SUR1 mutations and Subunit Associations in ATP-Sensitive Potassium Channels

描述(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分析的起点，了解ATP敏感钾通道(KATP)的亚单位是如何相互作用的。KATP通道在β细胞生理学中表达，并对其负责。它们的电导状态受细胞内核苷酸水平的调节，因此是细胞代谢状态和细胞兴奋性之间的重要联系。KATP通道由两种亚基组成：Kir6.2和SUR1，但这些亚基之间如何相互通信-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的生物发生和活性-尚不清楚。亚基和KATP复合体都不存在高分辨晶体结构；因此，需要功能实验来回答这些重要的问题。我将对SUR1中的两个残基E128和R74进行详细研究。由于转运缺陷，这些残基与先天性高胰岛素血症有关，但它们的活性谱模仿新生儿糖尿病突变。这两个残基都是一个特定基团的成员，该基团的突变导致运输缺陷，这些缺陷可以通过磺脲类药物(SU)治疗来“挽救”。根据任一残基的突变导致KATP生物发生和活性缺陷的事实，我假设E128和R74参与了SUR1-Kir6.2的相互作用。具体目标1利用生化实验阐述这两个残基如何促进适当的KATP生物发生。我将确定这些残基的系统突变如何影响SUR1-Kir6.2结合(免疫共沉淀)和向质膜运输(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。特殊目的2利用相同的突变SUR1亚基，并考虑它们如何影响KATP通道活性，包括ATP和SU敏感性。最后，在特定的目标3，1将通过对Kir6.2的系统突变扫描来鉴定Kir6.2与SUR1 E128和R74相互作用的残基。SUR1-Kir6.2相互作用对的验证将通过研究双突变的电生理特性和预期的对在半胱氨酸置换和氧化后形成二硫键的能力来实现。这项工作具有特别重要的公共卫生意义，因为被广泛用于糖尿病治疗的药物磺脲类药物最近被发现改变了胰岛素分泌的重要调节因素-KATP离子通道。研究这些药物如何发挥作用，有可能发现一种不同疾病的新疗法，即先天性高胰岛素血症。此外，这项工作将进一步加深我们对KATP通道的基础生物学的理解。

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.