Product(s) used in this publication: Reference Peptides for Targeted Proteomics - SpikeTides™ & SpikeMix™
Mechanosensitive ion channels are implicated in the biology of touch, pain, hearing and vascular reactivity; however, the identity of these ion channels and the molecular basis of their activation is poorly understood. We previously found that transient receptor potential vanilloid 4 (TRPV4) is a receptor operated ion channel that is sensitised and activated by mechanical stress. Here, we investigated the effects of mechanical stimulation on TRPV4 localisation and activation in native and recombinant TRPV4-expressing cells. We used a combination of total internal reflection fluorescence microscopy, cell surface biotinylation assay and Ca(2+) imaging with laser scanning confocal microscope to show that TRPV4 is expressed in primary vascular endothelial cells and that shear stress sensitises the response of TRPV4 to its agonist, GSK1016790A. The sensitisation was attributed to the recruitment of intracellular pools of TRPV4 to the plasma membrane, through the clathrin and dynamin-mediated exocytosis. The translocation was dependent on ILK/Akt signalling pathway, release of Ca(2+) from intracellular stores and we demonstrated that shear stress stimulated phosphorylation of TRPV4 at tyrosine Y110. Our findings implicate calcium-sensitive TRPV4 translocation in the regulation of endothelial responses to mechanical stimulation.
Ca2+; Endothelium; Mechanotransduction; Shear stress; TRPV4; Translocation