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. ? ? ?
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