Assessment of Swelling-Activated Cl − Channels Using the Halide-Sensitive Fluorescent Indicator 6-Methoxy- N-(3-Sulfopropyl)quinolinium

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl − channels, using the halide-sensitive fluorescent dye 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO 3 − Ringer's were exposed to I − pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I − influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential ( E m) was −58 and −32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I − permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I −) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I − influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I − influx, together with E m under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl − channels with ER = 4.9 when the hyposmotic shock was 110 ± 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl − channels in monolayers.