Role of ion channels in the response of conjunctival goblet cells to dry eye

Abstract

Purpose : Although ion channels impact the function of virtually all cells, their role in the response of the ocular surface to dry eye is poorly understood. Here, the goal was to elucidate the role of ion channels in the response of the conjunctiva to extracellular hyperosmolarity, which is the hallmark of dry eye. I focused on conjunctival goblet cells whose exocytosis of tear-stabilizing mucin is triggered by a cholinergic neural reflex.

Methods : Perforated-patch pipettes were sealed onto goblet cells located in freshly isolated rat conjunctival specimens suprafused with solutions of various osmolarities. pClamp software aided current-voltage analyses. Under voltage-clamp, the phase-tracking technique provided a high-sensitivity/high-temporal resolution assay of membrane capacitance, which is an indicator of cell surface area and thus, increases as exocytosising granules fuse with the plasma membrane. Exocytosis was induced by the cholinergic agonist, carbachol (10 µM).

Results : Recordings revealed that as extracellular osmolarity rises above 330 mOsmol, the membrane potential of goblet cells rapidly hyperpolarizes from -38 ± 6 mV (SD; n = 32) to -53 ± 4 mV (n = 31; P < 0.0001). Current-voltage plots showed that this voltage increase is due to the activation of a conductance that has a reversal potential (-56 ± 4 mV; n = 17) near E_K (-58 mV) and is inhibited by the K_ATP channel blocker, glibenclamide (0.5 µM; n = 8; P < 0.0001). What is the functional impact of the hyperosmolar-induced K_ATP-driven hyperpolarization? Since hyperpolarization enhances the electro-gradient for calcium influx, which is a key step in cholinergic-induced mucin secretion, I postulated that exocytosis is boosted under hyperosmotic conditions. Supporting this scenario, the increase in membrane capacitance induced by carbachol was ~10-fold larger at 360 mOsmol than at 300 mOsmol (P < 0.0001); 8700 ± 1035 fF (n = 17) versus 855 ± 350 fF (n = 12). As expected for a K_ATP-driven mechanism, the addition of glibenclamide to the hyperosmotic solution resulted in a 92 ± 8% (n = 9; P < 0.0001) diminution in the carbachol-induced increase in membrane capacitance.

Conclusions : A previously unappreciated adaptive mechanism by which goblet cells respond to hyperosmolarity is the generation of a K_ATP-driven hyperpolarization that in turn, markedly boosts the cholinergic-induced exocytosis of tear-stabilizing mucin.

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