Regulation of Vascular BK Channels in Diabetes

描述(申请人提供)：大电导钙激活钾(BK)通道是介导血管松弛的主要离子决定因素，也是内皮衍生超极化因子(EDHFs)的靶标。我们发现在糖尿病动物中，EDHFs对BK通道的调节是异常的，糖尿病冠状动脉中BK通道的固有特性发生了改变。糖尿病动物冠状动脉平滑肌细胞上的BK通道对钙和电压依赖的激活的敏感性降低。我们已经证明，1型和II型糖尿病患者血管BK通道调节改变的机制涉及高血糖诱导的氧化应激，其中BK通道中的半胱氨酸残基是氧化还原调节的靶点。BK通道孔孔亚基的C911残基对高血糖诱导的氧化应激的调节特别敏感。此外，BK-�亚单位在糖尿病中的表达显著下调，这是由于高血糖诱导的F-Box Only蛋白上调，F-Box Only蛋白是SCF型泛素连接酶复合体的组成部分，促进BK-�的降解。本项目的目标是进一步阐明糖尿病患者中活性氧簇(ROS)调节BK通道的分子机制。由于Nrf2(核因子E-2相关因子2)已经成为细胞解毒和氧化还原状态的主要调节因子，我们将检验Nrf2信号在糖尿病BK通道功能调节中发挥核心作用的假设。提出了三个具体目标。目的1研究糖尿病中NRF2信号对BK-�的调节机制。我们将确定NRF2下调在糖尿病中对BK-�氧化和功能的作用，以及小凹靶向在这种调节中的作用。目的2研究糖尿病中NRF2信号对BK-�的调节机制。我们将通过泛素-蛋白酶体系统确定NRF2在糖尿病中下调对BK-�降解的作用。目的3研究Nrf2激动剂对糖尿病动物模型的治疗作用及其对血管BK通道功能和血管反应性的影响。这些研究将使用糖尿病的体外和体内模型进行。全细胞和单通道膜片钳技术、生化、药理学、钙成像以及生理和分子生物学方法，包括使用特定的转基因小鼠，将被用来确定Nrf2信号的作用以及糖尿病对BK通道功能和降解的影响。该项目的结果可能为BK通道功能改变的分子机制提供重要的新见解，BK通道功能改变可能导致糖尿病血管功能障碍。这一结果也可能允许开发新的方法来治疗糖尿病中的BK通道病变和血管异常。

DESCRIPTION (provided by applicant): Large conductance calcium-activated potassium (BK) channels are major ionic determinants in mediating vasorelaxation and are the target of endothelium-derived hyperpolarizing factors (EDHFs). We have found that regulation of BK channels by EDHFs is abnormal in diabetic animals and the intrinsic properties of BK channels are altered in diabetic coronary arteries. BK channels in coronary arterial smooth muscle cells from diabetic animals have reduced sensitivity to calcium- and voltage-dependent activation. We have demonstrated that the mechanism by which vascular BK channel regulation is altered in Type 1 and Type II diabetes involves hyperglycemia-induced oxidative stress, where the cysteine residues in BK channels are targets of redox modulation. The C911 residue of the BK channel pore subunit is particularly sensitive to modulation by hyperglycemia-induced oxidative stress. In addition, the BK-�subunit is significantly down regulated in diabetes as a result of hyperglycemia- induced up regulation of F-Box Only proteins, which are components of the SCF-type ubiquitin ligase complex that facilitates the degradation of BK-� The goal of this project is to further delineate the molecular mechanisms through which reactive oxygen species (ROS) modulate BK channels in diabetes. Since Nrf2 (Nuclear factor E-2 related factor 2) has emerged as a master regulator of cellular detoxification and redox status, we will test the hypothesis that Nrf2 signaling plays a central role in the regulation of BK channel function in diabetes. Three specific aims are proposed. Aim 1 will examine the mechanism of BK-�hannel regulation by Nrf2 signaling in diabetes. We will determine the role of Nrf2 down regulation in diabetes on BK-�xidation and function, as well as the role of caveolae targeting in such regulation. Aim 2 will examine the mechanism of BK-�regulation by Nrf2 signaling in diabetes. We will determine the role of Nrf2 down regulation in diabetes on BK-�degradation through the ubiquitin- proteasome system. Aim 3 will examine the treatment of diabetic animal models by Nrf2 activators and their effects on vascular BK channel function and vasoreactivity. These studies will be performed using in vitro and in vivo models of diabetes. Whole-cell and single channel patch clamp techniques, biochemical, pharmacological, Ca2+ imaging, and physiological and molecular biological approaches, including the use of specific transgenic mice, will be employed to determine the role of Nrf2 signaling and the effects of diabetes on BK channel function and degradation. The results of this project may provide important novel insights into the molecular mechanisms of altered BK channel function that may contribute to vascular dysfunction in diabetes. The results may also allow the development of novel approaches in the treatment of BK channelopathy and vascular abnormalities in diabetes.