Human salty taste receptor and methods of modulating salty taste perception

Abstract

Methods for identifying modulators of the epithelial sodium ion channel and for identifying modulators of salty taste perception are described. Also featured are isolated human salty taste receptors, artificial lipid bilayers comprising an epithelial sodium ion channels, and kits for practicing the claimed methods.

1 . A method for identifying modulators of epithelial sodium ion channels, comprising:
 assembling at least one epithelial sodium ion channel in an artificial lipid membrane, wherein the epithelial sodium ion channel comprises at least three subunits, wherein the subunits are independently an alpha subunit, a beta subunit, a gamma subunit, a delta subunit or an epsilon subunit;   contacting the ion channel with a test compound in the presence of sodium or lithium; and   determining a modulation of the biological activity of the epithelial sodium ion channel in the presence of the test compound relative to the biological activity of the epithelial sodium ion channel in the absence of the test compound.   
     
     
         2 . The method of  claim 1  wherein the epithelial ion channel comprises at least one delta subunit, at least one beta subunit, and at least one gamma subunit. 
     
     
         3 . The method of  claim 1 , wherein the epithelial ion channel comprises at least one alpha subunit, at least one beta subunit, at least one delta, and at least one gamma subunit. 
     
     
         4 . The method of  claim 3 , further comprising at least one epsilon subunit. 
     
     
         5 . The method of  claim 1 , further comprising contacting the epithelial sodium ion channel with an epithelial sodium ion channel antagonist. 
     
     
         6 . The method of  claim 1 , wherein the artificial lipid membrane is a micelle, liposome, or lipid bilayer. 
     
     
         7 . The method of  claim 1 , wherein at least two subunits of an epithelial sodium ion channel are present in the lipid membrane at differing ratios relative to each other. 
     
     
         8 . The method of  claim 1 , wherein the epithelial sodium ion channel comprises at least one biological activity of a functional human salty taste receptor. 
     
     
         9 . A method according to  claim 1 , adapted for high throughput screening. 
     
     
         10 . An artificial lipid membrane comprising at least one type of phospholipid and an epithelial sodium ion channel or specific ratios of epithelial sodium ion channel subunits, wherein the subunits are independently an alpha subunit, a beta subunit, a gamma subunit, a delta subunit, or an epsilon subunit. 
     
     
         11 . The artificial lipid membrane of  claim 10 , wherein the membrane is a micelle, liposome, or lipid bilayer. 
     
     
         12 . The artificial lipid membrane of  claim 10 , comprising at least one epithelial sodium ion channel comprising at least one alpha subunit, at least one beta subunit, at least one delta subunit, and at least one gamma subunit. 
     
     
         13 . The artificial lipid membrane of  claim 12 , further comprising at least one epsilon subunit. 
     
     
         14 . The artificial lipid membrane of  claim 10 , comprising at least one epithelial sodium channel comprising at least one delta subunit, at least one beta subunit, and at least one gamma subunit. 
     
     
         15 . A method for preparing the artificial lipid membrane of  claim 10 , comprising:
 admixing a micelle or liposome comprising at least one phospholipid with an epithelial sodium ion channel or specific ratios of epithelial sodium ion channel subunits, wherein the epithelial sodium ion channel or epithelial sodium ion channel subunits are dissolved in a suitable aqueous buffer comprising at least one surfactant;   incubating said micelle or liposome with the epithelial sodium ion channel or epithelial sodium ion channel subunit for a sufficient amount of time; and   removing the at least one surfactant.   
     
     
         16 . The method of  claim 15 , further comprising reconstituting the proteo-liposome into a planar lipid bilayer. 
     
     
         17 . A method for identifying modulators of salty taste perception, comprising:
 assembling at least one epithelial sodium ion channel in an artificial lipid membrane, wherein the epithelial sodium ion channel comprises at least one beta subunit, at least one gamma subunit, and at least one delta subunit;   contacting the ion channel with a test compound in the presence of sodium or lithium;   determining a modulation of the biological activity of the epithelial sodium ion channel in the presence of the test compound relative to the biological activity of the epithelial sodium ion channel in the absence of the test compound; and   administering the test compound to a subject and determining a modulation of salty taste perception in the subject relative to the level of salty taste perception in the subject in the absence of the test compound.   
     
     
         18 . The method of  claim 17  wherein the delta subunit comprises the amino acid sequence of SEQ ID NO:12. 
     
     
         19 . The method of  claim 17  further comprising the step of screening positive candidate compounds in a cell-based assay for epithelial sodium channel activity. 
     
     
         20 . An isolated human salty taste receptor comprising at least one beta polypeptide subunit, at least one gamma polypeptide subunit and at least one delta polypeptide subunit, wherein said delta polypeptide subunit comprises the amino acid sequence of SEQ ID NO:12. 
     
     
         21 . The isolated human salty taste receptor of  claim 20  wherein said delta polypeptide subunit has the amino acid sequence of SEQ ID NO:9. 
     
     
         22 . A kit for identifying modulators of the human salty taste receptor comprising:
 at least one phospholipid;   substantially purified epithelial sodium ion channel subunits comprising delta subunits, beta subunits, and gamma subunits;   optionally comprising an epithelial sodium ion channel modulator, sodium or lithium; and   instructions for using the kit in a method for identifying modulators of the human salty taste receptor.   
     
     
         23 . The kit of  claim 22  wherein the subunits are admixed in known ratios in a single container. 
     
     
         24 . The kit of  claim 22  wherein at least two subunits are present at differing ratios relative to each other. 
     
     
         25 . The kit of  claim 22 , wherein the modulator is amiloride, phenamil, benzamil, chlorhexidine or a source of guanidinium ion or an organic polyamine. 
     
     
         26 . A compound identified by the method of  claim 1 , wherein the compound activates the epithelial sodium ion channel and induces a salty taste perception when administered to the mouth of a subject, provided that the compound is not sodium or lithium.