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 - 29 . (canceled)
30 . A method for identifying compounds having potential in vivo application for modulating taste comprising:
(i) contacting a cell that expresses a nucleic acid encoding a human epithelial sodium channel (“hENaC”) channel comprising a delta subunit polypeptide possessing at least 90% sequence identity to the polypeptide identical to the polypeptide of SEQ ID NO: 10 with one or more putative hENaC enhancer compounds;
(ii) conducting an electrophysiological assay which detects sodium conductance in the presence and absence of said one or more putative hENaC enhancer compounds;
(iii) based on the results of step (ii) identifying the compound as putatively modulating human taste if the compound increases sodium conductance; and
(iv) assaying the effect(s) of one or more compounds or derivatives thereof which are identified in step (iii) to increase sodium conductance in a taste test.
31 . The method of claim 30 , wherein the taste test is effected in a human subject.
32 . The method of claim 32 , wherein the taste test determines the effect if any of the compound or a derivative thereof on salty taste perception.
33 . The method of claim 30 , wherein the cell further expresses human beta and gamma hENaC subunits or variants thereof.
34 . The method of claim 30 , wherein the delta hENaC is expressed in an Xenopus oocyte or a mammalian cell.
35 . The method of claim 34 , wherein said mammalian cell is selected from the group consisting of a Swiss3T3, CHO, BHK, NIH3T3, and a COS cell.
36 . The method of claim 30 , wherein said electrophysiological assay of (ii) comprises the use of a sodium or voltage sensitive dye.
37 . The method of claim 30 , wherein said electrophysiological assay of (ii) is a patch clamp or two electrode voltage clamp assay.
38 . The method of claim 36 , wherein said sodium or voltage sensitive 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-benzenedicarboxylic acid, 4,4-(1,4,10-trioxa-7,13-diazacylopentadecane-7,13-diyibis(5-methoxy-6,1,2-berizofurandiyl))bis-tetrakis[(acetyloxy)methyl]ester.
39 . The method of claim 30 , wherein said electrophysiological assay of (ii) detects delta hENaC activity by an ion flux assay.
40 . The method of claim 39 , wherein said ion flux is detected by atomic absorption spectroscopy.
41 . The method of claim 30 , wherein the delta hENaC subunit is expressed under the control of a regulatable promoter.
42 . The method of claim 30 , wherein detection of hENaC activity in step (ii) comprises the use of a fluorescence plate reader or by the use of a voltage imaging plate reader.
43 . The method of claim 30 , wherein the effect of the selected putative enhancer compound on sodium ion transport into taste bud cells is further assayed.
44 . The method of claim 42 , wherein detection of hENaC activity comprises the use of 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.
45 . The method of claim 30 , wherein said cell stably or transiently expresses said delta hENaC.
46 . The method of claim 30 , wherein in the electrophysiological assay in step (ii) delta hENaC activity is effected using a Xenopus oocyte that expresses said delta hENaC by patch clamping or two electrode voltage clamping.
47 . The method of claim 30 , wherein said delta subunit polypeptide possesses at least 95% sequence identity to the polypeptide identical to SEQ ID NO: 10.
48 . The method of claim 30 , wherein said nucleic acid encoding said delta subunit polypeptide possesses a single nucleotide polymorphism (“SNP”).