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 NO3- 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 (Em) 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 Em 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.
Srinivas
S P
SP
Morton D. Sarver Center for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley, California 94720, USA. srinivas@uclink.bekeley.edu
Bonanno
J A
JA
Hughes
B A
BA
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D002463:Cell Membrane Permeability; D048429:Cell Size; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007454:Iodides; D008564:Membrane Potentials; D008954:Models, Biological; D009566:Nitrates; D009997:Osmotic Pressure; D011806:Quinolinium Compounds
D018118:Chloride Channels; D005456:Fluorescent Dyes; D007454:Iodides; D009566:Nitrates; D011806:Quinolinium Compounds; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium