SUR1 mutations and Subunit Associations in ATP-Sensitive Potassium Channels

ATP 敏感钾通道中的 SUR1 突变和亚基关联

• 批准号: 8037030
• 负责人: Emily B Pratt
• 金额: $ 4.49万
• 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/8037030
• 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; Co-Immunoprecipitations; Complement; Complex; 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; Pancreas; Persistent Hyperinsulinemia Hypoglycemia of Infancy; Pharmaceutical Preparations; Potassium; Process; Property; Proteins; Public Health; 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; 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）：这项研究的目的是了解ATP敏感钾通道（KATP）的亚基如何通过利用已知的致病突变作为生化和电生理分析的起点来相互作用。 KATP通道在β细胞生理中表达并负责。它们的电导态受细胞内核苷酸水平的调节，因此是细胞代谢状态与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，但是这些亚基如何与每个特定的亚基间氨基酸相互作用进行通信，以及这些相互作用如何指定KATP生物发生和活动 - 尚不清楚。对于亚基或KATP复合物都不存在高分辨率的晶体结构。因此，需要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。这些残基与流量缺陷引起的先天性高胰岛素有关，但其活性谱模仿新生儿糖尿病突变。这两个残留物都是精选组的成员，其突变导致贩运缺陷，可以被磺胺尿素（SU）处理“营救”。基于这两个残基的突变导致KATP生物发生和活性缺陷，我假设E128和R74有助于SUR1-KIR6.2相互作用。具体目标1解决了这两个残基如何使用生化实验促进正确的KATP生物发生。我将确定这些残基的系统诱变如何影响SUR1-KIR6.2关联（共免疫沉淀）和贩运到质膜（Western blot＆Chemilumeilumeiliencence）。此外，将测量SU对两个过程的影响。特定目标2利用相同的突变体SUR1亚基，并考虑它们如何影响KATP通道活动，包括ATP和SU敏感性。最后，在特定的目标中，3,1将通过系统的KIR6.2的系统诱变扫描与SUR1 E128和R74相互​​作用的KiR6.2残基。通过研究双突出电生理特性以及前瞻性对在半胱氨酸替代和氧化后形成二硫化纤维桥的能力，可以实现SUR1-KIR6.2相互作用对的验证。这项工作特别重要，因为最近发现在糖尿病治疗中广泛使用的药物改变了胰岛素分泌的重要调节剂KATP ION通道。研究这些药物的工作原理有可能发现针对另一种疾病，先天性高胰岛素主义的新疗法。此外，这项工作将进一步了解KATP渠道的基本生物学。