Trabecular Meshwork TREK-1 Channels Function as Polymodal Integrators of Pressure and pH

Yarishkin, Oleg; Phuong, Tam T. T.; Križaj, David

doi:10.1167/iovs.19-26851

Cited by 18 publications

(20 citation statements)

References 52 publications

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“…Anterior chambers were fixed in 4% para-formaldehyde for one hour, cryoprotected in 15 and 30% sucrose gradients, embedded in Tissue-Tek® O.C.T. (Sakura, 4583), and cryosectioned at 12 µm, as described (32) Steps of positive (in whole-cell recording) and negative (in single-channel recording) pressure were delivered via High-Speed Pressure Clamp (ALA Scientific) as described ( Figure 1A) (17,34). Outflow Facility Measurements.…”

Section: See Si Experimental Procedures For More Detailsmentioning

confidence: 99%

“…The cells expressed standard TM markers, including MYOC, TIM3, AQP1, MGP and ACTA2 and responded to steroid DEX with MYOC upregulation ( Supplementary Fig. 1) (34). Stretch-activated currents 6 were measured under voltage clamp in the whole-cell configuration, at the holding potential of -40 mV that approximates the cells' resting potential (17).…”

Section: Sac Activity In Tm Cells Involves Kinetically Separable Compmentioning

confidence: 99%

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Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Yarishkin

Phuong

Baumann

et al. 2020

Preprint

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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.

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Section: See Si Experimental Procedures For More Detailsmentioning

confidence: 99%

Section: Sac Activity In Tm Cells Involves Kinetically Separable Compmentioning

confidence: 99%

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Yarishkin

Phuong

Baumann

et al. 2020

Preprint

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“…Human trabecular meshwork (hTM cells), isolated from the juxtacanalicular and corneoscleral regions of the human eye (ScienCell Research Laboratories) were cultured in Trabecular Meshwork Cell Medium (ScienCell Research Laboratories) supplemented with 2% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, at 37 o C and pH 7.4. In accordance with consensus characterization recommendations (23,104,24), the phenotype was periodically validated by profiling for markers MYOC, TIMP3, AQP1, MGP, ACTA2 (α-smooth muscle actin, α Technologies. Salts were purchased from Sigma or VWR.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we tested the hypothesis that force-induced remodeling requires collaboration between transient receptor potential vanilloid isoform 4 (TRPV4), a calcium-permeable polymodal SAC that mediates the responses to strain, swelling, pressure and polyunsaturated fatty acids in TM cells (23)(24)(25), and Rho signaling, the actin cytoskeleton and FA pathways. TRPV4 is prominently expressed in mouse and human TM membrane and primary cilia (23,26) and is often targeted to actin-enriched regions and areas of high strain such as FAs (27)(28)(29)(30).…”

Section: Introductionmentioning

confidence: 99%

Mechanically induced cytoskeletal remodeling in trabecular meshwork cells requires TRPV4 - Rho signaling interactions

Lakk¹,

Križaj

2020

Preprint

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Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor outflow from the anterior chamber of the eye. IOP-dependent increases in TM stiffness and contractility drive open angle glaucoma but the mechanotransduction mechanisms that regulate these processes remain poorly understood. We used fluorescence imaging and biochemical analyses to investigate cytoskeletal and focal adhesion remodeling in human TM cells stimulated with cyclic strain. The cells showed enhanced F-actin polymerization, increased number and size of focal adhesions, and activation of the Rho-associated protein kinase (ROCK). Stretch-induced activation of the small GTPase RhoA, and tyrosine phosphorylations of focal adhesion proteins paxillin, focal adhesion kinase (FAK), vinculin and zyxin were time-dependently inhibited by HC-067047, an antagonist of transient receptor potential vanilloid 4 (TRPV4) channels, and the ROCK inhibitor Y-27632. TRPV4 and ROCK activation were required for zyxin translocation and increase in the number/size of focal adhesions in stretched cells. Y-27632 blocked actin polymerization without affecting calcium influx induced by membrane stretch and the TRPV4 agonist GSK1016790A. These results reveal that mechanical tuning of TM cells requires parallel activation of TRPV4, integrins and ROCK, with chronic stress leading to sustained remodeling of the cytoskeleton and focal complexes.

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“…Whole-cell patch-clamp measurements from primary CECs were conducted with Multiclamp 700B amplifiers, pClamp 10.7 acquisition software, and DigiData 1550 (Molecular Devices (San Jose, CA, USA)) as described in published protocols. 20,35 The standard extracellular recording solution contained (mM): 140 NaCl, 2.5 KCl, 1.5 MgCl 2 , 1.8 CaCl 2 , 10 HEPES, 5.5 D-glucose. The pipette solution contained (mM): 120 K-gluconate, 10 KCl, 10 HEPES, 1 MgCl 2 , 4 Mg-ATP, 0.6 Na-GTP, 0.5 EGTA, pH was adjusted to 7.3 with potassium hydroxide (KOH).…”

Section: Patch Clamp and Pressure Clamp Experimentsmentioning

confidence: 99%

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

Lapajne

Lakk

Yarishkin

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. Contact lenses, osmotic stressors, and chemical burns may trigger severe discomfort and vision loss by damaging the cornea, but the signaling mechanisms used by corneal epithelial cells (CECs) to sense extrinsic stressors are not well understood. We therefore investigated the mechanisms of swelling, temperature, strain, and chemical transduction in mouse CECs. METHODS. Intracellular calcium imaging in conjunction with electrophysiology, pharmacology, transcript analysis, immunohistochemistry, and bioluminescence assays of adenosine triphosphate (ATP) release were used to track mechanotransduction in dissociated CECs and epithelial sheets isolated from the mouse cornea. RESULTS. The transient receptor potential vanilloid (TRPV) transcriptome in the mouse corneal epithelium is dominated by Trpv4, followed by Trpv2, Trpv3, and low levels of Trpv1 mRNAs. TRPV4 protein was localized to basal and intermediate epithelial strata, keratocytes, and the endothelium in contrast to the cognate TRPV1, which was confined to intraepithelial afferents and a sparse subset of CECs. The TRPV4 agonist GSK1016790A induced cation influx and calcium elevations, which were abolished by the selective blocker HC067047. Hypotonic solutions, membrane strain, and moderate heat elevated [Ca 2+ ] CEC with swelling-and temperature-, but not strain-evoked signals, sensitive to HC067047. GSK1016790A and swelling evoked calcium-dependent ATP release, which was suppressed by HC067027 and the hemichannel blocker probenecid. CONCLUSIONS. These results demonstrate that cation influx via TRPV4 transduces osmotic and thermal but not strain inputs to CECs and promotes hemichannel-dependent ATP release. The TRPV4-hemichannel-ATP signaling axis might modulate corneal pain induced by excessive mechanical, osmotic, and chemical stimulation.

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Trabecular Meshwork TREK-1 Channels Function as Polymodal Integrators of Pressure and pH

Cited by 18 publications

References 52 publications

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Mechanically induced cytoskeletal remodeling in trabecular meshwork cells requires TRPV4 - Rho signaling interactions

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

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