Patch-Clamp Recording of K+ Current from Lacrimal Gland Duct Cells

Abstract

AbstractElectrophysiologic studies have characterized ion channels in lacrimal gland acinar cells, but due to relative paucity and inaccessibility, such studies on lacrimal gland duct cells are challenging. The duct cells are believed to secrete the high level of K+ that is present in tears. The goal of this project was to develop a method for isolation of viable, single duct cells, demonstrate their utility for patch-clamp recording and characterize the K+ channels expressed by duct cells. Exorbital lacrimal gland slices from Sprague-Dawley rats were incubated with collagenase. Under a microscope, the ducts were microdissected from the glands and then incubated with elastase and collagenase. Dispersed duct cells were plated on a cover slip coated with BD Cell-Tak. Duct cells were distinguished from acinar cells by their smaller size and lack of granularity. Whole-cell K+ currents were recorded from duct cells using the perforated-patch technique and pipettes with resistances of less than 10 MΩ. EGTA and CaCl2 in the pipette solution were adjusted to give 1 uM free Ca++. When held at −80 mV, duct cells showed K+ currents that activated at command voltages near 0 mV and reached amplitudes near 1 nA at +100 mV. Currents reached peak amplitude less than 20 ms after depolarization and did not inactivate. These currents were inactivated by holding the cells at 0 mV. Currents were blocked reversibly by TEA in the presence of Ca2+, but were not blocked by TEA in the absence of Ca2+. Currents were not affected by clotrimazole (10 uM) or Ba2+ (5 mM). A method has been established for isolation and dissociation lacrimal gland duct cells for electrophysiologic studies. These cells express a voltage-activated K+ channel that is dependent on the presence of intracellular Ca2+ and may correspond to the IKCa1 channel expressed on the apical membranes of lacrimal gland ducts.