TY - JOUR
T1 - Right-angle light scattering to assay basal and regulated plasma membrane Cl- conductances
AU - Dho, S.
AU - Chou, S.
AU - Chang, X. B.
AU - Rommens, J. M.
AU - Foskett, J. K.
PY - 1992
Y1 - 1992
N2 - We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca2+-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.
AB - We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca2+-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.
KW - chloride channel
KW - cystic fibrosis
KW - cystic fibrosis transmembrane conductance regulator
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U2 - 10.1152/ajpcell.1992.263.2.c530
DO - 10.1152/ajpcell.1992.263.2.c530
M3 - Article
C2 - 1381149
AN - SCOPUS:0026742852
SN - 0002-9513
VL - 263
SP - C530-C534
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 2 32-2
ER -