SUR1 mutations and Inter-Subunit Associations In ATP-Sensitive Potassium Channels

ATP 敏感钾通道中的 SUR1 突变和亚基间关联

• 批准号: 7487588
• 负责人: Emily B Pratt
• 金额: $ 4.41万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7487588
• 关键词: ATP sensitive potassium channel complex; Address; Affect; Amino Acid Substitution; Amino Acids; B-Lymphocytes; Beta Cell; Biochemical; Biogenesis; Biological Assay; Biology; Blood Glucose; Categories; Cell membrane; Cell physiology; Cells; Charge; Chemicals; Classification; Co-Immunoprecipitations; Complement; Complex; Condition; Coupling; Cysteine; Data; Defect; Development; Diabetes Mellitus; Disease; Disulfides; Event; Exposure to; Goals; Homology Modeling; Hydrophobicity; Insulin; Ion Channel; Lead; Ligands; Link; Measures; Metabolic; Molecular Chaperones; Mutagenesis; Mutate; Mutation; Nature; Nucleotides; Numbers; Pancreas; Persistent Hyperinsulinemia Hypoglycemia of Infancy; Pharmaceutical Preparations; Potassium; Process; Property; Proteins; Public Health; Purpose; Recovery; Reporting; Research; Resolution; Scanning; Severities; Signal Transduction; Specific qualifier value; Structure; Structure-Activity Relationship; Sulfonylurea Compounds; Testing; Transmembrane Domain; Western Blotting; Work; analog; base; chemical property; disease-causing mutation; falls; insight; insulin secretion; member; mutant; neonatal diabetes mellitus; oxidation; prospective; protein folding; research study; size; sulfonylurea receptor; trafficking

DESCRIPTION (provided by applicant): The goal of this research is to understand how the subunits of ATP sensitive potassium channels (KATP) interact by utilizing known disease-causing mutations as starting points for biochemical and electrophysiological analysis. KATP channels are expressed in and responsible for beta-cell physiology. Their conductance state is regulated by intracellular nucleotide levels, and thus is an important link between cellular metabolic status and cell excitability. KATP channels are composed of two types of subunits: Kir6.2 and SUR1, But how these subunits communicate with each other-their specific inter-subunit amino acid interactions, and how these interactions specify KATP biogenesis and activity-is not known. High- resolution crystal structures do not exist for either subunit or for the KATP complex; therefore, functional experiments are needed to answer these important questions. I will perform a detailed study of two residues within SUR1, E128 and R74. These residues are associated with congenital hyperinsulinism due to trafficking defects, but their activity profiles mimic neonatal diabetes mutations. Both residues are members of a select group, mutation of which leads to trafficking defects that can be 'rescued' by sulfonylurea (SU) treatment. Based on the fact that mutation of either residue leads to KATP biogenesis and activity defects, I hypothesize that E128 and R74 contribute to SUR1-Kir6.2 interactions. Specific Aim 1 addresses how these two residues facilitate proper KATP biogenesis using biochemical experiments. I will determine how systematic mutagenesis of these residues effect SUR1-Kir6.2 associations (co-immunoprecipitation) and trafficking to the plasma membrane (western blot & chemiluminescence). Further, the influence of SU on both processes will be measured. Specific Aim 2 utilizes the same mutant SUR1 subunits and considers how they affect KATP channel activity, both ATP and SU sensitivities. Finally, in Specific Aim 3,1 will identify the residues of Kir6.2 that interact with SUR1 E128 and R74 by a systematic mutagenesis scan of Kir6.2. Verification of SUR1-Kir6.2 interacting pairs will be achieved by study of double-mutant electrophysiological properties and the ability of prospective pairs to form disulphide-bridges following cysteine-replacement and oxidation. This work is of particular public health importance because drugs widely used in diabetes treatment, sulfonylureas, have recently been found to change an important regulator of insulin secretion, the KATP ion channel. Studying how these drugs work has the potential for discovery of new treatments for a different disease, congenital hyperinsulinism. In addition, this work will further our understanding of the basic biology of the KATP channel.

描述（由申请人提供）：本研究的目标是通过利用已知的致病突变作为生化和电生理学分析的起点，了解 ATP 敏感钾通道 (KATP) 的亚基如何相互作用。 KATP 通道在 β 细胞生理学中表达并负责。它们的电导状态受细胞内核苷酸水平调节，因此是细胞代谢状态和细胞兴奋性之间的重要联系。 KATP 通道由两种类型的亚基组成：Kir6.2 和 SUR1，但是这些亚基如何相互通信（它们特定的亚基间氨基酸相互作用，以及这些相互作用如何指定 KATP 生物发生和活性）尚不清楚。无论是亚基还是 KATP 复合物都不存在高分辨率晶体结构；因此，需要功能实验来回答这些重要问题。我将对 SUR1、E128 和 R74 中的两个残基进行详细研究。这些残留物与由于运输缺陷而导致的先天性高胰岛素血症有关，但它们的活性特征模仿了新生儿糖尿病突变。这两个残基都是选定群体的成员，该群体的突变会导致运输缺陷，而这些缺陷可以通过磺酰脲（SU）治疗来“挽救”。基于任一残基的突变都会导致 KATP 生物发生和活性缺陷的事实，我假设 E128 和 R74 有助于 SUR1-Kir6.2 相互作用。具体目标 1 阐述了这两个残基如何通过生化实验促进正确的 KATP 生物合成。我将确定这些残基的系统诱变如何影响 SUR1-Kir6.2 关联（免疫共沉淀）和运输到质膜（蛋白质印迹和化学发光）。此外，还将测量 SU 对这两个过程的影响。具体目标 2 利用相同的突变型 SUR1 亚基，并考虑它们如何影响 KATP 通道活性（ATP 和 SU 敏感性）。最后，在具体目标 3,1 中，将通过 Kir6.2 的系统诱变扫描来识别与 SUR1 E128 和 R74 相互作用的 Kir6.2 残基。 SUR1-Kir6.2相互作用对的验证将通过研究双突变体电生理特性和预期对在半胱氨酸取代和氧化后形成二硫桥的能力来实现。这项工作对于公共卫生具有特别重要的意义，因为最近发现广泛用于糖尿病治疗的药物磺酰脲类药物可以改变胰岛素分泌的重要调节剂 KATP 离子通道。研究这些药物的作用机制有可能发现针对另一种疾病（先天性高胰岛素血症）的新疗法。此外，这项工作将进一步加深我们对 KATP 通道基础生物学的理解。