Tandem-pore domain potassium channels are components of intracellular signaling pathways, determinants of pH sensitivity and mechanosensitivity of human trabecular meshwork cells.

Abstract

Purpose : Our goal is to identify and characterize the mechanotransducers in the trabecular meshwork (TM), a pressure-sensitive tissue that regulates the dynamics of aqueous fluid outflow in the eye. Here, we studied ion channels that regulate the membrane potential, excitability and pressure sensitivity in TM cells. We sought to establish the molecular link between mechanotransduction, calcium homeostasis and TM contractility that underpin the dynamic regulation of intraocular pressure (IOP).

Methods : Transcript and protein levels in primary human TM cells were assessed with PCR, Western blot and immunocytochemistry. Patch-clamp, high-speed pressure clamp and calcium imaging were used to characterize the electrophysiological features of the mechanotransducers. Outflow facility of human TM was assessed in nano-scaffold model of TM.

Results : TM cells express multiple putative mechanogated channels, among which TRPV4, TREK-1 and TASK-1 mRNA and protein predominate. The membrane potential and steady-state whole-cell current were sensitive to K2P blockers, external pH and short hairpin RNA (shRNA) that target specific K2P isoforms. shRNAs attenuated the whole-cell conductance whereas K2P channel agonists hyperpolarized the membrane potential and increased the transmembrane conductance. Patch clamp and HSPC experiments conducted in the presence of TRPV4 antagonists showed robust activation of outward currents in response to physiological steps of positive pressure. These responses were suppressed by quinine and reduced in cells that overexpressed K2P-specific, but not scrambled, shRNAs. K2P inhibition elevated intracellular Ca²⁺ levels, an effect that was attenuated by high-K⁺. The outflow facility in the nano-scaffold model of the healthy human TM was, however, unaffected by quinine.

Conclusions : This study suggests that tensile homeostasis in human TM cells is maintained by the opposing dynamic activation of pressure-sensitive TRPV4 and K2P channels. Mechanical stretch and/or pressure evoke hyperpolarizing shifts that are linked to K2P activation, intracellular Ca²⁺ homeostasis and thus TM contractility. The pressure sensitivity of the conventional outflow pathway may be, under certain conditions, negatively regulated by K2P channels.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.