Na–K–Cl cotransporter 1(NKCC1) cotransports Na+, K+, and Cl-ions across the plasma membrane into cells. Accumulation of Cl-ions in dorsal root ganglion neurons induces depolarizing GABAA receptors, which mediate pre...Na–K–Cl cotransporter 1(NKCC1) cotransports Na+, K+, and Cl-ions across the plasma membrane into cells. Accumulation of Cl-ions in dorsal root ganglion neurons induces depolarizing GABAA receptors, which mediate presynaptic inhibition and filtration of sensory noise. The activity of the Na–K–Cl cotransporter is modulated by high-dose loop diuretics, such as furosemide and bumetanide. To identify NKCC1 modulators, we developed a functional cell-based assay feasible for highthroughput screening(HTS), in which the activity of NKCC1 was detected by a BTC-AM dye-based thallium transportation assay. We demonstrated that the influx of Tl?was mediated by NKCC1, which required the existence of Cl-ions and could be inhibited by bumetanide and furosemide. Our results demonstrated that the assay was stable, reproducible, and suitable for HTS of negative modulators for NKCC1.展开更多
基金supported by ‘‘Significant new drugs creation’’ science and technology major special ‘‘Twelfth Five’’planKey Technology Research of GPCR targeting Drug Screening and Mechanism of Action Studies Project(2012ZX09301003-003)
文摘Na–K–Cl cotransporter 1(NKCC1) cotransports Na+, K+, and Cl-ions across the plasma membrane into cells. Accumulation of Cl-ions in dorsal root ganglion neurons induces depolarizing GABAA receptors, which mediate presynaptic inhibition and filtration of sensory noise. The activity of the Na–K–Cl cotransporter is modulated by high-dose loop diuretics, such as furosemide and bumetanide. To identify NKCC1 modulators, we developed a functional cell-based assay feasible for highthroughput screening(HTS), in which the activity of NKCC1 was detected by a BTC-AM dye-based thallium transportation assay. We demonstrated that the influx of Tl?was mediated by NKCC1, which required the existence of Cl-ions and could be inhibited by bumetanide and furosemide. Our results demonstrated that the assay was stable, reproducible, and suitable for HTS of negative modulators for NKCC1.
