Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca²⁺]_TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl^- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl^- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

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