Evidence for functional and dynamic microcompartmentation of Cav-1/TRPV4/KCa in caveolae of endothelial cells

Ca2+-activated K+ channels (K-Ca) play a pivotal role in the endothelium-dependent hyperpolarization and regulation of vascular tone and blood pressure. For activation, K-Ca depend on an increase of intracellular calcium which is substantially mediated by Ca2+-permeable cation channels including the transient receptor potential V4 (TRPV4). It has been proposed that K-Ca and Ca2+-permeable cation channels may be clustered in localized positions within the cell membrane to form functional units and that caveolae may constitute the scaffolding for such microcompartmental organization. Here, we sought to elucidate the composition and functional relevance of these microcompartments in vitro and in vivo. We show that TRPV4 and small-conductance K(Ca)2.3 are enriched in caveolae of human microvascular endothelial cells. Using immunoprecipitation, immunocytology and superresolution microscopy, we found a caveolae-dependent association between caveolin-1, TRPV4 and small conductance K(Ca)2.3, but not intermediate conductance K(Ca)3.1, in endothelial cells under static condition. Mechanical stimulation of cells via exposure to shear stress led to a partial de-novo colocalization of K(Ca)3.1 with Cav-1 and TRPV4. In a mouse model of genetic Cav-1 deficiency, we found significantly reduced K-Ca-mediated currents as determined by patch-clamping in carotid artery endothelial cells (CAEC) from Cav-1(-/-) mice compared to wildtype. Functionally, Cav-1 deficiency was associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation in response to shear stress and acetylcholine. In summary, our findings provide evidence for a dynamic microcompartmentation of TRPV4/K-Ca in caveolae of endothelial cells and highlight the importance of Cav-1 for endothelial K-Ca functions and flow-induced vasodilation. (C) 2015 Elsevier GmbH. All rights reserved.