NOVEL CELL LINES EXPRESSING ENaC and METHODS USING THEM

Abstract

Cell lines that stably express ENaC and methods for using those cell lines are disclosed herein. The invention includes cell lines that express various subunit combinations and various proteolyzed isoforms of ENaC and techniques for creating cell lines. The ENaC-expressing cell lines are highly sensitive, physiologically relevant and produce consistent results.

1 - 40 . (canceled) 
     
     
         41 . An engineered cell or cell line stably expressing
 (a) an epithelial sodium channel (ENaC) alpha or delta subunit;   (b) an ENaC beta subunit; and   (c) an ENaC gamma subunit;   wherein the cell or cell line is a mammalian cell or cell line; and   wherein a functional ENaC is stably expressed.   
     
     
         42 . The cell or cell line of  claim 41 , wherein at least one subunit is expressed from an introduced nucleic acid encoding it. 
     
     
         43 . The cell or cell line of  claim 41 , wherein the cell or the cells in the cell line do not express ENaC endogenously. 
     
     
         44 . The cell or cell line of  claim 41 , wherein the cell is or the cells in the cell line are CHO cells. 
     
     
         45 . The cell or cell line of  claim 41 , wherein the cells in the cell line are capable of forming polarized monolayers. 
     
     
         46 . The cell or cell line of  claim 41 , wherein the ENaC:
 (a) is mammalian;   (b) is human;   (c) comprises subunit from different species;   (d) comprises one or more sub units that are chimeric;   (e) is proteolyzed; or   (f) any combination of (a)-(e).   
     
     
         47 . The cell or cell line of  claim 41 , which produces a 71 value of:
 (a) at least 0.3 in a membrane potential dye assay using NaCl as an agonist;   (b) at least 0.5 in a membrane potential dye assay using NaCl as an agonist;   (c) at lease 0.6 in a membrane potential dye assay using NaCl as an agonist; or   (d) at least 0.7 in a membrane potential dye assay using NaCl as an agonist.   
     
     
         48 . The cell or cell line of  claim 41 , which stably expresses ENaC in culture media without antibiotics. 
     
     
         49 . The cell or cell line of  claim 41 , wherein the ENaC alpha subunit is selected from the group consisting of:
 (a) an ENaC alpha polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21;   (b) an ENaC alpha polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOS: 7, 10, 19, 20 and 21; and   (c) an ENaC alpha polypeptide that is an allelic variant of any one of SEQ ID NOS: 7, 10, 19, 20 and 21.   
     
     
         50 . The cell or cell line of  claim 41 , wherein the ENaC alpha subunit is encoded by a nucleic acid selected from the group consisting of:
 (a) a nucleic acid comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 22;   (b) a nucleic acid that encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 7, 10, 19, 20 and 21;   (c) a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOS: 1, 4 and 22; and   (d) a nucleic acid that is an allelic variant of any one of SEQ ID NOS: 1, 4 and 22.   
     
     
         51 . The cell or cell line of  claim 41 , wherein the ENaC comprises an ENaC beta subunit is selected from the group consisting of:
 (a) an ENaC beta subunit polypeptide that comprises the amino acid sequence set forth in SEQ ID NOS: 8 or 11;   (b) an ENaC beta subunit polypeptide that comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NOS: 8 or 11; and   (c) an ENaC beta subunit polypeptide that is an allelic variant of SEQ ID NOS: 8 or 11.   
     
     
         52 . The cell or cell line of  claim 41 , wherein the ENaC beta subunit is encoded by a nucleic acid selected from the group consisting of:
 (a) a nucleic acid comprising the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 23;   (b) a nucleic acid that encodes the polypeptide of SEQ ID NOS: 8 or 11;   (c) a nucleic acid comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOS: 2, 5 and 23; and   (d) a nucleic acid that is an allelic variant of any one of SEQ ID NOS: 2, 5 and 23.   
     
     
         53 . The cell or cell line of  claim 41 , wherein the ENaC gamma subunit is selected from the group consisting of:
 (a) an ENaC gamma subunit polypeptide comprising the amino acid sequence set forth in SEQ ID NOS: 9 or 12;   (b) an ENaC gamma subunit polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NOS: 9 or 12; and   (c) an ENaC gamma subunit polypeptide that is an allelic variant of SEQ ID NOS: 9 or 12.   
     
     
         54 . The cell or cell line of  claim 41 , wherein the ENaC gamma subunit is encoded by a nucleic acid selected from the group consisting of:
 (a) a nucleic acid comprising the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 24;   (b) a nucleic acid that encodes the polypeptide of SEQ ID NOS: 9 or 12;   (c) a nucleic acid with at least 95% sequence identity to any one of SEQ ID NOS: 3, 6 and 24; and   (d) a nucleic acid that is an allelic variant of any one of SEQ ID NOS: 3, 6 and 24.   
     
     
         55 . The cell or cell line of  claim 41 , wherein the ENaC delta subunit is encoded by a nucleic acid selected from the group consisting of:
 (a) a nucleic acid comprising the sequence set forth in SEQ ID NO: 25;   (b) a nucleic acid with at least 95% sequence identity to SEQ ID NO: 25; and   (c) a nucleotide that is an allelic variant of SEQ ID NO: 25.   
     
     
         56 . The cell or cell line of  claim 41 , wherein the cell or cell line has been treated with a protease. 
     
     
         57 . The cell or cell line of  claim 56 , wherein said protease is trypsin. 
     
     
         58 . The cell or cell line of  claim 41 , wherein at least one ENaC subunit is a proteolyzed subunit. 
     
     
         59 . A collection of cell lines comprising two or more cell lines of  claim 41  that have been proteolyzed and express different proteolyzed forms of ENaC. 
     
     
         60 . A method for identifying a modulator of an ENaC function comprising the step of exposing at least one cell or cell line of  claim 41  to a test compound and detecting a change in an ENaC function. 
     
     
         61 . The method of  claim 60 , wherein the detecting step utilizes a membrane potential assay, electrophysiology assay, a binding assay or an Ussing chamber assay. 
     
     
         62 . The method of  claim 60 , wherein the modulator is selected from the group consisting of an ENaC inhibitor, an ENaC agonist or an ENaC potentiator. 
     
     
         63 . The method of  claim 60 , wherein the test compound is a small molecule, a chemical moiety, a polypeptide, or an antibody. 
     
     
         64 . The method of  claim 60 , wherein the test compound is a library of compounds. 
     
     
         65 . The method of  claim 64 , where the library is a small molecule library, a combinatorial library, a peptide library or an antibody library. 
     
     
         66 . The method of  claim 60 , wherein the modulator is selective for an enzymatically modified form of ENaC. 
     
     
         67 . The method of  claim 60 , wherein the cell or cell line is grown in reduced sodium media. 
     
     
         68 . The method of  claim 67 , wherein the sodium concentration in the media is between 9 and 150 mM sodium. 
     
     
         69 . The method of  claim 68 , wherein the sodium concentration in the media is 90 mM or less.