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

Abstract

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. The assays described herein have advantages over existing cellular expression systems. In the case of mammalian cells, such assays can be run in standard 96 or 384 well culture plates in high-throughput mode with enhanced assay results being achieved by the use of a compound that inhibits ENaC function, preferably an amiloride derivative such as Phenamil. In the case of the inventive oocyte electrophysiological assays (two-electrode voltage-clamp technique), these assays facilitate the identification of compounds which specifically modulate human ENaC. The assays of the invention provide a robust screen useful to detect compounds that facilitate (enhance) or inhibit hENaC function. Compounds that enhance or block human ENaC channel activity should thereby-modulate salty taste in humans.

1 - 110 . (canceled) 
     
     
         111 . An isolated mammalian cell or cell population that expresses a functional hENaC comprising a delta subunit which is at least 95% identical to the polypeptide in SEQ ID NO:7 and an alpha and beta subunit which are respectively at least 95% identical to the polypeptides in SEQ ID NO: 1 and 2. 
     
     
         112 . The isolated cell or cell population of  claim 111 , which is human. 
     
     
         113 . The isolated cell or cell population of  claim 111 , which is selected from the group consisting of MDCK, BHK, COS, NIH3T3, Swiss3T3 and CHO cells. 
     
     
         114 . The isolated cell or cell population of  claim 111  wherein said cell or cells transiently express the alpha (or delta), beta and gamma hENaC subunits. 
     
     
         115 . The isolated cell or cell population of  claim 111 , wherein said cell or cells stably express the alpha (or delta), beta and gamma hENaC subunits. 
     
     
         116 . The isolated cell or cell population of  claim 111 , wherein said cell or cells are comprised in a multi-well test plate device. 
     
     
         117 . The isolated cell or cell population of  claim 111 , wherein said cell or cells are loaded with a membrane potential dye. 
     
     
         118 . The isolated cell or cell population of  claim 111 , wherein said cells are grown to about 80% confluence. 
     
     
         119 . The isolated cell or cell population of  claim 118 , wherein the membrane potential dye is CC2-DMPVE, DiSBAC2(3) or ESS-CY4. 
     
     
         120 . The isolated cell or cell population of  claim 111 , wherein the delta subunit has the sequence in SEQ ID NO:7. 
     
     
         121 . The isolated cell or cell population of  claim 111 , wherein the alpha and beta subunits respectively contain the polypeptides in SEQ ID NO: 1 and 2. 
     
     
         122 . A method of identifying a compound which modulates taste by: (i) contacting a recombinant or isolated eukaryotic cell or cell population that expresses a functional human Epithelial Sodium Channel (hENaC) comprising a delta subunit which is at least 95% identical to the polypeptide encoded by SEQ ID NO:7 and an alpha and beta subunit which are respectively at least 95% identical to the polypeptides encoded by SEQ ID NO: 1 and 2 with at least one compound, (ii) identifying whether the compound modulates the activity of said functional hENaC; and (iii) based on whether the compound modulates activity of said hENaC identifying the compound as one potentially modulating taste. 
     
     
         123 . The method of  claim 122 , wherein the eukaryotic cell is selected from mammalian cells, yeast,  Xenopus  oocytes and insect cells. 
     
     
         124 . The method of  claim 122 , which includes testing the identified compound in a taste test. 
     
     
         125 . The method of  claim 122 , wherein the isolated eukaryotic cell or cell population is human. 
     
     
         126 . The method of  claim 122 , wherein the isolated eukaryotic cell or cell population is selected from the group consisting of MDCK, BHK, COS, NIH3T3, Swiss3T3 and CHO cells. 
     
     
         127 . The method of  claim 122 , wherein the isolated eukaryotic cell or cell population transiently or stably expresses the alpha (or delta), beta and gamma hENaC subunits. 
     
     
         128 . The method of  claim 122 , wherein the isolated eukaryotic cell or cell population are comprised in a multi-well test plate device and the isolated eukaryotic cell or cell population are loaded with a membrane potential dye. 
     
     
         129 . The method of  claim 122 , wherein the expressed delta subunit is encoded by the sequence in SEQ ID NO:7 and the expressed alpha and beta subunits respectively contain the polypeptides encoded by SEQ ID NO: 1 and 2.