Sour/acid taste receptors assays, genes and proteins

This application claims priority to and benefit of U.S. Ser. No. 60/741,352, TASTE RECEPTOR GENES AND PROTEINS by Zuker and Huang, Filed Nov. 30, 2005. This application is a CIP of and claims priority to U.S. Ser. No. 11/483,423 MAMMALIAN SOUR/ACID TASTE AND CSF RECEPTOR GENES, POLYPEPTIDES AND ASSAYS by Zuker and Huang filed Jul. 6, 2006. Each of these prior applications are incorporated herein by reference in their entirety.

This invention was partially supported by grant NIH R01 DC04861. The United States government may have certain rights in the invention.

The invention includes the surprising discovery that PKD1-L3, previously implicated as a potential polycystic kidney disease gene, encodes a taste receptor protein (denoted polycystin 1-like 3, or “PC-1-L3”). PC-1-L3 is a transmembrane ion channel protein that is co-expressed with taste receptor protein polycystin 2-like 1, (“PC-2-L1”), encoded by PKD2-L1, in taste receptor cells.

Taste transduction is one of the most sophisticated forms of chemotransduction in animals (Avenet and Lindemann, 1989; Margolskee, 1993; Lindemann, Physiol. Rev. 76:718-766, 1996; Kinnamon et al., Annu. Rev. Physiol. 54:715-731, 1992; and Gilbertson et al., Curr. Opin. Neurobiol. 10: 519-527, 2000). Gustatory signaling is found throughout the animal kingdom, from simple metazoans to the most complex of vertebrates; its main purpose is to provide a reliable signaling response to non-volatile ligands.

Mammals are believed to have five basic types of taste modalities: salty, sour, sweet, umami (the taste of MSG), and bitter. Each of these is thought to be mediated by distinct signaling pathways leading to receptor cell depolarization, generation of a receptor or action potential and release of neurotransmitter and synaptic activity (Roper (1989) Ann. Rev. Neurosci. 12:329-353).

In general, the identification of new taste receptors is highly desirable. The identification of a taste receptor provides methods and systems for screening for new tastants, such as the identification of new artificial tastants (sweeteners, sour flavors, salt substitutes, etc.) and for the identification of activity modulators that produce a greater receptor response to specified amounts of a tastant. For example, the use of sour or other flavor enhancers may be useful in reducing the amount of sour or other flavoring needed to provoke, enhance, reduce or eliminate a sour receptor taste cell response, which may thus be useful as a flavor modulator. Similarly, acid is used as a preservative; the ability to reduce the flavor impact of such preservatives can be useful in food storage and packaging applications.

Relatively recently, the receptors for bitter, sweet and umami were cloned and shown to be encoded by two families of G-protein coupled receptors (Nelson et al., 2000; Nelson et al., 2001; Zhang et al., 2003; Zhao et al., 2003; Mueller et al., 2005). In contrast, most of the molecular components of the sour pathways are previously unknown. Electrophysiological studies suggested that sour tastants modulate taste cell function by direct entry of H⁺ and Na⁺ ions through specialized membrane channels on the apical surface of the cell. Thus, ion channels selectively expressed in taste receptor cells could be candidates for mediators of sour/acid tastes. Alternatively, ion channels can function as a final critical signaling component in the activation of taste cells (akin to the role of TRPM5 in sweet, umami and bitter cells; Zhang et al., 2003).

Many other families of cell receptors are also known to function in a variety of signal transduction events associated with cell sensation. For example, the polycystins (e.g., polycystin-1, or “PC-1” and polycystin-2, or “PC-2,” encoded by PKD1 and PKD2, respectively) are integral membrane proteins with large extracellular N termini that are thought to possess a number of functions, including mechanosensation in renal and nodal cilia (reviewed in Nauli and Zhou 2004 “Polycystins and Mechanosensation in renal and nodal cilia” Bioessays 26.8 844-856 Wiley Periodicals). The polycystins fall into two basic classes of proteins, the PC-1-like proteins, which are receptor-like molecules and the PC-2-like proteins, which are ion channels (these proteins can also collectively form ion channel pore complexes). Several studies have found overlapping and interdependent roles for these proteins in various systems, particularly in kidney cells. Mutations in various of these genes cause polycystic kidney disease.

The present invention includes the surprising discovery that certain of the polycystin genes encode taste receptor proteins.

The invention includes the surprising discovery that PKD1-L3, encoding polycystin 1-L3 (PC-1-L3), is a sour/acid taste receptor protein. Applicants previously described the surprising discovery that PKD2-L1, which encodes polycystin 2-L1 (PC-2-L1), is a sour/acid taste receptor protein. See, co-pending application U.S. Ser. No. 11/176,958 and U.S. Ser. No. 11/483,423, incorporated herein by reference for all purposes. PC-1-L3 has been found to be a likely partner for polycystin-2L1 in some, though not all tissues expressing PKD2L1, including certain taste receptor cells. PKD1-L3 and PKD2-L1 are co-expressed in taste receptor cells in vivo and their products interact in sour/acid taste signaling in those cells. The surprising discovery that PC-1-L3 and PC-2-L1 are specifically co-expressed in certain taste receptor cells, suggesting that they form taste receptor protein complexes (e.g., including PC-1-L3 and/or PC-2-L1) in those cells (e.g., in the form of receptors and/or ion channels and/or receptor/channel complexes) provides receptor targets for tastant and activity modulator identification and for studies on any taste-related physiological or behavioral effects mediated by either of these polypeptides, separately, and/or in combination.

Previously, PC-1-L3 and PC-2-L1 were though to be primarily involved in kidney function, as defects in various closely related PKD1 and PKD2 genes (encoding PC-1 and PC-2 proteins) are known to cause polycystic kidney disease. The surprising discovery that PC-1-L3 and PC-2-L1 are specifically co-expressed in taste receptor cells, suggesting that they form taste receptor protein complexes (e.g., including PC-1-L3 and/or PC-2-L1) in those cells (e.g., in the form of receptors and/or ion channels and/or receptor/channel complexes) provides receptor targets for tastant and activity modulator identification and for studies on any taste-related behavioral effects mediated by these proteins, separately, and/or in combination. The PC-2-L1 receptor protein has been definitively assigned as the sour/acid receptor (see, e.g., U.S. Ser. No. 11/483,423), as well as having a role in CNS acid receptor sensation. Based on the association between PC-2-L1 and PC-1-L3 in certain taste receptors, PC-1-L3 is assigned as a component of the sour/acid sensation pathway.

Assays of the invention can be cell or tissue based, e.g., screening of natural or transgenic cells, or transfected cells or tissues expressing PKD1-L3, PKD2L1, PC-2-L1 and/or PC-1-L3 for activity in response to test compounds, or can be behaviorally based on whole animal studies. For animal studies, transgenic non-human animals (e.g., mice) can be produced, including PKD1-L3 and/or PKD2-L1 knock-outs and transgenic animals comprising heterologous PKD1-L3 and/or PKD2-L1 genes, e.g., to facilitate behavioral and tastant studies for PKD1-L3 and/or PKD2-L1 gene(s) and encoded proteins of interest. For example, a PKD1-L3 and/or PKD2-L1 knock-out mouse can be made transgenic with the PKD2-L1 and/or PKD1-L3 gene from a human, and the resulting transgenic mouse used to study responses to putative human PC-2-L1 and/or PC-1-L3 binders and activity modulators. In addition, the invention provides for the identification of taste-receptor defects at the molecular level (e.g., thorough detection of PKD1-L3 and/or PKD2-L1 polymorphisms) and for the correction of these defects by gene therapy. Corresponding systems and kits are also included. Further details regarding these and other features of the invention are found herein.

Thus, in a first aspect, the invention provides methods of identifying a compound that binds to and/or modulates an activity of a PC-1-L3 receptor polypeptide, or a PC-2-L1/PC-1-L3 polypeptide complex. Typically, the method includes contacting a biological or biochemical sample comprising the polypeptide or complex with a test compound. Binding of the test compound to the PC-1-L3 receptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex, and/or modulation of the activity of the polypeptide or polypeptide complex by the test compound is detected. This identifies the compound that binds to and/or modulates the activity of the PC-1-L3 receptor polypeptide and/or complex.

In a closely related aspect, the invention provides methods of screening for a compound that binds to and/or modulates an activity of a PC-1-L3 taste receptor polypeptide, or a PC-2-L1/PC-1-L3 polypeptide complex. The method includes contacting a biological or biochemical sample comprising the PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex with a test compound, and determining whether the test compound binds to the PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex, and/or modulates an activity of the polypeptide or polypeptide complex by the test compound, thereby screening the compound for binding to and/or modulation of the PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex.