Assays and enhancers of the human delta ENaC sodium channel
This invention relates to electrophysiological assays that measure sodium conductance activity of a delta human epithelial sodium channel (ENaC) in the presence and absence of delta hENaC enhancers. Also, the invention generally relates to assays for identifying compounds that enhance the activity of delta hENaC, especially in an oocyte expression system. These compounds have potential application in modulating (enhancing) salty taste perception.
1. An electrophysiological assay for identifying a compound having potential in vivo application for modulating human salty taste comprising the following:
(i) contacting a cell that expresses a human epithelial sodium channel (“hENaC”) channel containing a delta subunit polypeptide possessing at least 90% sequence identity to the polypeptide identical to the polypeptide of SEQ ID NO:10 with at least one putative hENaC enhancer compound;
(ii) assaying sodium conductance in the presence and absence of said putative hENaC enhancer; and
(iii) identifying the compound as a potential salty taste enhancer based on whether it increases sodium conductance.
2. The method of claim 1 which further includes determining the effect on salty taste perception of said compound or a derivative thereof in a human taste test.
3. The method of claim 1 wherein the delta hENaC further comprises human beta and gamma subunits or variants thereof.
4. The method of claim 1 wherein the delta hENaC is expressed in an amphibian oocyte.
5. The method of claim 1 wherein the delta hENaC is expressed in a mammalian cell.
6. The method of claim 1 wherein said electrophysiological assay uses a sodium sensitive dye.
7. The method of claim 1 wherein said assay is a two electrode voltage clamping assay.
8. The method of claim 7 wherein the cell is a Xenopus oocyte or a mammalian cell.
9. The method of claim 8 wherein said mammalian cell is selected from the group consisting of a Swiss3T3, CHO, BHK, NIH3T3, and COS cell.
10. The method of claim 8 wherein the cell is a Xenopus oocyte.
11. The method of claim 1 wherein said assay is a patch clamp assay.
12. The method of claim 6 wherein said dye is selected from the group consisting of voltage-sensitive blue dye, 4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)hydroxide, inner salt, DiSBACC4(2)(bis-(1,2-dibabituric acid)-triethine oxanol), Cc-2-DMPE, 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt and SBFI-AM (1,3-benzenedicrboxylic acid, 4,4-(1,4,10-trioxa-7,13-diazacylopentadecane-7,13-diyibis(5-methoxy-6,1,2-berizofurandiyl))bis-tetrakis[(acetyloxy)methyl]ester.
13. The method of claim 6 which uses a sodium sensitive dye.
14. The method of claim 1 wherein delta hENaC activity is measured by an ion flux assay.
15. The method of claim 14 which uses atomic absorption spectroscopy to detect ion flux.
16. The method of claim 1 wherein the delta hENaC subunit is expressed under the control of a regulatable promoter.
17. The method of claim 1 which uses a fluorescence plate reader.
18. The method of claim 1 which uses a voltage imaging plate reader.
19. The method of claim 1 wherein the selected compound promotes sodium ion transport into taste bud cells.
20. The method of claim 1 which uses a membrane potential dye selected from the group consisting of voltage-sensitive blue dye, Di-4-ANEPPS (pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalen-yl)ethenyl)-1-(3-sulfopropyl)-hydroxide, inner salt); DiSBACC4(2)(bis-(1.2-dibarbituric acid)-trimethine oxanol); DiSBAC4(3) (bis-(1,3-dibarbituric acid)-trimethine oxanol); CC-2-DPME (1,2-dietradecanoyl-sn-glycerol-3-phosphoethanolamine, triethylammonium salt) and SBFI-AM (1,3-Benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,1,2-benzofurandiyl)]bis-tetrakis[(acetyloxy)methyl]ester.
21. The method of claim 1 wherein said cell stably expresses said delta hENaC.
22. The method of claim 1 wherein said cell transiently expresses said delta hENaC.
23. The method of claim 21 wherein ENaC activity is monitored using a sodium sensitive dye.
24. The method of claim 1 wherein delta hENaC activity is assayed in a frog oocyte that expresses said delta hENaC electrophysiologically by patch clamping or two electrode voltage clamping.
25. The method of claim 24 which uses an automatic imaging instrument.
26. The method of claim 25 wherein said instrument is a fluorescence plate reader.
27. The method of claim 25 wherein said instrument is a voltage imaging plate reader.
28. The method of claim 1 , wherein said cell is selected from the group consisting of BHK, CHO, COS, monkey L cell, African green monkey kidney cell, Ltk-cell and an oocyte.
29. The method of claim 17 , wherein said cell is selected from the group consisting of BHK, CHO, COS, monkey L cell, African green monkey kidney cell, Ltk-cell and an oocyte.
30. The method of claim 1 , wherein said delta subunit polypeptide possesses at least 95% sequence identity to the polypeptide identical to SEQ ID NO: 10.
31. The method of claim 1 , wherein a nucleic acid encoding said delta subunit polypeptide possesses a single nucleotide polymorphism (“SNP”).