Right-angle light scattering to assay basal and regulated plasma membrane Cl- conductances.
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 Ca(2+)-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.
Dho
S
S
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Chou
S
S
Chang
X B
XB
Rommens
J M
JM
Foskett
J K
JK
D000818:Animals; D002118:Calcium / Q000502:physiology; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000502:physiology*; D002712:Chlorides / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D004553:Electric Conductivity; D006096:Gramicidin / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008027:Light; D008565:Membrane Proteins / Q000235:genetics; D012016:Reference Values; D012542:Scattering, Radiation
D002712:Chlorides; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006096:Gramicidin; D015759:Ionomycin; D004247:DNA; D000242:Cyclic AMP; D002118:Calcium