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

描述（由申请人提供）：本研究的目的是了解ATP敏感性钾通道（KATP）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进行相互作用。KATP通道在β细胞中表达并负责β细胞生理学。它们的传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Kir6.2和SUR 1两种亚基组成，但这些亚基之间如何相互通讯，即它们之间特定的氨基酸相互作用，以及这些相互作用如何决定KATP的生物合成和活性，目前尚不清楚。高分辨率的晶体结构不存在任何亚基或KATP复合物，因此，功能实验需要回答这些重要的问题。我将对SUR 1中的两个残基E128和R74进行详细研究。这些残基与由于运输缺陷引起的先天性高胰岛素血症相关，但其活性谱模拟新生儿糖尿病突变。这两个残基是一个选择组的成员，其突变导致可以通过磺酰脲（SU）治疗“拯救”的运输缺陷。基于任一残基突变导致KATP生物合成和活性缺陷的事实，我假设E128和R74有助于SUR 1-Kir6.2相互作用。具体目标1使用生化实验解决这两个残基如何促进适当的KATP生物合成。我将确定这些残基的系统诱变如何影响SUR 1-Kir6.2协会（免疫共沉淀）和运输到质膜（蛋白质印迹和化学发光）。此外，SU对这两个过程的影响将被测量。特异性目标2利用相同的突变体SUR 1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的系统诱变扫描鉴定与SUR 1 E128和R74相互作用的Kir6.2残基。通过研究双突变体的电生理特性和预期对在半胱氨酸置换和氧化后形成二硫键的能力，将实现SUR 1-Kir6.2相互作用对的验证。这项工作具有特别的公共卫生重要性，因为广泛用于糖尿病治疗的药物磺酰脲类药物最近被发现可以改变胰岛素分泌的重要调节因子KATP离子通道。研究这些药物的作用机制，有可能发现治疗另一种疾病--先天性高胰岛素血症的新方法。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.