Hinweise für die funktionelle und dynamische Mikrokompartimentierung von Caveolin-1, TRPV4 und Calcium-aktivierten Kaliumkanälen in den Caveolae von Endothelzellen

Cardiovascular diseases are the major cause of death worldwide. Especially arterial hypertension contributes as a risk factor for cardiovascular diseases. Hypertension is often associated with endothelial dysfunction, athero- and arteriosclerosis, and pathological vascular remodeling. For blood pressure regulation, the endothelium releases relaxing and contracting factors and thereby adjusts smooth muscle contractility. Prostacyclin (PGI2) and nitric oxide (NO) were the earliest identified endothelium-dependent vasodilators. Next to them, the endothelium possess a third vasodilator system, the endothelium-derived hyperpolarizing factor (EDHF). While the role of endothelial NO and PGI2 for local and systemic blood pressure control is well established, the nature of EDHF and EDHF-signalling pathways is not fully understood. However, Ca2+-activated K+ channels (KCa) play a pivitol role in the endothelium-dependent hyperpolarization and regulation of vascular tone and blood pressure. For activation, KCa 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 KCa- and Ca2+-permeable cation channels may be spatially associated to form functional units, and caveolae of the cell membrane have been suggested as possible scaffolds for such microcompartmental organization. Caveolae are small, omega-shaped membrane invaginations present on the surface of many mammalian cell types. They are enriched in cholesterol and sphingolipids and therefore represent lipid-raft microdomains. Caveolae function in a variety of cellular and physiological processes including endocytosis, transcytosis, lipid regulation, signaling, pathogen entry and cancer. Caveolin-1, an integral membrane protein, is the major coat protein of caveolae. In this study we asked the question whether endothelial KCa and TRPV4 are enriched in caveolae and whether deletion of caveolin 1 (Caveolin-1), the major structural protein of caveolae, affects KCa-currents in vivo. We show that TRPV4 and small-conductance KCa2.3 are enriched in caveolae of human microvascular endothelial cells. Using immunoprecipitation, immunocytology and superresolution microscopy, we found that a close and caveolae-dependent association exists between caveolin-1, TRPV4 and small conductance KCa2.3, but not intermediate conductance KCa3.1, in endothelial cells under static condition. Also we could show an interaction und colocalization between KCa2.3 and TRPV4. Mechanical stimulation of cells via exposure to continuous shear stress led to a de-novo colocalization of KCa3.1 with Caveolin-1 and TRPV4. Patch-Clamp measurements of freshly isolated carotid artery endothelial cells (CAEC) from Caveolin-1-/- mice demonstrated a ~ 45 % reduction of KCa-mediated potassium currents (-54 % for KCa2.3 and -40 % for KCa3.1) when compared to wildtype controls. In summary, present work provide evidence for a dynamic microcompartimentation of TRPV4/KCa in caveolae of endothelial cells and highlight the importance of Caveolin-1 for endothelial KCa functions.