Regulation of Vascular BK Channel in Diabetes

描述（由申请人提供）：大电导钙活化钾（BK）通道是介导血管舒张的主要离子决定因素，是内皮衍生超极化因子（EDHFs）的靶标。我们发现EDHFs对BK通道的调节在糖尿病动物中是异常的，并且BK通道的固有特性在糖尿病冠状动脉中发生了改变。糖尿病动物冠状动脉平滑肌细胞中的BK通道对钙和电压依赖性激活的敏感性降低。我们已经证明血管BK通道调节在1型和2型糖尿病中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目标。BK通道孔亚基的C911残基对高血糖和过氧化氢的调节特别敏感，因为C911A突变对过氧化氢和高葡萄糖的影响不敏感。该项目的目标是进一步描述通过活性氧（ROS）调节糖尿病BK通道的分子机制。拟验证的假设有：1)糖尿病血管BK通道功能异常是由于活性氧对特定半胱氨酸残基的氧化还原调节所致。2) ROS修饰的BK通道是泛素化和蛋白酶体降解的底物。提出了三个具体目标。目的1将探讨糖尿病诱导的氧化应激对BK通道调节的机制。目的2将研究预防特定半胱氨酸残基的氧化调节是否会维持糖尿病患者BK通道功能的完整性。目的3将确定氧化还原调节的BK通道是否是泛素化和蛋白酶体降解的目标。这些研究将在体外和体内糖尿病模型中进行。采用全细胞和单通道膜片钳技术、抗氧化酶体外基因转移和特异性转基因小鼠来确定糖尿病诱导的ROS产生对BK通道功能和降解的影响。该项目的结果可能为BK通道功能改变的电生理和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中内皮依赖性和非依赖性血管功能障碍。公共卫生相关性：糖尿病已成为一种流行病，心血管疾病是糖尿病患者发病和死亡的最重要原因。BK通道是调节血管张力的蛋白质，是重要器官血液流动的关键决定因素。我们发现糖尿病冠状动脉中BK通道功能异常。这个项目的目标是描述这些变化背后的分子机制。我们假设糖尿病中的BK通道由于氧化应激增强而异常，而“氧化”BK通道的命运是加速降解。该项目的结果可能为糖尿病导致心血管系统功能异常的机制提供重要的新见解。

DESCRIPTION (provided by applicant): The 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 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. The 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 whereby 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 and by hydrogen peroxide, as the C911A mutation is insensitive to the effects of hydrogen peroxide and to high glucose. The goal of this project is to further delineate the molecular mechanisms through which reactive oxygen species (ROS) modulate BK channels in diabetes. The hypotheses to be tested are: 1) Abnormal vascular BK channel function in diabetes is due to redox modulation of specific cysteine residues by ROS. 2) BK channel modified by ROS is a substrate for ubiquitination and proteasomal degradation. Three specific aims are proposed. Aim 1 will examine the mechanism of BK channel modulation by diabetes-induced oxidative stress. Aim 2 will examine whether prevention of oxidative modulation of specific cysteine residues would maintain the integrity of BK channel function in diabetes. Aim 3 will determine whether redox-modulated BK channels are targets for ubiquitination and proteasomal degradation. These studies will be performed using in vitro and in vivo models of diabetes. Whole-cell and single channel patch clamp techniques, antioxidant enzyme in vitro gene transfer, and specific transgenic mice will be employed to determine the effects of diabetes-induced ROS production on BK channel function and degradation. The results of this project may provide important novel insight into the electrophysiological and molecular mechanisms of altered BK channel function that may contribute to both endothelium-dependent and -independent vascular dysfunction in diabetes. PUBLIC HEALTH RELEVANCE: Diabetes has become a disease of epidemic proportions and cardiovascular diseases are the most important causes of morbidity and mortality in diabetic patients. BK channels are proteins that regulate vessel tone and are critical determinants of blood flow in vital organs. We have found that the function of BK channels is abnormal in diabetic coronary arteries. The goal of this project is to delineate the molecular mechanisms underlying these changes. We hypothesize that BK channels in diabetes are abnormal as a result of enhanced oxidative stress and the fate of "oxidized" BK channel is accelerated degradation. The results of this project may provide important novel insights into the mechanisms through which diabetes leads to abnormal function in the cardiovascular system.