Regulation of Vascular BK Channels in Diabetes

大电导钙激活钾(BK)通道是 介导血管松弛，是内皮衍生超极化因子(EDHFs)的靶标。我们 发现糖尿病动物EDHFs对BK通道的调节异常 糖尿病冠状动脉中BK通道的特性发生改变。冠状动脉中的BK通道 糖尿病动物的平滑肌细胞对钙和电压依赖的敏感性降低 激活。我们已经证明了血管BK通道调节的机制是 1型和II型糖尿病的改变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标。BK通道的C911残基 孔亚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影响不敏感。这样做的目的是 该项目旨在进一步阐明活性氧自由基(ROS) 调节糖尿病患者的BK通道。需要检验的假设是：1)血管BK通道异常 糖尿病的功能是由于ROS对特定半胱氨酸残基的氧化还原调节。2)BK频道 ROS修饰是泛素化和蛋白酶体降解的底物。三个具体目标是 建议。目标1将研究糖尿病引起的氧化作用对BK通道的调节机制 压力。目标2将研究防止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。目标3将确定氧化还原调制是否 BK通道是泛素化和蛋白酶体降解的靶标。这些研究将被执行 使用体外和体内糖尿病模型。全细胞和单通道膜片钳技术， 体外抗氧化酶基因转移，以及特定的转基因小鼠将被用来确定 糖尿病引起的ROS产生对BK通道功能和降解的影响。这样做的结果 该项目可能为电生理和分子机制提供重要的新见解。 BK通道功能改变可能与内皮依赖和非内皮依赖有关 糖尿病患者的血管功能障碍。

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.