Mechanotransduction by the trabecular meshwork (TM) is an essential component of intraocular pressure regulation in the vertebrate eye. This process is compromised in glaucoma but is poorly understood. In this study, we identify transient receptor potential vanilloid isoform 4 (TRPV4) and TWIK-related potassium channel-1 (TREK-1) as key molecular determinants of TM membrane potential, pressure sensitivity, calcium homeostasis, and transcellular permeability. We show that resting membrane potential in human TM cells is unaffected by “classical” inhibitors of voltage-activated, calcium-activated, and inwardly rectifying potassium channels but is depolarized by blockers of tandem-pore K+ channels. Using gene profiling, we reveal the presence of TREK-1, TASK-1, TWIK-2, and THIK transcripts in TM cells. Pressure stimuli, arachidonic acid, and TREK-1 activators hyperpolarize these cells, effects that are antagonized by quinine, amlodipine, spadin, and short-hairpin RNA–mediated knockdown of TREK-1 but not TASK-1. Activation and inhibition of TREK-1 modulates [Ca2+]TM and lowers the impedance of cell monolayers. Together, these results suggest that tensile homeostasis in the TM may be regulated by balanced, pressure-dependent activation of TRPV4 and TREK-1 mechanotransducers.

Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. r Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. r We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. r Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. r This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye.

Over- and underexposure to cholesterol activates glia in neurodegenerative brain and retinal diseases but the molecular targets of cholesterol in glial cells are not known. Here, we report that disruption of unesterified membrane cholesterol content modulates the transduction of chemical, mechanical and temperature stimuli in mouse Müller cells. Activation of TRPV4 (transient receptor potential vanilloid type 4), a nonselective polymodal cation channel was studied following the removal or supplementation of cholesterol using the methyl-beta cyclodextrin (MβCD) delivery vehicle. Cholesterol extraction disrupted lipid rafts and caveolae without affecting TRPV4 trafficking or membrane localization of the protein. However, MβCD suppressed agonist (GSK1016790A)- and temperature-evoked elevations in [Ca2+]i, and suppressed transcellular propagation of Ca2+ waves. Lowering the free membrane cholesterol content markedly prolonged the time-course of the glial swelling response, whereas MβCD:cholesterol supplementation enhanced agonist- and temperature-induced Ca2+ signals and shortened the swelling response. Taken together, these data show that membrane cholesterol modulates polymodal transduction of agonists, swelling and temperature stimuli in retinal radial glia and suggest that dyslipidemic retinas might be associated with abnormal glial transduction of ambient sensory inputs.

Retinal ganglion cells (RGCs) are projection neurons that transmit the visual signal from the retina to the brain. Their excitability and survival can be strongly influenced by mechanical stressors, temperature, lipid metabolites, and inflammatory mediators but the transduction mechanisms for these non-synaptic sensory inputs are not well characterized. Here, we investigate the distribution, functional expression, and localization of two polymodal transducers of mechanical, lipid, and inflammatory signals, TRPV1 and TRPV4 cation channels, in mouse RGCs. The most abundant vanilloid mRNA species was Trpv4, followed by Trpv2 and residual expression of Trpv3 and Trpv1. Immunohistochemical and functional analyses showed that TRPV1 and TRPV4 channels are expressed as separate molecular entities, with TRPV1-only (∼10%), TRPV4-only (∼40%), and TRPV1 + TRPV4 (∼10%) expressing RGC subpopulations. The TRPV1 + TRPV4 cohort included SMI-32-immunopositive alpha RGCs, suggesting potential roles for polymodal signal transduction in modulation of fast visual signaling. Arguing against obligatory heteromerization, optical imaging showed that activation and desensitization of TRPV1 and TRPV4 responses evoked by capsaicin and GSK1016790A are independent of each other. Overall, these data predict that RGC subpopulations will be differentially sensitive to mechanical and inflammatory stressors.

No abstract

AbstractMechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca2+]i elevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests a novel potential therapeutic target for treating glaucoma.Significance StatementTrabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts, and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

No abstract