Compositions and methods for the identification of salt alternatives

Abstract

The present invention comprises methods of identifying a compound that modulates taste, such as salty taste, the methods comprising: contacting a test compound with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof; and measuring a response of the taste receptor to the test compound by comparing activity of the taste receptor in the presence and the absence of the test compound; wherein said test compound modulates taste when a change is observed in the activity of the taste receptor in the presence of the test compound.

Description

[0001] COMPOSITIONS AND METHODS FOR THE IDENTIFICATION OF SALT ALTERNATIVES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority of US Provisional application number 63 / 592,421 filed October 23, 2023, the entire contents of which being incorporated herein by reference as though set forth in full.

[0004] GRANT SUPPORT STATEMENT

[0005] This invention was made with government support under DC000882, DC017693, and DCO 18042 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL SUMBITTED IN ELECTRONIC FORM

[0007] The content of the electronic sequence listing (M0N-M118.xml; Size: 59,450 bytes; and Date of Creation: October 23, 2023) is herein incorporated by reference in its entirety.

[0008] BACKGROUND OF THE INVENTION

[0009] The perception of salt taste is a complex process with appetitive (lower- concentration; positive) and aversive (higher-concentrations; negative) components. In mice, the receptors for the appetitive response to very low sodium chloride concentrations (sweet; heterodimer Taslr2 / Taslr3) and low-salt concentrations (salty; ENaC epithelial sodium channels) are known. The fruit fly receptor for the appetitive sensation of salt is also known, but it is different than those in mice (Ir94b; ionotropic glutamate receptor, iGluR).

[0010] In mice and humans, the receptor responsible for the aversive or high-intensity salt perception is still not identified. In mice, the unknown receptor is believed to travel aversive sour and bitter taste pathways. In humans, some people consistently report that high concentrations of sodium chloride are bitter, and this trait is heritable. These data together suggest there is a currently unknown taste receptor for sodium chloride that conveys an aversive or bitter sensation and that genetic variation in this receptor or pathway explains person-to-person differences in the unpleasant sensation evoked by high salt solutions. In the fly, Ir94b may also be part of the aversive high salt system (as well as the appetitive system), depending on the sub-type of sensory cell expressing it.

[0011] Salt substitutes that do not contain sodium play a critical role in addressing the health concerns of many individuals worldwide. High sodium intake is linked to various health issues, including hypertension, heart disease, and stroke. For those who need to reduce sodium consumption due to medical conditions or for preventive health measures, these substitutes provide a way to enjoy flavorful foods without the associated risks of traditional salt. Moreover, they offer an alternative for individuals who have been advised to follow a low-sodium diet. By using salt substitutes, these individuals can still experience the sensory pleasure of seasoned food while maintaining their health. Furthermore, the development and promotion of sodium-free salt substitutes can contribute to a broader public health initiative, encouraging healthier dietary choices and potentially reducing the prevalence of diet-related diseases.

[0012] As the excessive intake of sodium chloride in foods is a human health problem that could be solved in part by the discovery of non-sodium salt substitutes, there is clearly an urgent need for screening assays to identify salt substitutes.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention comprises methods of identifying a compound that modulates taste, the methods comprising: contacting a test compound with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof; and measuring a response of the taste receptor to the test compound by comparing activity of the taste receptor in the presence and the absence of the test compound; wherein said test compound modulates taste when a change is observed in the activity of the taste receptor in the presence of the test compound.

[0015] When the activity of the taste receptor is increased in the presence of the test compound, the test compound produces or enhances the salty taste. When the activity of the taste receptor is decreased in the presence of the test compound, the test compound reduces or inhibits an unpleasant taste. In another aspect of the invention, methods of identifying compounds that modulate salty taste without unpleasant taste, the methods comprising: contacting a known salt substitute with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof; and measuring a response of the taste receptor to the salt substitute by comparing activity of the taste receptor in the presence and the absence of the salt substitute; wherein the salt substitute modulates salty taste without unpleasant taste when no increase in activity of the taste receptor is observed in the presence of the test compound are provided.

[0016] In certain embodiments, the GRIK2 polypeptide, or fragment thereof, is expressed by a cell. In certain embodiments, the GRIK2 polypeptide has the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the response is selected from comparing the observed current of the receptor before and after contacting the cell with the test substance, or comparing observed cation influx into the cell when the test substance is contacted with the cell with cation influx into the cell observed when the test substance is not contacted with the cell. In another embodiment, the response is measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium. In certain embodiments, the cation influx is measured by measuring cell membrane current of the cell in the presence or absence of sodium ions.

[0017] In another embodiments, the GRIK2 polypeptide, or fragment thereof, is comprised within an artificial lipid membrane. In certain embodiments, the GRIK2 polypeptide has the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the response is selected from comparing the observed current of the receptor before and after contacting the membrane with the test substance, or comparing observed cation influx into the membrane when the test substance is contacted with the membrane with cation influx into the membrane observed when the test substance is not contacted with the membrane. In another embodiment, the response is measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium. In certain embodiments, the cation influx is measured by measuring membrane current in the presence or absence of sodium ions.

[0018] Also provided herein are methods of modulating taste in an ingestible composition, the methods comprising administering a flavor-modifying composition comprising the compound identified in the preceding methods. In another aspect, methods for reducing an amount of sodium in an ingestible composition, without reducing a salty taste of a flavored product, comprising administering the compound identified in the preceding methods are provided.

[0019] In certain aspects of the invention, methods of imparting or enhancing a salty taste in an ingestible composition, comprising increasing the expression, levels, or activity of GRIK2 are provided. In other aspects of the invention, methods for reducing an amount of sodium in an ingestible composition, without reducing a salty taste of a flavored product, comprising increasing the expression, levels, or activity of GRIK2

[0020] In certain embodiments, the flavor-modifying composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof. In certain embodiments, the flavor-modifying compound reduces the amount of sodium by at least 20%, or at least 25%, or at least 30%, or by at least 40%, or by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

[0021] In another aspect of the invention, salt receptor agonists and salt receptor inhibitors identified by the methods described herein are provided.

[0022] Still other aspects and advantages of these compositions and methods for making the compositions and using the compositions are described further in the following detailed description of the preferred embodiments thereof.

[0023] BRIEF DESCRIPTION OF THE FIGURES

[0024] FIGs. 1 A-1C. Person-to-person differences in salt bitterness are associated with alleles in the GRIK2 gene. 312 people from four populations worldwide rated the bitterness of 256 mM sodium chloride. These taste data were associated with genetic variants from the Global Diversity Array from Illumina, imputed to -6.4M markers using the Michigan Imputation Server. FIG. 1 A: The red dot (upper center) is within the GRIK2 gene and shows the most statistically significant result from the genome-wide association study; the dashed horizontal line is the threshold for significant association (p<5xl0‘8). FIG. IB: Fine-mapping plot. Each dot represents data from one genetic variant (singlenucleotide polymorphism (SNP). The statistical strength is plotted on the left y-axis, and the recombination fraction is traced with a blue line and plotted relative to the right y-axis. The r2refers to the amount of linkage disequilibrium between genetic variants. Genes and genome locations are located on the x-axis. FIG. 1C: Allelic effects. People with one or two G genotypes report high-salt bitterness. The high-bitterness allele is rare in people of European ancestry but less rare in people of African ancestry. In previous studies, this association was missed because most participants were of European ancestry.

[0025] FIG. 2. Replication of Person-to-person differences in the bitterness of a high- concentration of sodium chloride with alleles in the GRIK2 gene. Adult twins of predominantly European descent ratings 263 mM sodium chloride for bitterness. Those people homozygous for the high-risk genotype (G:G) rated sodium chloride as more bitter on average than did those with the heterozygous genotype (A:G) or homozygous for the low-risk genotype (A: A).

[0026] FIGs. 3A-3J. Two genetic mapping approaches show a sodium chloride (NaCl) taste QTL that contains the Grik2 gene. The average taste preference for NaCl was measured over a range of concentrations (1, 3, 10, and 30 mM) in mice from an informative breeding cross (A / J x B6 F2). Identified in FIG. 3 A is a large QTL region that decomposes into two smaller QTLs, one of which contains the Grik2 gene (QTL2). FIGs. 3B and 3C show allelic effect plots (% preference and threshold) for the three possible genotypes within this F2 population for QTL2 (e.g., A / A means both alleles are originally from the A / J inbred strain). Preference = percentage of salty water mice drank when offered a choice between plain water or NaCl in water. Threshold = lowest NaCl concentration eliciting salty taste. FIGs. 3D and 3E show the data generated using the same experiment for QTL1. Bars that do not share letters (e.g.,aorb) are statistically different, rs numbers refer to the single-nucleotide variants. The markers nearest the QTL peaks are identified herein. FIG. 3F: Five congenic strains (C1-C5) were created that contain a part of mouse chromosome 10 from the A / J strain, with all other parts of the genome from the B6 strain. The names of these congenic strains are on the y-axis, and the number of markers typed to show which regions are from A / J and which are from the B6 strain are on the top x-axis, with the genomic location shown relative to GRCm38 / mml0. The genomic range is shown in the top blue bar, and ranges of the decomposed QTLs are shown in the bottom blue bars annotated by QTL2 (left) and QTL1 (right). For each congenic strain, the chromosome region from the A / J strain is shown in black, and the breakpoints are shown in gray. White parts of the bar are from the B6 strain. FIGs. 3G and 3H show the allelic effects for NaCl preference (measured in the same way as for FIGs. 3B and 3D) and threshold (as for FIGs. 3C and 3E) by comparing each congenic strain with one copy of donor fragment from A (B6 / A) to the control strain without the donor fragment (B6 / B6). FIG. 31 shows the NaCl preference for each concentration tested, with 1, 3, 10 and 30 mM being most informative. At these concentrations, most or all of these mice avoid 150 mM NaCl when offered as a choice with plain water. FIG. 3 J shows the same data from the same congenic mice as in FIG. 31, analyzed with a method to better calculate the NaCl threshold (the horizontal line is the EC50). A / J and B6 mice differ in genotype in at least two locations of chromosome 10, one near or within the Grik2 gene.

[0027] FIGs. 4A-4D. FIG. 4A and 4B: Mice from the F2 population of a 129 x B6 cross do not differ for QTL1 (see FIG. 3) but do for QTL2, the region containing the Grik2 gene. In this cross, the 129 allele suppresses the intake of 75 mM sodium chloride relative to the B6 strain. FIGs. 4C and 4D are similar to FIGs. 4A and 4B but contain data from the measurement of the chorda tympani activity. We recorded its activity while 100 mM sodium chloride was flowed over the mouse tongue. The sodium chloride was mixed with amiloride, a drug used to distinguish among different salt-taste pathways. The nerves of mice with 129 allele are more reactive to the effects of 100 mM sodium chloride + amiloride, which in turn reduces preference compared to the B6 allele.

[0028] FIG. 5. GRIK2 is expressed in human taste receptor cells from fungiform papillae. GRIK2 is expressed in about the same amount and in the same cell type (x-axis) as other taste receptors or taste signaling molecules (e.g., TAS1R3 and PLCB2'). ATP1A3 and GAD1 are type III taste cell markers; TAS1R3 and GNAT3 are type II markers. GRIK3 is also expressed in human taste receptor cells. Avg Exp = average expression, Pert Exp = percentage of cells expressing the mRNA.

[0029] FIG. 6. Open chromatin in the mouse Grik2 gene in taste receptor and other cell types. The y-axis is the signal indicating open chromatin, with larger numbers meaning higher openness. The x-axis is the chromosomal location, with the gene location at the bottom, indicated by a blue line. There are longer and shorter forms of Grik2, and the area of most open chromatin is near the start site of the shorter form (NM001111268).

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The present subject matter may be understood more readily by reference to the following detailed description which forms part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.

[0032] Here we offer evidence that a human salt receptor, responsible at least in part for its negative quality, is a protein derived from the Glutamate receptor ionotropic, kainate 2 (GRIK2) gene, an ionotropic glutamate receptor. Also provided herein is the use of GRIK2 to screen cell-based chemical libraries to find ligands that would mimic the response to high concentrations of sodium chloride.

[0033] This invention will be used, inter alia, in high-throughput cell screening assays using chemical libraries to identify salt substitutes, including known agonists of GRIK2.

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition to definitions included in this sub-section, further definitions of terms are interspersed throughout the text.

[0035] In this invention, “a” or “an” means “at least one” or “one or more,” etc., unless clearly indicated otherwise by context. The term “or” means “and / or” unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term “or” refers back to more than one preceding claim in the alternative only.

[0036] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from the wild type or a comprises non naturally occurring components.

[0037] A “salty compound” refers to a compound that elicits a detectable salty flavor in a subject, e.g., a compound that activates a salty taste receptor in vitro in the presence of the taste receptor. In certain embodiments, the salty taste receptor is GRIK2. A “salty flavor enhancer” refers to a compound that agonizes the activation of one or more salty taste receptors in vitro.

[0038] The terms “agent” and “test compound” refers to any purified molecule, substantially purified molecule, molecules that are one or more components of a mixture of compounds, or a mixture of a compound with any other material that can be analyzed using the methods of the present invention. Test compounds can be organic or inorganic chemicals, or biomolecules, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Biomolecules include proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Test compounds can be of natural or synthetic origin, and can be isolated or purified from their naturally occurring sources, or can be synthesized de novo. Test compounds can be defined in terms of structure or composition, or can be undefined. The compound can be an isolated product of unknown structure, a mixture of several known products, or an undefined composition comprising one or more compounds. Examples of undefined compositions include cell and tissue extracts, growth medium in which prokaryotic, eukaryotic, and archaebacterial cells have been cultured, fermentation broths, protein expression libraries, and the like.

[0039] In certain embodiments, the test compound is a salt substitute. The phrase “salt substitute” refers to a compound that contains no sodium or small amounts of sodium and replaces salt in ingestible compositions.

[0040] It is also contemplated that the term “compound” or “compounds” refers to the compounds discussed herein and includes precursors and derivatives of the compounds, and pharmaceutically acceptable salts of the compounds, precursors, and derivatives.

[0041] In certain embodiments, the test compound is a known GRIK family agonist. The term GRIK family agonist as used herein is meant a compound, molecule (synthetic or natural) or a composition comprising multiple compounds or molecules that modifies the activation, nucleic acid expression, protein expression, signaling or activity of at least one GRIK receptor or an intermediate in the signaling pathway thereof. In certain embodiments, the GRIK agonists or antagonist provides, increases, or up-regulates the activation, expression, signaling or activity of at least one GRIK receptor. In certain embodiments, the GRIK agonist is a known GRIK agonist such as such as CNQX, NBQX, or derivatives thereof.

[0042] SCREENING METHODS

[0043] The present disclosure provides certain compounds and polypeptide sequences that are useful in simulating in vitro the response that certain human salt taste receptors would exhibit towards such compounds when ingested orally. In certain embodiments, these polypeptide sequences simulate at least one salt taste receptor.

[0044] As used herein, the terms “modulate” means any change, increase, or decrease in the amount, quality, or effect of a particular activity or protein. “Modulators” refer to any inhibitory or activating molecules identified using in vitro and in vivo assays for, e.g., agonists, antagonists, and their homologs, including fragments, variants, and mimetics, as defined herein, that exert substantially the same biological activity as the molecule. “Inhibitors” or “antagonists” are modulating compounds that reduce, decrease, block, prevent, delay activation, inactivate, desensitize, or downregulate the biological activity or expression of a molecule or pathway of interest. “Inducers,” “activators,” or “agonists” are modulating compounds that increase, induce, stimulate, open, activate, facilitate, enhance activation, sensitize, or upregulate a molecule or pathway of interest. In some preferred aspects of the invention, the level of inhibition or upregulation of the expression or biological activity of a molecule or pathway of interest refers to a decrease (inhibition or downregulation) or increase (upregulation) of greater than from about 50% to about 99%, and more specifically, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,

[0045] 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%,

[0046] 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

[0047] 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. The inhibition or upregulation may be direct, i.e., operate on the molecule or pathway of interest itself, or indirect, i.e., operate on a molecule or pathway that affects the molecule or pathway of interest.

[0048] In the present disclosure, “identification”, “separation”, “sorting”, “isolation”, “purification”, or “screening” has a meaning commonly used in the art, and typically refers to sorting of a target such as an organism, a cell, a substance, data, or the like that has certain targeted characteristics from a population containing a large number of cells by a specific manipulation / evaluation method.

[0049] In certain embodiments, the salt taste receptor is a beneficial salt receptor. A beneficial salt receptor is a receptor that does not trigger an unpleasant sensation in a subject when exposed to a salt. In certain embodiments, the unpleasant sensation is a bitter or sour taste. In certain embodiments, the unpleasant sensation is a bitter taste. Various beneficial salt receptors are known by those skilled in the art. Exemplary beneficial salt receptors include, without limitation, ENaCs and TRPV1.

[0050] In certain embodiments, the salt taste receptor is an aversive salt receptor. An aversive salt receptor is a receptor that triggers an unpleasant sensation in a subject when exposed to high concentrations of salt. In certain embodiments, the unpleasant sensation is a bitter or sour taste. In certain embodiments, the unpleasant sensation is a bitter taste. In certain embodiments, the aversive salt receptors do not trigger an unpleasant sensation in a subject when exposed to lower concentrations of salt. In certain embodiments, the receptor is a member of the GRIK family. In certain embodiments, the salt taste receptor is a polypeptide sequence of a GRIK family protein, or a functional fragment thereof. In certain embodiments, the salt receptor is selected from GIRK2, GIRK3, GIRK5, GRID1, and GRID2.

[0051] In certain embodiments, a salt taste receptor may be an aversive salt receptor when exposed to high concentrations of salt and a beneficial salt receptor when exposed to low concentrations of salt.

[0052] In certain embodiments, the receptor is GRIK2. The term “GRIK2”, “glutamate ionotropic receptor kainite type subunit 2”, refers to a member of the kainite family of glutamate receptors, which are composed of four subunits and function as ligand-activated ion channels. The subunit encoded by this gene is subject to RNA editing at multiple sites within the first and second transmembrane domains, which is thought to alter the structure and function of the receptor complex. Alternatively spliced transcript variants encoding different isoforms have also been described for this gene. The amino acid sequence and nucleotide sequence for GRIK2 are publicly available, see, e.g., GENBANK Accession No. NP_068775.1 (SEQ ID NO: 1) and NM_021956.5 (SEQ ID NO: 2).

[0053] In certain embodiments, the receptor is GRIK3. The term “GRIK3”, “glutamate ionotropic receptor kainite type subunit 3”, refers to a member of the kainite family of glutamate receptors, which are composed of four subunits and function as ligand-activated ion channels. A Ser310Ala polymorphism has been associated with schizophrenia, and there are conflicting reports of its association with the pathogenesis of delirium tremens in alcoholics. Alternatively spliced transcript variants encoding different isoforms have also been described for this gene. The amino acid sequence and nucleotide sequence for GRIK3 are publicly available, see, e.g., GENBANK Accession No. NP_000822.2 (SEQ ID NO: 3) and NM_000831.4 (SEQ ID NO: 4). In certain embodiments, the receptor is GRIK5. The term “GRIK5”, “glutamate ionotropic receptor kainite type subunit 5”, refers to a member of the kainite family of glutamate receptors, which are composed of four subunits and function as ligand-activated ion channels. The protein encoded by this gene forms functional heteromeric kainate- preferring ionic channels with the subunits encoded by related gene family members. Alternative splicing results in multiple transcript variants. The amino acid sequence and nucleotide sequence for GRIK2 are publicly available, see, e.g., GENBANK Accession No. NP_001287959.1 and NP_002079.3 (SEQ ID NOs: 5 and 6) and NM_001301030.2 and NM_002088.5 (SEQ ID NO: 7 and 8).

[0054] In certain embodiments, the receptor is GRID1. The term “GRIDl”, “glutamate ionotropic receptor delta type subunit 1”, refers to a subunit of glutamate receptor channels. These channels mediate most of the fast excitatory synaptic transmission in the central nervous system and play key roles in synaptic plasticity. The amino acid sequence and nucleotide sequence for GRIDl are publicly available, see, e.g., GENBANK Accession No. NP_060021.1 (SEQ ID NO: 9) and NM_017551.3 (SEQ ID NO: 10).

[0055] In certain embodiments, the receptor is GRID2. The term “GRID2”, “glutamate ionotropic receptor delta type subunit 2”, refers to a member of the family of ionotropic glutamate receptors which are the predominant excitatory neurotransmitter receptors in the mammalian brain This protein also plays a role in synapse organization between parallel fibers and Purkinje cells. Alternate splicing results in multiple transcript variants encoding distinct isoforms. The amino acid sequence and nucleotide sequence for GRID2 are publicly available, see, e.g., GENBANK Accession No. NP_001273767.1 and NP_001501.2 (SEQ ID NO: 11 and 12) and NM_001286838.1 and NM_001510.4 (SEQ ID NO: 13 and 14).

[0056] For purposes of the invention, “nucleic acid”, “nucleotide sequence” or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5’ to 3’ direction. With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Alternatively, this term may refer to a DNA that has been sufficiently separated from (e.g., substantially free of) other cellular components with which it would naturally be associated. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification. When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0057] The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0058] As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0059] In certain aspects, the amino acid or nucleic acid sequence encoding human GRIK2 is provided. In certain embodiments, the sequences herein relate to the known full length amino acid sequences as well as at least 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 265 contiguous amino acids selected from such amino acid sequences, or biologically active variants thereof.

[0060] A “derivative” of a polypeptide, polynucleotide or fragments thereof means a sequence modified by varying the sequence of the construct, e.g., by manipulation of the nucleic acid encoding the protein or by altering the protein itself. “Derivatives” of a gene or nucleotide sequence refers to any isolated nucleic acid molecule that contains significant sequence similarity to the gene or nucleotide sequence or a part thereof. In addition, “derivatives” include such isolated nucleic acids containing modified nucleotides or mimetics of naturally-occurring nucleotides.

[0061] The term “functional” as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.

[0062] According to the present invention, an isolated or biologically pure molecule or cell is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.

[0063] Variants which are biologically active, refer to those, which maintains the native function of the protein. In the case of the salt receptors described herein, biologically active variants include sequences which are activated by the presence of high levels of sodium. Preferably, naturally or non-naturally occurring polypeptide variants have amino acid sequences which are at least about 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98, 99, or 100% identical to the full-length amino acid sequence or a fragment thereof. Percent identity between a putative polypeptide variant and a full-length amino acid sequence is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes). Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and / or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

[0064] The term “identity” as used herein and as known in the art, is the relationship between two or more oligo sequences, and is determined by comparing the sequences. Identity also means the degree of sequence relatedness between oligo sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While a number of methods to measure identity between two polynucleotide sequences are available, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskovm, M. and Devereux, J., eds., M. Stockton Press, New York (1991)). Methods commonly employed to determine identity between oligo sequences include, for example, those disclosed in Carillo, H., and Lipman, D., Siam J. Applied Math. (1988) 48: 1073. In certain embodiments, the present invention may have 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the SEQ ID NOs disclosed herein.

[0065] Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active polypeptide can readily be determined by assaying for native activity, as described for example, in the specific Examples, below.

[0066] Reference to genetic sequences herein refers to single- or double-stranded nucleic acid sequences and comprises a coding sequence or the complement of a coding sequence for polypeptide of interest. Degenerate nucleic acid sequences encoding polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 60, preferably about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98, 99, or 100% identical to the DNA may be used in accordance with the teachings herein polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of nucleic acid sequences which encode biologically active polypeptides also are useful polynucleotides.

[0067] Variants and homologs of the nucleic acid sequences described above also are useful nucleic acid sequences. Typically, homologous polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions: 2*SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2*SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2*SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

[0068] In certain aspects, the disclosure provides methods of identifying compounds that modulate taste. In certain aspects, the methods comprise introducing a test compound to a taste receptor, and measuring response of the taste receptor to the test compound by comparing activity of the taste receptor in the presence and the absence of the test compound. In certain embodiments, the taste receptor is a salt taste receptor. In certain embodiments, the salt taste receptor is selected from GRIK2, GRIK3, GRIK5, GRID1, and GRID2. In certain embodiments the taste receptor is GRIK2. The test compound produces or enhances the salty taste when the activity of the taste receptor is increased in the presence of the test compound. The test compound reduces or inhibits an unpleasant taste when the activity of the taste receptor is decreased in the presence of the test compound.

[0069] Also provided herein are methods of modulating salty taste without an unpleasant taste. In certain aspects, the methods comprise introducing known salt substitute with a taste receptor, and measuring a response of the taste receptor to the test compound by comparing activity of the taste receptor in the presence and the absence of the test compound. The salt substitute modulates salty taste without unpleasant taste when no increase in activity of the taste receptor is observed in the presence of the test compound.

[0070] In certain embodiments, the screening method involves the salt taste receptor present in a lipid bilayer in order to identify compounds that modulate the biological activity of the salt taste receptor, such as GRIK2. In particular it is an object of the present invention to use the precise molar ratios of the salty taste receptor subunits to reconstitute the salty taste receptor in the lipid bilayer in order to identify compounds that modulate the biological activity of the salty taste receptor and to identify compounds that modulate salty taste perception in human beings. Without intending to be limited to any particular theory or mechanism of action, it is believed that a passive influx of sodium ions through epithelial sodium channels in certain taste receptor cells causes a change in intracellular ion balance leading to a depolarization, ultimately resulting in neurotransmitter release, which in turn produces a perception of salty taste.

[0071] The assays further identify compounds that bind and / or modulate the human salty taste receptor. The methods comprise assembling at least one epithelial sodium ion channel in a lipid membrane, wherein the epithelial sodium ion channel comprises an alpha, beta, gamma, or delta subunit, contacting the at least one ion channel with a test compound in the presence of sodium or lithium, and determining a modulation of the biological activity of the at least one epithelial sodium ion channel in the presence of the test compound relative to the biological activity of the at least one subunit in the absence of the test compound.

[0072] Where the biological activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound is an agonist. If the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound is an antagonist.

[0073] In a related aspect, the invention also provides for an in vitro screening method for identifying salty compounds or an GRIK family agonist. The method comprising contacting a cell comprising a salt taste receptor, with a test compound and measuring the response of the cell to the test compound. In certain embodiments, the response is cation influx into the cell. In certain embodiments, the cation influx is measured by measuring cell membrane current of the cell in the presence of sodium ions or absence of sodium ions. The response may also be measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium.

[0074] The effect of the test compound on the biological activity of the receptor can be determined by any means suitable in the art. The test compound can be assessed at multiple concentrations. In some aspects, the test compound is assessed for its ability to modulate at least one biological activity of the receptor.

[0075] The biological activity of the salt taste receptor can be determined by measuring the current of receptor assembled in the lipid membrane. Voltage clamping is one preferable technique to measure current. Voltage clamp techniques are well known in the art. (Nagel, G et al. (2005) J. Physiol. 564(Pt 3):671-82; Staruschenko, A et al. (2004) J. Biol. Chem. 279:27729-34; Tong, Q et al. (2004) J. Biol. Chem. 279:22654-63; Sheng, S et al. (2000) J. Biol. Chem. 275:8572-81). The following parameters can be measured using a voltage clamp: single channel conductance, channel open time, voltage dependence, blockade induced by application of a particular compound, and activation induced by application of a particular compound. Other suitable techniques for measuring the biological activity of salt taste receptors include flux assays, patch clamping, voltagesensitive dyes, and ion-sensitive dyes. In certain embodiments, receptor activity is measured by membrane electrophysiology or by assessing the change in fluorescence of a membrane potential dye in response to sodium or lithium, or analogs thereof (e.g., isotopes). All such assays are well known in the art. (Gill, S et al. (2003) Assay Drug Dev. Technol. 1 :709-17, flux assay; Caldwell, R A et al. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 288:L813-9, patch clamp). A variety of voltage sensitive dyes are commercially available, including without limitation styryl dyes, oxonol dyes, and merocyanine- rhodanine dyes. Selection of the appropriate voltage sensitive dye is within the relevant skill in the art. Similarly, a variety of ion sensitive dyes are commercially available, including single excitation dyes, dual excitation radiometric dyes, and dual emission radiometric dyes.

[0076] The salty taste receptor is responsive to sodium and lithium ions. However, the human salty taste receptor is not sensitive to amiloride. Thus, amiloride should not inhibit or stimulate the salty taste receptor. Conversely, chlorhexidine acts as an inhibitor of the salty taste response in humans, and may be used in assays to identify salty taste modifiers. One may assess specificity of stimulation of the salty taste receptor with test compounds by showing that the effect is inhibited by chlorhexidine. Moreover, test compounds that can overcome the effect of chlorhexidine (and stimulate the salty taste receptors in the membrane systems of the invention) are strong salty taste enhancers. Basic compounds containing guanidinium ions as well as certain amines act as salty taste enhancers. These include guanidine, arginine, and homoarginine. Both L- and D-arginine are equally effective. While not wishing to be bound by any particular theory of operation, this lack of enantiomeric specificity suggests that the primary enhancing effect derives from a compact, basic moiety, in this case the guanidinium ion. Thus, salty taste receptors in the membrane system of the invention may be stimulated by contacting them with a source of guanidinium ions. It may be assumed that these enhancing compounds interact directly with the human salty taste ion channel, since most sodium channel blockers and enhancers contain guanidinium groups that interact with acidic moieties inside the channel pore lumen. Thus, the molecular mechanisms of human salty taste share selected functional features in common with known sodium channels but also have unique pharmacological attributes.

[0077] Amiloride and amiloride derivatives (e.g., phenamil, benzamil and the like) may be useful in assessing the salt taste receptor. Amiloride and derivatives may also be used in assays to inhibit the background if the purity of the salt taste receptor is low such that host cell are contaminating the preparation. Thus, in some aspects of the invention, the methods further comprise contacting the salt taste receptor with a sodium ion channel antagonist. Such antagonists are well-known to those of skill in the art. Preferably, the antagonist is amiloride, chlorhexidine, or a homolog, analog, or derivative thereof.

[0078] In another aspect of the invention, methods of modulating taste of an ingestible composition are provided. As used herein, “ingestible composition” means a composition that is intended to be consumed or ingested through the mouth. Examples of forms of ingestible compositions include, but are not limited to, foods, tablets, pills, capsules, powders, granules, solutions or suspensions, and drops. Ingestible compositions do not include compositions intended to be topically administered to the skin, eyes, nostrils, tympanic cavity, vaginal cavity, or rectal cavity.

[0079] The methods of modulating taste comprises administering the compound identified above in an ingestible composition. This ingestible composition may further comprise a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof. Also provided herein are methods of reducing the amount of sodium present in an ingestible composition. These methods comprise administering the compound identified above in an ingestible composition. In certain embodiments, the compound reduces sodium by at least 20%, or at least 25%, or at least 30%, or by at least 40%, or by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. This ingestible composition may further comprise a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof.

[0080] Additionally, methods of imparting salty taste in an ingestible composition or reducing the amount of sodium in an ingestible composition without reducing a salty taste are provided. These methods comprise increasing the expression, levels, or activity of GRIK2.

[0081] HIGH-THROUGHPUT SCREENING ASSAYS

[0082] The invention also includes within its scope high-throughput screening assays to identify compounds that modulate the biological activity of the salty taste receptor. High- throughput screening assays permit screening of large numbers of test compounds in an efficient manner. For example, but not by way of limitation, lipid membranes comprising an assembled salt taste receptor can be dispersed throughout a multi-well plate such as a 96-well microtiter plate. Each well of the microtiter plate can be used to run a separate assay against a candidate modulator. A microtiter plate permits screening of multiple concentrations of a test compound, multiple test compounds, alone or in combination with other test compounds, and multiple salt taste receptors. In other aspects of the invention, planar lipid bilayers containing the receptor of interest is contacted with a test compound and a measurement is taken. The solution on one or both sides of the planar bilayer is changed and the bilayer is contacted with a second test compound. This may be continuously used as a high-throughput assay. The assays may take place in the presence of additional agonists or antagonists. Data obtained for the test compounds are compared with measurements taken in the presence of known agonists or antagonists and / or to control samples (such as a non-stimulatory / non-inhibitory medium).

[0083] Serial assays may be performed to narrow down the pool of test compounds that act as salty taste modifiers. For example, the in vitro assays of the invention may be combined with cell-based assays as a secondary or confirming screening step. Such assays have been described, for example, in published U.S. Patent Application 2005 / 0059094 to Servant et al.

[0084] LIPID MEMBRANE AND HOST CELLS

[0085] In certain aspects, the invention includes the lipid membranes produced with the salty taste receptors discussed herein. These salty taste receptors include members of the GRIK family of taste receptors. In preferred aspects, the taste receptor is GRIK2.

[0086] The salty taste receptors that are to be assembled into the lipid membrane can be obtained from any source suitable in the art. For example, a salty taste receptor can be freshly isolated from any cell that expresses the receptor, including cell lines and stable cell lines. For example, but not by way of limitation, salty taste receptors are expressed in neural tissue, the pancreas, testes, ovaries, tongue, colon, kidneys, lungs, sweat glands, and the like. In some aspects, the salt taste receptor is isolated from the papillae of the tongue. In other aspects, a salt taste receptor can be recombinantly expressed, purified and used to reconstitute a lipid membrane to form the functional receptor, or may be expressed recombinantly in a cell line for use herein.

[0087] In certain aspects, the receptor contains subunits and each subunit of the receptor is separately expressed in a recombinant expression system such as, but not limited to host cells. A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, HEK cells, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293 -6E and DG44 cells, respectively. In certain embodiments, the cell is a host cell is transfected with the vector described above. As discussed at length below, methods for delivering vectors / constructs or other nucleic acids (such as in vitro transcribed RNA) into host cells can include electroporation, transformation, transfection or transduction.

[0088] The expressed protein is purified by standard biochemical means as is well-known in the art. Alternatively, expressed protein may be immunopurified using immobilized antibodies that specifically bind the salt taste receptor. Methods for purifying proteins by immunoaffinity (using antibodies that specifically bind the receptor of interest). In other aspects, the receptors are expressed as a fusion protein with a polypeptide that allows for rapid purification and subsequent cleavage from the expressed protein. Such purification systems include, but are not limited to the pGEX system (glutathione-S-transferase fusion proteins) and multi-histidine fusion proteins (for nickel binding affinity purification). These and other types of purification are described in numerous references and are well- known to those of skill in the art.

[0089] In certain aspects, the subunits of the receptors are substantially purified prior to incorporation into the membrane. As used herein, “substantially purified” refers to subunits that are at least 80% free of contaminating material (e.g., proteins, polysaccharides, and lipids) derived from the cells from which they are obtained. Preferably, the subunits are at least about 85% free of contaminating material. More preferably, the subunits are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, free of contaminating material.

[0090] The concentration of affinity-purified protein can be determined by measuring the total nitrogen content of the protein eluate and comparing the nitrogen content with the total protein content of the eluate. Nitrogen content can be determined by any means suitable in the art, such as the well-known Kjeldahl Nitrogen Method. Protein concentration can be determined, for example, by spectrophotometry whereby a protein sample is analyzed for its absorption of light at 280 nm to derive an absorption coefficient. Any means known in the art for assessing concentration and / or purity of protein may be used.

[0091] The invention thus provides artificial membrane systems containing substantially purified salt taste receptor proteins. Specifically, the invention provides artificial membrane systems containing substantially purified human salty taste receptor. These membrane systems permit analysis of the receptor, including, but not limited to the salty taste receptor apart from contaminating proteins. The invention permits the assembly of receptors in which the subunits are added at known ratios to permit the assembly of precise ratios of selected subunits. The lipid membrane can comprise any combination of lipids. Non-limiting examples of suitable lipids include phosphatidylcholine, phoshpatidylethanolamine, phostphatidylserine, phosphatidylglyine, phosphatidylinositol, sphingomyelin, cholesterol, cardiolipin, or a homolog, analog, or derivative thereof.

[0092] Phospholipids are preferred, and can be obtained from any source suitable in the art. For example, the phospholipids can be extracted from a cell, or can be synthetic phospholipids, which are commercially available.

[0093] The lipid membrane can be in any conformation or phase, including without limitation, liposomes, a lipid bilayer, or the hexagonal phase. Liposomes and lipid bilayers are particularly preferred.

[0094] In one aspect, an artificial lipid membrane is prepared by admixing a liposome that comprises at least one phospholipid with a salt taste receptor that has dissolved in a suitable aqueous buffer. The aqueous buffer comprises at least one detergent. Suitable detergents are well known in the art, and include without limitation, Tween, Triton, CHAPS, sodium cholate, and octyl-glucoside. After mixing the phospholipids and receptor, the mixture is allowed to incubate for several minutes, preferably at least about 20 minutes, to permit assembly of the receptor into a lipid membrane. Following the incubation, the detergent is removed according to any means suitable in the art, such as those described and exemplified herein. Other methods known in the art of preparing lipids and liposomes containing proteins may be used to produce the lipids and liposomes containing the receptor.

[0095] In some aspects, the salt taste receptor is assembled into a liposome. The liposome can be converted into a planar lipid bilayer by use of techniques that are well known and routine in the art, including those that are described and exemplified herein. In some aspects, the liposomes contain a substance other than found in the surrounding milieu. For example, but not by way of limitation, the liposomes may contain a fluorescent voltagesensitive or membrane potential dye that is responsive to sodium or lithium, to indicate a change in sodium content as a marker of sodium flow upon stimulation with a test compound.

[0096] VECTORS

[0097] Also described herein are vectors for directing expression the salt taste receptors described above. In certain embodiment, the vectors comprise a polynucleotide encoding GRIK2.

[0098] The term “delivery” as used herein refers to the introduction of foreign molecule (i.e., salt taste receptor nucleotide containing nanoparticle) into cells. The term “administration” as used herein means the introduction of a foreign molecule into a cell. The term is intended to be synonymous with the term “delivery”.

[0099] A “vector” as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate host cell for replication or expression of said nucleic acid sequence. Common vectors include non-viral vectors and viral vectors. As used herein, a non-viral system might be selected from nanoparticles, electroporation systems and novel biomaterials, naked DNA, phage, transposon, plasmids, cosmids (Phillip McClean, www.ndsu.edu / pubweb / ~mcclean / - plsc731 / cloning / cloning4.htm) and artificial chromosomes (Gong, Shiaoching, et al. “A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.” Nature 425.6961 (2003): 917-925).

[0100] As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and / or expression of the nucleic acid sequence and its gene product.

[0101] As used herein, “operably linked” sequences include both regulatory sequences that are contiguous with the nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region comprising a polyadenylation site, among other elements. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell. Typically, such an expression cassette for generating a viral vector contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

[0102] In addition to the coding sequence, in certain embodiments the vector includes regulatory sequences which direct expression in a host cell. In certain embodiments, the regulatory elements include a promoter. In addition to a promoter, the vector may contain one or more appropriate “regulatory elements” or “regulatory sequences”, which comprise but are not limited to an enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, e.g., the alpha fetoprotein enhancer, the TTR minimal promoter / enhancer, LSP (TH-binding globulin promoter / alphal-microglobulin / bikunin enhancer), amongst others. These control sequences or the regulatory sequences are operably linked to the nuclease coding sequence or transgene coding sequence.

[0103] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory compounds to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIB TECH 11 :211-217 (1993); Mitani & Caskey, TIB TECH 11 : 162-166 (1993); Dillon, TIBTECH 11 : 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1 : 13-26 (1994).

[0104] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, poly cation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptorrecognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0105] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817- 4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0106] EXAMPLES

[0107] The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

[0108] EXAMPLE 1 : IDENTIFICATION OF GRIK2 AS A SALT RECEPTOR

[0109] In flies, Ir94a and Ir94h have been identified as a potential part of the aversive high salt system. Accordingly, a search was conducted for human orthologues of Ir94a and Ir94h using an online tool called the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT). Several genes were identified as mouse and human orthologs for Ir94a and Ir94h (Table 1). From these orthologs, GIRK2 was analyzed as a plausible high-salt taste receptor due to its similarity to Ir94h. Additionally, GRIK2 responds to sodium and chloride; the locations of the cation and anion binding domains are known. Although studies showed that this class of receptor is expressed and functional in the expected taste cell types, prior to the experiments described herein, evidence of GRIK2 as a human averisive salt receptor or receptor component was unknown.

[0110] EXAMPLE 2: GENOME-WIDE ASSOCIATION STUDIES OF GRIK2

[0111] To analyze the role of GRIK2 as an aversive salt receptor, a genome-wide association study (GWAS) was performed by having individuals rate the bitterness of high-salt solutions and measuring the person-to-person differences and their statistical association with the alleles of the GRIK2 gene.

[0112] More specifically, 312 people from four populations worldwide were given a 256 mM sodium chloride. These people then rated the bitterness of the solution. These taste data were associated with genetic variants from the Global Diversity Array from Illumina, imputed to -6.4M markers using the Michigan Imputation Server. These data demonstrate that ratings of the bitterness of a high-salt solution are statistically associated with the GRIK2 gene, thereby supporting the hypothesis that the G allele of GRIK2 changes the aversive qualities of a high-salt solution. (FIG. 1)

[0113] Next, additional data from human twins (N=1212 subjects) was collected. (FIG. 2). The G form of the allele for the top hit is very rare in people of European descent and more common in people of African ancestry. Therefore, this data set included many people who are homozygous for the high-aversive allele (G) of GRIK2. Those homozygous for the high-aversive allele at rs75643080 (G:G) indicated that the sodium chloride was more bitter than the other genotype groups.

[0114] EXAMPLE 3: ANALYSIS OF GRIK2 IN MURINE MODEL

[0115] Next, studies were conducted in mice using strains that differ in their behavior toward sodium chloride (quantitative trait loci (QTL), congenic mapping studies). The approach to the murine studies was similar to the GWAS study in Example 2. Two large breeding studies were performed to understand mouse-to-mouse variation in the intake of sodium chloride solutions (expressed as a preference compared to plain water) and the lowest concentration that can be detected (threshold).

[0116] Generation ofF2 mice

[0117] For each study, we started with two inbred strains of mice that differ in their behavioral taste and taste nerve responses to sodium chloride. We interbred these parental strains (A / J x B6 and B6 x 129) to create two large second filial generations (F2: A.B6F2 and B6.I29F2). A large region on mouse chromosome 10 was associated with salty taste. The trace of the linkage in the A.B6F2 mice indicates that there are two QTL regions (FIG. 3 A). QTL2 contains the Grik2 gene.

[0118] Congenics

[0119] In addition to the F2 studies, in the A / J x B6 pairing (but not the 129 x B6 pairing), we bred five congenic mouse strains using methods that introgress large genomic regions containing the A / J version of Grik2 onto the background B6 strain. In Figure 3, the results of the A / J x B6 F2 QTL mapping and the congenic analysis from the region of the mouse genome, on chromosome 10, that contains the Grik2 gene are shown.

[0120] The average taste preferences for NaCl over a range of concentrations (1, 3, 10, and 30 mM) was measured. The allele effect plots show that QTL2 (FIG. 3B, 3C), which is very near or within Grik2, is not statistically significant, but when QTL2 is captured in congenic strains, those mice with the A / J allele of Grik2 drink more significantly more sodium chloride and have a higher threshold for detecting it.

[0121] These data indicate that mice with a B6 allele near or within Grik2 can taste the aversive aspects of sodium chloride at lower concentrations (as measured by threshold) and avoid it more (as measured by preference) than do mice with the A / J allele. To determine whether this B6 allele is also responsible for similar behavior in a different mouse cross, we analyzed a new set of data from a B6 x 129 strain F2 cross (Figure 4).

[0122] Comparing Figures 3 and 4 shows that the A / J strain of mice has a genomic region containing a Grik2 allele that increases the intake of sodium at 1, 3, 10, and 30 mM relative to the B6 strain. Additionally, these data indicate that the 129 strain has a Grik2 allele that decreases the intake of 75 mM sodium chloride and increases the responsiveness of the chorda tympani nerve to the amiloride-insensitive aspects of 100 mM sodium chloride. EXAMPLE 4: ANALYSIS OF GRIK2 USING TASTE RECEPTOR CELL GENE EXPRESSION

[0123] Next, GRIK2 was analyzed by measuring GRIK2 mRNA in human and mouse taste receptor cells (single-cell RNASeq) and measuring the open pattern of chromatin in mouse taste receptor cells (ATAC-Seq).

[0124] As that data above indicate that GRIK2 is an aversive sodium chloride taste receptor, it is expected to be expressed at low abundance in a subset of taste receptor cells in humans and mice, as is true for other taste receptors. Furthermore, as taste cells can be either selective or generally responsive to stimuli, not all receptors are present in all taste receptor cells.

[0125] Figure 5 shows data from the mRNA expression of single taste receptor cells removed from the fungiform papillae of a human research participant (young adult female). 48 out of 1,856 cells were classified as taste receptor cells because they had one or more taste receptor cell markers. These data show that GRIK2 is expressed at low levels in about 5-10 (10-20%) taste receptor cells, about the same percentage as other taste receptors like TAS1R3.

[0126] Similar results are shown in Table 2.

[0127] Type=taste receptor cell type,

[0128] N Exp = mRNA is expressed at 50 mRNA copies or more. Gust=cell is positive for the gustducin gene; Taslr3+ is positive for the sweet and umami receptor.

[0129] Sour is a cell with sour-markers.

[0130] Gadl is a marker for Type III taste cells.

[0131] Al means the cell is not responsive to the ability of amiloride to suppress bitterness, amiloride insensitive.

[0132] We used a cut-off of 50 mRNA transcript to consider a cell positive. The total number of cells studied per cell type is given in the last column.

[0133] Grik2 mRNA is co-expressed in about 10-20% of mouse taste receptor cells. These particular cells were selected for study as they either expressed taste cell markers tagged with a fluorescent protein (type II; Taslr3 and gustducin) or were identified as type III taste receptor cells using the marker Gadl. In all cases cells were counted as positive only if they expressed 50 or more Grik2 mRNA transcripts, to avoid counting cells that express only one or two transcripts due to amplification noise or contamination.

[0134] Other cells were identified because they responded to sodium chloride equally well after application of amiloride. The Grik2 gene is not expressed in these amiloride- insensitive taste cells. This result is surprising because amiloride is a drug used to distinguish among different salt-taste pathways, and the aversive pathway is thought to be amiloride insensitive. This result could be explained in several ways: (i) Grik2 was expressed but below the level of detection in this experiment, or (ii) the Grik2 protein is stable and there is low mRNA in these amiloride-insensitive taste cells. These explanations are supported as 8 of 14 amiloride-sensitive cells had between 0.1 and 2.1 normalized Grik2 transcripts (data not shown). In a separate experiment in mice using taste cells taken from the circumvallate taste papillae, we examined whether the chromatin was open near the Grik2 gene in multiple cell types. The data in FIG. 6 indicates that the chromatin is open at several locations in different types of taste receptors and other cells, but with a few open locations that are more specific to taste receptor cells.

[0135] These data indicate that that GRIK2 and other GRIKs expressed in human taste cells could be transfected into immortalized cell lines (e.g., HEK cells) and used to screen for GRIK2 agonist activity. These agonists would likely have a salty taste without containing sodium and could be suitable as salt substitutes. This is necessary as the only widely available salt substitute is potassium chloride, which has an off taste and is not suitable for people with certain health conditions.

[0136] REFERENCES

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[0155] EMBODIMENTS

[0156] 1. A method of identifying a compound that modulates taste, the method comprising:

[0157] (a) contacting a test compound with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof;

[0158] (b) measuring a response of the taste receptor to the test compound by comparing activity of the taste receptor in the presence and the absence of the test compound; wherein said test compound modulates taste when a change is observed in the activity of the taste receptor in the presence of the test compound. The method of embodiment 1, wherein said test compound produces or enhances the salty taste when the activity of the taste receptor is increased in the presence of the test compound. The method of embodiment 1, wherein the test compound reduces or inhibits an unpleasant taste when the activity of the taste receptor is decreased in the presence of the test compound. A method of identifying a compound that modulates salty taste without unpleasant taste, the method comprising:

[0159] (a) contacting a known salt substitute with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof;

[0160] (b) measuring a response of the taste receptor to the salt substitute by comparing activity of the taste receptor in the presence and the absence of the salt substitute; wherein the salt substitute modulates salty taste without unpleasant taste when no increase in activity of the taste receptor is observed in the presence of the test compound. A method of modulating taste in an ingestible composition, the method comprising administering a compound identified or selected according to the method of any one of embodiments 1 to 4 in the ingestible composition. The method of embodiment 5, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof. A method for reducing an amount of sodium in an ingestible composition without reducing a salty taste of a flavored product, comprising administering a compound identified or selected according to the method of any one of embodiments 1 to 4 in the ingestible composition. The method of embodiment 7, wherein the compound reduces the amount of sodium by at least 20%, or at least 25%, or at least 30%, or by at least 40%, or by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. The method of embodiment 7 or embodiment 8, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof. The method of any one of embodiments 1 to 4, wherein the GRIK2 polypeptide, or fragment thereof, is expressed by a cell. The method of embodiment 10, wherein the response is measured by (i) comparing the observed current of the receptor before and after contacting the cell with the test substance, or (ii) comparing observed cation influx into the cell when the test substance is contacted with the cell with cation influx into the cell observed when the test substance is not contacted with the cell. The method of embodiment 11, wherein the response is measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium. The method of embodiment 11, wherein the cation influx is measured by measuring cell membrane current of the cell in the presence of sodium ions. The method of embodiment 11, wherein the cation influx is measured by measuring cell membrane current of the cell in the absence of sodium ions. The method of any one of the preceding embodiments, wherein the GRIK2 protein has at least 80% identity with the amino acid sequence of SEQ ID NO:2. A method of imparting or enhancing a salty taste in an ingestible composition, comprising administering a flavor-modifying composition that increases the expression, level, or activity of GRIK2 in the ingestible composition. The method of embodiment 16, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof. A method for reducing an amount of sodium in an ingestible composition without reducing a salty taste of a flavored product, comprising administering a flavormodifying composition that increases the expression, level, or activity of GRIK2 in the ingestible composition. The method of embodiment 18, wherein the flavor-modifying compound reduces the amount of sodium by at least 20%, or at least 25%, or at least 30%, or by at least 40%, or by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. The method of embodiment 18 or embodiment 19, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof. The method of any one of the preceding embodiments, wherein the GRIK2 protein is comprised within an artificial lipid membrane. The method of embodiment 21, wherein the response is selected from comparing the observed current of the receptor before and after contacting the membrane with said test substance, or comparing observed cation influx into the membrane when the test substance is contacted with the membrane with cation influx into the cell observed when the test substance is not contacted with the membrane. The method of embodiment 22, wherein the response is measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium. 24. The method of embodiment 23, wherein the cation influx is measured by measuring membrane current in the presence of sodium ions.

[0161] 25. The method of embodiment 23, wherein the cation influx is measured by measuring membrane current in the absence of sodium ion.

[0162] 26. The method of any one of the preceding embodiments, wherein the GRIK2 protein has the amino acid sequence of SEQ ID NO:2.

[0163] 27. A salt receptor agonist identified by the method of any one of embodiments 1, 2, 4, 10 to 15, or 21 to 26.

[0164] 28. An aversive salt receptor inhibitor identified by the method of any one of embodiments 1, 3, 10 to 15, or 21 to 26.

[0165] Each and every patent, patent application, and publication, including publications listed herein and publicly available nucleic acid and amino acid sequences cited throughout the disclosure, is expressly incorporated herein by reference in its entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention are devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include such embodiments and equivalent variations.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a compound that modulates taste, the method comprising:(a) contacting a test compound with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof;(b) measuring a response of the taste receptor to the test compound by comparing activity of the taste receptor in the presence and the absence of the test compound; wherein said test compound modulates taste when a change is observed in the activity of the taste receptor in the presence of the test compound.

2. The method of claim 1, wherein said test compound produces or enhances the salty taste when the activity of the taste receptor is increased in the presence of the test compound.

3. The method of claim 1, wherein the test compound reduces or inhibits an unpleasant taste when the activity of the taste receptor is decreased in the presence of the test compound.

4. A method of identifying a compound that modulates salty taste without unpleasant taste, the method comprising:(a) contacting a known salt substitute with a taste receptor comprising a GRIK2 polypeptide, or a functional fragment thereof;(b) measuring a response of the taste receptor to the salt substitute by comparing activity of the taste receptor in the presence and the absence of the salt substitute; wherein the salt substitute modulates salty taste without unpleasant taste when no increase in activity of the taste receptor is observed in the presence of the test compound.

5. A method of modulating taste in an ingestible composition, the method comprising administering a compound identified or selected according to the method of claim 1 in the ingestible composition.

6. The method of claim 5, wherein the ingestible composition further comprises a bitterblocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof.

7. A method for reducing an amount of sodium in an ingestible composition without reducing a salty taste of a flavored product, comprising administering a compound identified or selected according to the method of claim 1 in the ingestible composition.

8. The method of claim 7, wherein the compound reduces the amount of sodium by at least 20%, or at least 25%, or at least 30%, or by at least 40%, or by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

9. The method of claim 7, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof.

10. The method of claim 1, wherein the GRIK2 polypeptide, or fragment thereof, is expressed by a cell.

11. The method of claim 10, wherein the response is measured by (i) comparing the observed current of the receptor before and after contacting the cell with the test substance, or (ii) comparing observed cation influx into the cell when the test substance is contacted with the cell with cation influx into the cell observed when the test substance is not contacted with the cell.

12. The method of claim 11, wherein the response is measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium.

13. The method of claim 11, wherein the cation influx is measured by measuring cell membrane current of the cell in the presence of sodium ions.

14. The method of claim 11, wherein the cation influx is measured by measuring cellmembrane current of the cell in the absence of sodium ions.

15. The method of claim 1, wherein the GRIK2 protein has at least 80% identity with the amino acid sequence of SEQ ID NO:2.

16. A method of imparting or enhancing a salty taste in an ingestible composition, comprising administering a flavor-modifying composition that increases the expression, level, or activity of GRIK2 in the ingestible composition.

17. The method of claim 16, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof.

18. A method for reducing an amount of sodium in an ingestible composition without reducing a salty taste of a flavored product, comprising administering a flavormodifying composition that increases the expression, level, or activity of GRIK2 in the ingestible composition.

19. The method of claim 18, wherein the flavor-modifying compound reduces the amount of sodium by at least 20%, or at least 25%, or at least 30%, or by at least 40%, or by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

20. The method of claim 18, wherein the ingestible composition further comprises a bitter blocking compound, a mouthfeel enhancer, a salty enhancer, or any combination thereof.

21. The method of claim 1, wherein the GRIK2 protein is comprised within an artificial lipid membrane.

22. The method of claim 21, wherein the response is selected from comparing the observed current of the receptor before and after contacting the membrane with said test substance, orcomparing observed cation influx into the membrane when the test substance is contacted with the membrane with cation influx into the cell observed when the test substance is not contacted with the membrane.

23. The method of claim 22, wherein the response is measured using a flux assay, patch clamping, voltage sensitive dyes, ion-sensitive dyes, electrophysiology, or changes in fluorescence of a membrane potential dye in response to sodium or lithium.

24. The method of claim 23, wherein the cation influx is measured by measuring membrane current in the presence of sodium ions.

25. The method of claim 23, wherein the cation influx is measured by measuring membrane current in the absence of sodium ion.

26. The method of any one of the preceding claims, wherein the GRIK2 protein has the amino acid sequence of SEQ ID NO:2.

27. A salt receptor agonist identified by the method of any one of claims 1, 2, 4, 10 to 15, or 21 to 25.

28. An aversive salt receptor inhibitor identified by the method of any one of claims 1, 3,10 to 15, or 21 to 25.

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