Membrane cholesterol regulates TRPV4 function, cytoskeletal expression and the cellular response to tension

Despite the association of cholesterol with debilitating pressure-related diseases, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and activation of stretch- activated TRPV4 (transient receptor potential vanilloid isoform 4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time- dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depletion of free membrane cholesterol with m-{beta}-cyclodextrin (M{beta}CD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. M{beta}CD increased membrane expression of TRPV4, caveolin-1 and flotillin. Caveolin-1 antibody partially precipitated a truncated [~]75 kDa variant whereas the majority of TRPV4 did not colocalize or interact with caveolae or lipid rafts, indicating that TRPV4 is mainly localized outside of cholesterol-enrichedmembrane domains. M{beta}CD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, while the membrane C/P ratio reflects the biomechanical milieu, trabecular transduction of mechanical information is modulated by the membrane cholesterol content. Diet, cholesterol metabolism and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes.