AbstractCholesterol plays important roles in the regulation of membrane viscoelasticity, protein function and cellular health but despite its association with debilitating pressure-related diseases its role in mechanotransduction is not well understood. Here, we investigated how alterations in membrane cholesterol levels impact activation of TRPV4 (transient receptor potential vanilloid isoform 4), a stretch-activated cation channel, in trabecular meshwork (TM) cells that control intraocular pressure in the mammalian eye. Human TM cells responded to pharmacological depletion of free membrane cholesterol with augmented TRPV4 activation by agonist GSK1016790A, swelling and strain, and by increased membrane expression of TRPV4 immunoreactivity. Cyclic stretch increased the membrane cholesterol/phosphatidylcholine ratio and upregulated expression of F-actin, with the low-cholesterol effects reversed by cholesterol supplementation. Cholesterol depletion induced a constitutive current in TRPV4-expressing Xenopus laevis oocytes and obviated its activation by hypotonic swelling. Taken together, these findings suggest that cholesterol regulates translocation and activation of TRPV4, and guides its participation in macromolecular complexes, cytoskeletal architecture and cell-matrix contacts; in turn membrane cholesterol levels are dynamically regulated by the biomechanical milieu. By regulating TM pressure sensitivity, calcium homeostasis and contractility, cholesterol-TRPV4 interactions could influence IOP homeostasis and its role in glaucoma and diabetes.