Redox Regulation of ATP Sensitive Potassium Channels

描述(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。硫氧还蛋白是一种小蛋白，通过硫醇-二硫键交换反应减少二硫键，从而起到抗氧化剂的作用。硫氧还蛋白的主要功能是在氧化修饰的情况下维持正常的蛋白质功能。在我们的初步研究中，我们利用生物信息学的方法，在ATP敏感K+(KATP)通道的SuR亚基的核苷酸结合折叠(NBF)中发现了新的蛋白质-蛋白质相互作用结构域，即卷曲线圈(CC)结构域。以SUR1CC结构域为诱饵，在与大鼠心脏cDNA文库的双杂交筛选中，我们鉴定了硫氧还蛋白-2是一种可能的KATP通道相互作用蛋白。硫氧还蛋白与SURCC结构域的相互作用随后通过GST下拉实验得到证实。此外，免疫共沉淀分析表明，硫氧还蛋白-2和胞内亚型硫氧还蛋白1在异源表达系统中也直接与全长SUR1或SUR2亚基相互作用。推测硫氧还蛋白与SUR亚基接近，这表明硫氧还蛋白对于保护KATP通道免受氧化还原修饰是至关重要的。本文提出的实验将1)使用免疫共沉淀分析和分子技术来表征硫氧还蛋白和KATP通道亚基在异源表达系统和分离的心肌细胞中的相互作用；2)使用膜片钳技术在内向外构型和药理学研究中检验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有关。众所周知，KATP通道的开放在各种病理条件下对心肌细胞具有保护作用。该项目将为生理条件下心肌细胞KATP通道功能的氧化还原修饰以及这种修饰在病理状态中的参与提供新的见解。阐明硫氧还蛋白在病理条件下调节KATP通道功能的作用可能为促进治疗的发展提供有价值的信息。 公共卫生相关性： KATP通道活性对生理和病理条件下氧化还原状态的改变做出反应。我们的初步研究表明，硫氧还蛋白是还原蛋白的成员，与KATP通道相互作用。在这项研究中，我们将1)表征硫氧还蛋白与KATP通道之间的相互作用，以及2)在生理和病理条件下，这种相互作用在KATP通道活性的氧化还原调节中的功能相关性。

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.