| 摘要 |
<p>The present invention relates to a mammalian cell-based high-throughput assay for the profiling and screening of human epithelial sodium channel (hENaC) cloned from a human kidney c-DNA library and is also expressed in other tissues including human taste tissue. It is thought that ENaC is involved in mediating mammalian salty taste responses. Compounds that modulates ENaC function in a cell-based ENaC assay would be expected to affect salty taste in humans. The present invention also provides recombinant mammalian cells that express a functional hENaC. The assay described herein has major advantages over existing cellular expression systems, in that both mammalian cells are employed and the assay can be run in standard 96 or 384 well culture plates in high-throughput mode. In brief, the mammalian cell line HEK293T (a human embryonic kidney cell line expressing the SV40 large T-cell antigen) are transiently transfected with all three subunits of human ENaC (or and ) either by Ca2+ phosphate or lipid-based systems. Transfected cells are seeded into 96 or 384-well culture plates, and functional expression is allowed to proceed for a minimum of 24 hours. The cells are then incubated with a membrane-potential fluorescent dye or a sodium fluorescent dye (from Molecular Devices) that provides a high-throughput, fast, simple and reliable fluorescence-based method for detecting changes in voltage across the cell membrane. The assay of the invention can reliably detect both facilitation or inhibition of hENaC function, providing a robust screen for compounds that could either enhance or block channel activity, and thereby modulate salty taste in humans.</p> |
