Redox Regulation of ATP Sensitive Potassium Channels

描述（由申请人提供）：心血管系统在体内循环供氧，也是最容易受到氧化损伤的系统之一。氧化应激已被证明与多种心脏疾病有关，包括心肌缺血再灌注损伤和心力衰竭。硫氧还毒素是一种小蛋白质，通过巯基-二硫交换反应减少二硫键，起到抗氧化剂的作用。硫氧还毒素的主要功能是维持正常的蛋白质功能，防止氧化修饰。在我们的初步研究中，利用生物信息学方法，我们在atp敏感的K+ (KATP)通道SUR亚基的核苷酸结合折叠（NBF）中发现了新的假定的蛋白质-蛋白质相互作用结构域，即卷曲线圈（CC）结构域。利用SUR1 CC结构域作为诱饵，在针对大鼠心脏cDNA文库的双杂交筛选中，我们鉴定出硫氧还蛋白-2是推测的KATP通道相互作用蛋白。硫氧还毒素与SUR CC-结构域的相互作用随后通过GST下拉试验得到证实。此外，共免疫沉淀实验表明，在异源表达系统中，硫氧还蛋白-2和胞质异构体硫氧还蛋白1也直接与全长SUR1或SUR2亚基相互作用。假设硫氧还毒素接近SUR亚基，表明硫氧还毒素对保护KATP通道免受氧化还原修饰至关重要。本文提出的实验将1)使用共免疫沉淀法和分子技术来表征异源表达系统和分离的肌细胞中硫氧还蛋白和KATP通道亚基的相互作用2)使用膜片钳技术在内外配置和药理学研究中检测硫氧还蛋白结合是否在功能上与KATP通道活性和氧化还原诱导的通道修饰相关。众所周知，在各种病理条件下，KATP通道的开放可以保护肌细胞。该项目将为生理条件下心肌细胞中KATP通道功能的氧化还原修饰以及这种修饰在病理状态下的参与提供新的见解。阐明硫氧还毒素在病理条件下调节KATP通道功能的作用可能为促进治疗发展提供有价值的信息。

DESCRIPTION (provided by applicant): The cardiovascular system circulates oxygen supply in the body, and is also one of the most vulnerable systems to oxidative damage. Oxidative stress has been shown to be involved in various cardiac disorders, including myocardial ischemia-reperfusion injury and heart failure. Thioredoxins are small proteins that act as antioxidants by reducing disulfide bonds via a thiol-disulfide exchange reaction. The major functions of thioredoxins are to maintain normal protein functions against oxidative modification. In our preliminary studies, using bioinformatic approaches, we identified novel putative protein-protein interaction domains, the coiled-coil (CC) domain, in the nucleotide binding fold (NBF) of SUR subunits of ATP-sensitive K+ (KATP) channel. Using the SUR1 CC domain as the bait in a two-hybrid screen against a rat cardiac cDNA library, we identified thioredoxin-2 as a putative KATP channel interacting protein. Interaction of thioredoxins with the SUR CC- domain was subsequently confirmed using GST pull-down assays. Moreover, co-immunoprecipitation assays demonstrated that thioredoxin-2 and the cytosolic isoform thioredoxin 1 also direct interact with full-length SUR1 or SUR2 subunit in a heterologous expression system. The presumed proximity of thioredoxins to SUR subunits suggests that thioredoxins are crucial to protect KATP channels against redox modification. Experiments proposed here will 1) use co-immunoprecipitation assays and molecular techniques to characterize interaction of thioredoxins and KATP channel subunits in the heterologous expression system and isolated myocytes 2) use patch-clamp technique at inside-out configuration and pharmacological studies to examine whether thioredoxin binding is functionally relevant to KATP channel activity and to the redox-induced channel modification. It is known that opening of KATP channels protect myocytes under various pathological conditions. This project will provide novel insights in the redox modification of KATP channel functions in cardiac myocytes under physiological conditions as well as the involvement of this modification in pathological states. Elucidation of the role of thioredoxins in regulating KATP channel functions during pathological conditions might provide valuable information to facilitate therapeutic development. PUBLIC HEALTH RELEVANCE: The KATP channel activity responds to alterations in the redox state that occurs in physiological and pathological conditions. Our preliminary study demonstrates that thioredoxins, members of reducing proteins, interact with KATP channels. In this study, we will 1) characterize the interaction between thioredoxins and KATP channels, and 2) explore the functional relevance of this interaction in redox regulation of KATP channel activity under physiological and pathological conditions.