We have evidence that TRPV4 channels in the nonpigmented ciliary epithelium (NPE) interact withconnexin-50 to form a mechanism that responds to mechanical stimuli. Distortion of NPE cells iscapable of causing TRPV4 channel activation which in turn causes hemichannel opening at theaqueous humor-facing surface of the NPE. What this means is the ciliary body which secretesaqueous humor has a mechanism capable to sensing and responding to distortion caused by anincrease of intraocular pressure. Our working hypothesis is that mechanosensitive hemichannelopening and ATP release are steps in an autocrine feedback loop that reduces NaK-ATPase activityin the NPE. Here we propose studies on how the hemichannel opening mechanism senses andresponds to a mechanical stimulus (Aim 1). We will characterize how the hemichannel mechanismpivots on TRPV4 activation the role of connexin-50 vs pannexin-1 how TRPV4 channels respond tomechanical stimuli (cell swelling and stretch) and the electrical conductance signal of thehemichannels. Aim 2 studies will examine how hemichannel opening allows ATP to exit the cell thenactivate receptors and signaling pathways that change NaK-ATPase activity in an autocrine fashion.We also will study cAMP and melatonin release into the aqueous humor via hemichannels. Studiesin Aim 3 will determine the effect of intraocular pressure on the NPE hemichannel mechanism in anex vivo arterially perfused eye preparation and test whether the effect of hemichannel blockingmolecules and TRPV4-interacting drugs on the rate of aqueous humor formation. The concept ofmechanosensitive feedback regulation of NaK-ATPase activity in the NPE is significant becauseNaK-ATPase activity provides the driving force for aqueous humor secretion. The NPE forms acellular barrier between blood and aqueous and so is subjected to altered physical forces whenintraocular pressure changes in relation to hydrostatic pressure in the ciliary process stroma.