Ethanol-induced Cl- flux in rat cerebellar granule cells as measured by a fluorescent probe

Cl- fluxes through the GABAA receptor gated ion channels in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl- ions. The cells were bathed in a low Cl- medium so that the Cl- gradient was directed outward. Ethanol increased the SPQ fluorescence indicating a decrease in intracellular Cl- due to Cl- efflux. Picrotoxin inhibited the effect at low concentrations of ethanol (less than 50 mM) in a concentration dependent manner. The effects of ethanol were potentiated at low concentrations (less than 10 microM) of gamma-aminobutyric acid (GABA), but inhibited at higher concentrations (0.3-2.0 mM). The results support the hypothesis that ethanol may act via the GABAA receptor gated ion channel. The results also suggest that SPQ is a suitable probe for measuring GABAA receptor-coupled Cl- fluxes through the GABAA receptor-gated channels in living cells.