Voltage-gated, pH-sensitive anion channel and its novel splice variant involved in taste sensation
The invention provides a novel splice-variant of the ClC-4 protein, termed ClC-4A. ClC-4A is expressed in taste bud cells and is involved in sour taste perception. The invention provides ClC-4A polynucleotides and ClC-4A polypeptides, vectors, host cells and ClC-4A specific antibodies as well as designing high potency taste stimuli, determining taste preferences in animals, developing breed-specific foods, and modifying the taste of foods and medications.
1. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence of SEQ ID NO:11, or a sequence encoding a polypeptide wherein said polypeptide consists of the amino acid sequence of SEQ ID NO:8 or an amino acid sequence comprising up to 15 amino acid changes relative to SEQ ID NO: 8.
2. An expression vector comprising the polynucleotide of claim 1 operably linked to a promoter.
3. A host cell comprising the expression vector of claim 2 .
4. The host cell of claim 3 wherein said cell is a mammalian cell.
5. The host cell of claim 3 wherein said cell is from an animal selected from the group consisting of human, mouse, rat, dog, cow, pig, cat, ox, buffalo, llama, sheep, horse, goat, llama, monkey, and ape.
6. The host cell of claim 3 wherein said cell is from an animal selected from the group consisting of a frog, bird, bacterium, yeast, insect, and nematode.
7. A cell culture comprising at least one cell of claim 5 or 6 .
8. An isolated and purified ClC-4A polypeptide consisting of the amino acid sequence of SEQ ID NO:8 or an amino acid sequence comprising up to 15 amino acid changes relative to the amino acid sequence of SEQ ID NO:8 or comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO:11.
9. The polypeptide of claim 8 wherein said polypeptide consists of the amino acid sequence of SEQ ID NO:8.
10. A method for identifying taste modifiers comprising contacting a ClC-4A polypeptide, wherein said ClC-4A polypeptide is encoded by the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO: 11 or consists of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence comprising up to 15 amino acid changes relative to the amino acid sequence of SEQ ID NO: 8, with a potential taste modifier and detecting binding of said potential taste modifier to said ClC-4A polypeptide.
11. The method of claim 10 wherein said ClC4-A polypeptide is expressed on the surface of a host cell.
12. The method of claim 10 wherein said potential taste modifier modulates intracellular chloride concentration.
13. The method of claim 11 wherein said host cells are Xenopus oocytes.
14. The method of claim 11 wherein said host cells are mammalian cells.
15. The method of claim 14 wherein said mammalian cells are selected from the group consisting of human embryonic kidney cells, Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, monkey kidney cells, N1E-115 cells, and PC 12 human hepatocellular carcinoma cells.
16. The method of claim 10 wherein said ClC4-A polypeptide is immobilized on a solid substrate.