TREK-1 channels regulate pressure sensitivity and calcium signaling in trabecular meshwork cells

Abstract

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.

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

“…Cell culture. TM cells were isolated from juxtacanalicular and corneoscleral regions of the human donors' eyes, as described (15,17), in accordance with consensus characterization recommendations (31).…”

“…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.…”

“…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). The cell membrane was expanded by application of hydrostatic pressure (1.5 sec, 25 mm Hg) delivered by high-speed pressure clamp (HSPC) ( Fig.…”

“…Pressure homeostasis in the vasculature, lung, bladder and eye is dynamically regulated by stretch-activated ion channels (SACs) (2,14,15) that belong to Piezo, TRP (transient receptor potential) and two-pore potassium channel families (16). TRPV4, a polymodal nonselective cation channel, mediates the TM Ca 2+ influx in response to stretch and contributes to cell remodeling in the presence of chronic pressure/strain (15,17) but the mechanotransduction mechanism that mediates the homeostatic adjustment to pressure fluctuations has remained unknown. Potential candidates are Piezo channels, which have been recently implicated in dynamic regulation of pressure-induced lung vascular hyperpermeability and shear-stress induced endothelial signaling (18,19).…”

Polynuclear ruthenium amines inhibit K2P channels via a ‘finger in the dam’ mechanism

“…There are fifteen K2P subtypes comprising six subfamilies in which the channel monomers assemble into dimers wherein each subunit contributes two conserved pore forming domains to make the channel pore (Brohawn et al, 2012;Dong et al, 2015;Feliciangeli et al, 2014;Lolicato et al, 2017;Miller and Long, 2012;Rödström et al, 2019). A range of physical and chemical signals control K2P function (Enyedi and Czirjak, 2010;Feliciangeli et al, 2014;Renigunta et al, 2015) and various K2P subtypes have emerging roles in a multitude of physiological responses and pathological conditions such as action potential propagation in myelinated axons (Brohawn et al, 2019;Kanda et al, 2019), anesthetic responses (Heurteaux et al, 2004;Lazarenko et al, 2010), microglial surveillance (Madry et al, 2018), sleep duration (Yoshida et al, 2018), pain (Alloui et al, 2006;Devilliers et al, 2013;Vivier et al, 2017), arrythmia (Decher et al, 2017), ischemia (Heurteaux et al, 2004;Laigle et al, 2012;Wu et al, 2013), cardiac fibrosis (Abraham et al, 2018), depression (Heurteaux et al, 2006), migraine (Royal et al, 2019), intraocular pressure regulation (Yarishkin et al, 2018), and pulmonary hypertension (Lambert et al, 2018). Although there have been recent advances in identifying new K2P modulators (Bagriantsev et al, 2013;Lolicato et al, 2017;Pope et al, 2018;Su et al, 2016;Tian et al, 2019;Vivier et al, 2017;Wright et al, 2019) and in defining key structural aspects of K2P channel pharmacolog...…”