Regulation of Vascular BK Channel in Diabetes

描述（由申请方提供）：大电导钙激活钾（BK）通道是介导血管舒张的主要离子决定簇，是内皮源性超极化因子（EDHF）的靶点。我们发现糖尿病动物中EDHF对BK通道的调节是异常的，并且BK通道的内在特性在糖尿病冠状动脉中改变。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II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靶点。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糖的作用不敏感。本项目的目标是进一步阐明活性氧（ROS）调节糖尿病BK通道的分子机制。有待检验的假设是：1）糖尿病中血管BK通道功能异常是由于ROS对特定半胱氨酸残基的氧化还原调节。2)活性氧修饰的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探讨糖尿病诱导的氧化应激对BK通道的调节机制。目的2将检查是否阻止特定半胱氨酸残基的氧化调节将维持糖尿病中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。这些研究将使用体外和体内糖尿病模型进行。将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。该项目的结果可能提供重要的新的洞察力的电生理和分子机制的BK通道功能改变，可能有助于内皮依赖性和非依赖性血管功能障碍的糖尿病。

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.