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氧气供应，也是最容易受到氧化损伤的系统之一。氧化应激已被证明参与各种心脏疾病，包括心肌缺血-再灌注损伤和心力衰竭。硫氧还蛋白是通过巯基-二硫键交换反应还原二硫键而充当抗氧化剂的小蛋白质。硫氧还蛋白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，使用生物信息学方法，我们确定了新的推定的蛋白质-蛋白质相互作用结构域，卷曲螺旋（CC）结构域，在核苷酸结合折叠（NBF）的SUR亚基的ATP敏感性K+（KATP）通道。使用SUR 1 CC结构域作为诱饵，在双杂交筛选对大鼠心脏cDNA文库，我们确定硫氧还蛋白2作为一个假定的KATP通道相互作用蛋白。硫氧还蛋白与SUR CC-结构域的相互作用随后使用GST下拉测定法证实。此外，免疫共沉淀试验表明，硫氧还蛋白-2和胞质异构体硫氧还蛋白1也直接与全长SUR 1或SUR 2亚基在异源表达系统中相互作用。硫氧还蛋白与SUR亚基的假定接近性表明硫氧还蛋白对于保护KATP通道免受氧化还原修饰至关重要。本文拟进行的实验将1）使用免疫共沉淀分析和分子技术来表征异源表达系统和分离的心肌细胞中硫氧还蛋白和KATP通道亚基的相互作用2）使用膜片钳技术在由内而外的配置和药理学研究来检查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。已知KATP通道的开放在各种病理条件下保护肌细胞。该项目将提供新的见解在心肌细胞的KATP通道功能的氧化还原修饰在生理条件下，以及这种修饰在病理状态下的参与。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开发提供有价值的信息。 公共卫生相关性： KATP通道活性响应于在生理和病理条件下发生的氧化还原状态的改变。我们的初步研究表明，硫氧还蛋白，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）表征硫氧还蛋白和KATP通道之间的相互作用，2）探索这种相互作用在生理和病理条件下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.