Electrophysiological assays using oocytes that express human ENaC and the use of phenamil to improve the effect of ENaC enhancers in assays using membrane potential reporting dyes
In one aspect, 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. The present invention further relates to amphibian oocyte-based medium-throughput electrophysiological assays for identifying human ENaC modulators, preferably ENaC enhancers. Compounds that modulate ENaC function in a cell-based ENaC assay are expected to affect salty taste in humans.
1. A composition comprising an isolated mammalian cell or mammalian cell population selected from the group consisting of MDCK, BHK, COS, NIH3T3, Swiss3T3 and CHO cells that expresses a functional hENaC comprising a delta subunit having the sequence of in SEQ ID NO:7 and beta and gamma subunit which respectively having the sequences SEQ ID NO: 2 and 3, which cell or cell population is loaded with a membrane potential fluorescent dye or a sodium fluorescent dye and wherein the composition further comprises at least one ENaC inhibitor comprising 0.2 to 0.5 μM phenamil and at least one potential ENaC enhancer.
2. The composition of claim 1 , wherein the isolated mammalian cell or mammalian cell population transiently expresses delta, beta and gamma hENaC subunits.
3. The composition of claim 1 , wherein the isolated mammalian cell or mammalian cell population stably expresses said delta, beta and gamma hENaC subunits.
4. The composition of claim 1 , wherein the isolated mammalian cell or mammalian cell population is comprised in a multi-well test plate device.
5. The composition of claim 1 , wherein the isolated mammalian cell or mammalian cell population is loaded with a membrane potential dye.
6. The composition of claim 1 , wherein the isolated mammalian cell or mammalian cell population is grown to about 80% confluence.
7. The composition of claim 5 , wherein the isolated mammalian cell or mammalian cell population of claim 5 wherein the membrane potential dye is CC2-DMPE, DiSBAC2(3) or ESS-CY4.