1 突变。通道功能的紊乱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浆葡萄糖水平较低，但胰岛素仍持续分泌。导致通道活性增强的 Kir6.2 最近已被证明会导致新生儿糖尿病，包括细胞内 ATP、MgADP 和膜磷酸肌醇，尤其是 PI-4,5-P2 (PIP2)，它们可调节 KATP 的活性。然而，对于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，该应用的目的是利用正向深入了解 KATP 通道的结构-功能关系。通过研究疾病中发现的突变如何影响通道功能，我们将采用遗传学方法确定由 COS 细胞在先天性高胰岛素血症中发现的九种新的 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。然后我们将评估这些突变如何影响 ¿ -通过在大鼠胰腺中表达突变型Kir6.2，细胞生理学以及它们如何响应潜在的分子或药物治疗 ¿ -细胞系 INS-1 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，我们将确定对 Kir6.2 细胞质结构域很重要的亚基间相互作用。基于我们之前的发现，Kir6.2 中亚基间离子对的破坏会损害正常的通道门控，我们将重点关注由已被处理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 通道的结构与功能关系，而且有助于了解通道突变引起的胰岛素分泌疾病的分子基础。药物开发对于设计这些疾病的有效治疗策略至关重要。