The overall objective of our research program is to understand the regulation of ion channels in the cardiovascular system. We made a novel observation that the large conductance Ca2+-activated K+ (BK) channels are targeted to caveolae in bovine aortic endothelial cells (BAEC). Caveolae are specialized membrane microdomains that serve as platforms for integrating specific cellular signal transduction processes. The goal of this study is to determine the physical, molecular, and physiological interactions between caveolin-1 and BK channels, as well as the physiological relevance of such interactions. The hypotheses to be tested are: 1) BK channels are targeted to caveolae in vascular endothelial cells and interact directly with caveolin-1. 2) BK channel function is modulated by caveolae targeting in vascular endothelial cells. 3) beta2-Adrenergic receptors (beta2AR) are targeted to endothelial caveolae and are more efficient than beta1AR in regulating BK channel function. 4) Shear stress modulates caveolae function and up- regulates endothelial BK channel function.
Three specific aims are proposed.
Aim 1 is to determine the physical and molecular interactions between BK channels and caveolin-1. These will be assessed by cell fractionation, density gradient centrifugation, immunoprecipitation, immunofluorescence imaging, mutagenesis of hSIo and caveoline-1, and GST-fusion protein binding assays.
Aim 2 is to determine the physiological interaction between BK channels and caveolae. Whole-cell and single channel patch clamp recordings of wild-type and mutant hSIo and caveolin-1 will be performed. The effects of caveolin-1 on BK current densities, channel voltage dependence, Ca2+ dependence, single channel opening probability and kinetics will be determined. Three plausible mechanisms will be explored. The strategies include: A) Synthesis of cavtratin, a cell permeable caveolin-1 scaffolding domain peptide to determine whether caveolin-1 is a negative regulator of BK channel. B) Surface biotinylation of BK channels in BAEC to determine whether BK channel activation involves the trafficking of BK channels from intracellular sites to the membrane. C) Knockdown of caveolin-1 by siRNA to determine whether activation of BK channels by signaling pathways requires normal caveolae structure.
Aim 3 is to determine the physiological relevance of BK channel- caveolin-1 interaction. We plan to determine the role of caveolae in beta-adrenergic activation of BK channels in vascular endothelial cells and the effect of shear stress on caveolae and BK function. The betaAR subtype in BAEC and the mechanism through which they activate BK channels will be analyzed. Results of the proposed research will help us better understand not only the regulation of BK channels in endothelial cells, but also fundamental mechanisms that modulate endothelial function.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080118-05
Application #
7858357
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
OH, Youngsuk
Project Start
2006-07-01
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2012-06-30
Support Year
5
Fiscal Year
2010
Total Cost
$359,270
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Lu, Tong; Sun, Xiaojing; Li, Yong et al. (2017) Role of Nrf2 Signaling in the Regulation of Vascular BK Channel ?1 Subunit Expression and BK Channel Function in High-Fat Diet-Induced Diabetic Mice. Diabetes 66:2681-2690
Lu, Tong; Wang, Xiao-Li; Chai, Qiang et al. (2017) Role of the Endothelial Caveolae Microdomain in Shear Stress-Mediated Coronary Vasorelaxation. J Biol Chem :
Ling, Tian-You; Wang, Xiao-Li; Chai, Qiang et al. (2017) Regulation of cardiac CACNB2 by microRNA-499: Potential role in atrial fibrillation. BBA Clin 7:78-84
Chai, Qiang; Lu, Tong; Wang, Xaio-Li et al. (2015) Hydrogen sulfide impairs shear stress-induced vasodilation in mouse coronary arteries. Pflugers Arch 467:329-40
Yi, Fu; Ling, Tian-You; Lu, Tong et al. (2015) Down-regulation of the small conductance calcium-activated potassium channels in diabetic mouse atria. J Biol Chem 290:7016-26
Yi, Fu; Wang, Huan; Chai, Qiang et al. (2014) Regulation of large conductance Ca2+-activated K+ (BK) channel ?1 subunit expression by muscle RING finger protein 1 in diabetic vessels. J Biol Chem 289:10853-64
Chai, Qiang; Wang, Xiao-Li; Zeldin, Darryl C et al. (2013) Role of caveolae in shear stress-mediated endothelium-dependent dilation in coronary arteries. Cardiovasc Res 100:151-9
Wang, Xiao-Li; Ling, Tian-You; Charlesworth, M Cristine et al. (2013) Autoimmunoreactive IgGs against cardiac lipid raft-associated proteins in patients with postural orthostatic tachycardia syndrome. Transl Res 162:34-44
Ling, Tian-You; Wang, Xiao-Li; Chai, Qiang et al. (2013) Regulation of the SK3 channel by microRNA-499--potential role in atrial fibrillation. Heart Rhythm 10:1001-9
Wang, Xiao-Li; Chai, Qiang; Charlesworth, M Cristine et al. (2012) Autoimmunoreactive IgGs from patients with postural orthostatic tachycardia syndrome. Proteomics Clin Appl 6:615-25

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