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.
Diabetes has reached epidemic proportions, with cardiovascular diseases as the most important causes of morbidity and mortality in diabetic patients. BK channels are proteins that regulate vessel tone and serve as 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 suppressed expression of Nrf2, a critical factor that normally keeps the damaging effects of excess oxidation under control. We will determine the effects of diabetes on Nrf2 expression and on BK channel function and degradation. We will design strategies to activate Nrf2 activities to restore BK channel function and normal blood vessel activities. The results of this project may provide important novel insights into the mechanisms through which diabetes leads to abnormal function in the cardiovascular system and may suggest avenues for innovative therapies.
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