Patent Description:
Lipid bilayers are impermeable to protons, whose movement into and out of cells is tightly regulated by membrane proteins, including ion channels. For example, <CIT> discloses the proton channel HvI. As presented herein, the transmembrane protein Otopetrin <NUM> (Otop1) and certain related transmembrane proteins are identified as proton-selective ion channels.

The drawings illustrate embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

The present invention relates to a method of identifying a modulator of otopetrin-mediated proton translocation activity comprising: (a) contacting an otopetrin polypeptide, or functional portion thereof, or host cell expressing said polypeptide with a test compound, wherein the otopetrin polypeptide or functional portion thereof is integrated into a cell membrane, the host cell membrane, or a synthetic membrane; and (b) determining a proton translocation activity mediated by the otopetrin polypeptide, or the functional portion thereof; wherein an increase or decrease of at least <NUM>% of the proton translocation activity, compared to an amount of proton translocation activity determined in an absence of the test compound, identifies the test compound as a modulator of the proton translocation activity mediated by the otopetrin polypeptide, or the functional portion thereof. According to the invention, the term "functional portion" refers to a "portion having proton translocation activity".

In accordance with a preferred embodiment the otopetrin polypeptide comprises a mammalian otopetrin polypeptide, or functional portion thereof, optionally wherein the mammalian otopetrin polypeptide comprises a polypeptide sequence selected from Otop1, Otop2, Otop3, and functional portions thereof; the otopetrin polypeptide comprises an insect otopetrin polypeptide, or functional portion thereof; and/or the otopetrin polypeptide is: at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; or <NUM>%; identical to an otopetrin polypeptide sequence of any one of SEQ ID Nos: <NUM>-<NUM>.

In accordance with a further preferred embodiment determining the proton translocation activity comprises: measuring conductivity, current, resistance, voltage potential across a membrane comprising the otopetrin polypeptide, or functional portion thereof; or intracellular pH or intracellular calcium within a host cell expressing an otopetrin polypeptide, or functional portion thereof, optionally, wherein: the conductivity, voltage potential, current, resistance, intracellular pH, or the intracellular calcium is measured in response to a change in pH; determining the proton translocation activity comprises a patch clamp technique, voltage clamp technique, measurement of whole cell current, or use of a pH sensitive indicator or calcium-sensitive indicator; the pH sensitive indicator comprises pHluorin, pHrodo Red, DFFDA, or BCECF; the calcium-sensitive indicator comprises Fura-<NUM>; the change in pH is an extracellular change in pH or the change in pH is a change in intracellular pH; and the change in pH is an increase in pH or the change in pH is a decrease in pH.

In accordance with another preferred embodiment determining the proton translocation activity comprises expressing the otopetrin polypeptide, or functional portion thereof in a cell, optionally wherein: the cell is an animal cell; the cell is a mammalian cell; the cell is an oocyte; the cell is a HEK-<NUM> cell; or the cell is a CHO cell.

In accordance with a preferred embodiment the test compound is selected from the group consisting of a small organic molecule, small inorganic molecule, electrophile, polysaccharide, peptide, protein, antibody, nucleic acid, and an extract, wherein the extract is from a biological material is selected from a bacteria, plant, fungi, animal cell, and animal tissue, optionally wherein: the test compound has a molecular weight in a range of <NUM> to <NUM>,<NUM> Daltons; the test compound is at a concentration in a range of: <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; or <NUM> pM to <NUM>; and/or the test compound is at a concentration of: at least <NUM> pM; at least <NUM> pM; at least <NUM> pM; at least <NUM>; at least <NUM>; at least <NUM>; at least <NUM>; at least <NUM>; at least <NUM>; or at least <NUM>.

The present invention also relates to a method of identifying a modulator of otopetrin-mediated proton translocation activity comprising: measuring a proton translocation activity mediated by an otopetrin polypeptide, or a functional portion thereof, wherein the otopetrin polypeptide or functional portion thereof is expressed in a heterologous cell and integrated into the cell membrane, and proton translocation is measured (i) in the presence of a test compound, and (ii) in the absence of the test compound; and determining a difference between the proton translocation activity measured in (a)(i) compared to the proton translocation activity measured in (a)(ii), thereby identifying the test compound as a modulator of the proton translocation activity mediated by the otopetrin polypeptide, or the functional portion thereof, optionally wherein: the difference is at least a difference of <NUM>%; the proton translocation activity measured in (a)(i) is at least <NUM>% greater than the proton translocation activity measured in (a)(ii) and the test compound is identified in (b) as an agonist of the proton translocation activity mediated by the otopetrin polypeptide, or the functional portion thereof; and/or the proton translocation activity measured in (a)(i) is at least <NUM>% less than the proton translocation activity measured in (a)(ii) and the test compound is identified in (b) as an antagonist of the proton translocation activity mediated by the otopetrin polypeptide, or the functional portion thereof.

The present invention in addition relates to a method of identifying a modulator of otopetrin-mediated proton translocation activity comprising: measuring a proton translocation activity mediated by an otopetrin polypeptide, or a functional portion thereof, wherein the otopetrin polypeptide or functional portion thereof is expressed in a heterologous cell, and proton translocation is measured (i) in the presence of a test compound, and (ii) in the absence of the test compound; and determining a difference between the proton translocation activity measured in (a)(i) compared to the proton translocation activity measured in (a)(ii), thereby identifying the test compound as a modulator of the proton translocation activity mediated by the otopetrin polypeptide, or the functional portion thereof, optionally wherein: measuring the proton translocation activity comprises a patch clamp technique, measurement of whole cell currents, two-electrode voltage clamping, or a fluorescence assay using a voltage-sensitive or pH sensitive dye.

In accordance with a preferred embodiment: the otopetrin polypeptide comprises a mammalian otopetrin polypeptide, or functional portion thereof; the mammalian otopetrin polypeptide comprises a polypeptide sequence selected from a murine or human Otop1, Otop2, Otop3, and functional portions thereof; the otopetrin polypeptide comprises an insect otopetrin polypeptide, or functional portion thereof; and/or the otopetrin polypeptide is: at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; at least <NUM>%; or <NUM>%; identical to an otopetrin polypeptide sequence of any one of SEQ ID Nos: <NUM>-<NUM>.

In accordance with a further preferred embodiment: measuring the proton translocation activity comprises measuring conductivity, current, intracellular pH, intracellular calcium, or voltage potential or resistance across a membrane comprising the otopetrin polypeptide, or functional portion thereof in response to a change in pH.

In accordance with another preferred embodiment the method comprises inducing a change in pH on one side of the otopetrin polypeptide, optionally wherein: the change in pH is an extracellular or intracellular change in pH; and the change in pH is an increase in pH or the change in pH is a decrease in pH.

In accordance with a preferred embodiment: the heterologous cell is an animal cell; and/or the heterologous cell is an oocyte.

In accordance with a preferred embodiment the test compound is selected from the group consisting of a small organic molecule, small inorganic molecule, polysaccharide, peptide, protein, antibody, nucleic acid, and an extract, wherein the extract is from a biological material is selected from a bacteria, plant, fungi, animal cell, and animal tissue, optionally, wherein: the test compound has a molecular weight in a range of <NUM> to <NUM>,<NUM> Daltons; the test compound is at a concentration in a range of: <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; <NUM> pM to <NUM>; or <NUM> pM to <NUM>; and/or the test compound is at a concentration of: at least <NUM> pM; at least <NUM> pM; at least <NUM> pM; at least <NUM>; at least <NUM>; at least <NUM>; at least <NUM>; at least <NUM>; at least <NUM>; or at least <NUM>.

Proton channels that can transport protons into eukaryotic cells have not been molecularly identified. An unbiased screen based on transcriptome profiling of taste cells was used to identify the transmembrane protein Otopetrin1 (Otop1) as encoding a proton-selective ion channel with novel biophysical properties. The related murine genes, Otop2 and Otop3 as well as the Drosophila gene dmOTOPLc also encode proton channels. It was also determined herein that Otop1 is required for Zn<NUM>+-sensitive proton conductance.

In some implementations, presented herein is a method of identifying a modulator of an otopetrin-mediated proton translocation activity. In some implementations, the method comprises contacting a test compound with an otopetrin polypeptide, or functional portion thereof, and determining a proton translocation activity mediated by an otopetrin polypeptide, or the functional portion thereof. The term "determining" as used herein in the context of "determining a proton translocation activity" means and includes, but is not limited to "measuring", "detecting", and/or "obtaining".

An otopetrin polypeptide, or functional portion thereof, is a multi-pass integral membrane protein that forms a channel in a lipid bilayer. Accordingly, an otopetrin polypeptide, or functional variant thereof, as referred to herein is an otopetrin polypeptide that is integrated into a lipid bilayer (e.g., a cell membrane, or synthetic lipid bilayer). When integrated into a cell membrane, an otopetrin polypeptide comprises a first side (e.g., an extracellular side) and a second side (e.g., an intracellular side). As shown herein, an otopetrin polypeptide provides translocation of protons from one side of the otopetrin polypeptide (e.g., one side of a membrane) to the other side of the otopetrin polypeptide (e.g., the other side of the membrane), often in response to a pH change (e.g., an increase or decrease in protons) on one side of a membrane. In some implementations a change in pH is induced on one side of an otopetrin polypeptide (e.g., by addition of a base or an acid) followed by detecting proton translocation through the otopetrin polypeptide.

In some implementations, an otopetrin polypeptide comprises or consists of an otopetrin polypeptide that is obtained, expressed, or derived from any suitable biological organism, non-limiting examples of which include animals, plants, protists, cnidaria (aquatic freshwater or marine animals), arthropods, fungi, bacteria, annelids, echinoderms, chordates, and mollusks, and the like. An otopetrin polypeptide can be obtained, expressed, or derived from any suitable species. An otopetrin polypeptide, in some implementations comprises or consists of an insect derived otopetrin polypeptide (e.g., an otopetrin polypeptide, or functional portion thereof, derived from Drosophila). In certain implementations, an otopetrin polypeptide comprises or consists of an otopetrin polypeptide that is obtained, expressed, or derived from a suitable mammal. In some implementations, an otopetrin polypeptide comprises a mammal otopetrin polypeptide, non-limiting examples of which include Otopetrin-<NUM> (Otop1), Otopetrin-<NUM> (Otop2), and Otopetrin-<NUM> (Otop3). In some implementations, an otopetrin polypeptide comprises or consists of an otopetrin polypeptide that is obtained, expressed, or derived from a rodent, non-limiting examples of which include mice and rats. In certain implementations, an otopetrin polypeptide comprises or consists of an otopetrin polypeptide that is obtained, expressed, or derived from a suitable primate. A primate can be a non-human primate or may include humans. In certain implementations, an otopetrin polypeptide comprises, consists of, is obtained from, or is derived from a human otopetrin polypeptide, non-limiting examples of which include Otopetrin-<NUM> (Otop1; UniProtKB - Q7RTM1; SEQ ID NO: <NUM>), Otopetrin-<NUM> (Otop2; UniProtKB - Q7RTS6; SEQ ID NO:<NUM>), and Otopetrin-<NUM> (Otop3; UniProtKB - Q7RTS5; SEQ ID NO:<NUM>). In certain implementations, an otopetrin polypeptide comprises a mouse otopetrin polypeptide non-limiting examples of which include Otopetrin-<NUM> (Otop1; UniProtKB - Q80VM9; SEQ ID NO:<NUM>), Otopetrin-<NUM> (Otop2; UniProtKB - Q80SX5; SEQ ID NO:<NUM>), Otopetrin-<NUM> (Otop3; UniProtKB - Q80UF9; SEQ ID NO:<NUM>), or a functional portion thereof. In certain implementations, an otopetrin polypeptide comprises or consists of an otopetrin polypeptide that comprises, consists of, a Drosophila Otop polypeptide, non- limiting examples of which include Drosophila OTOP variant D (NM_001272325. <NUM> (CG42492))(SEQ ID NO: <NUM>), Drosophila OTOP variant A (NM_134914. <NUM> (CG332))(SEQ ID NO: <NUM>), and Drosophila OTOP variant D (NM_001144688. <NUM> (CG42265))(SEQ ID NO: <NUM>). An otopetrin polypeptide can be expressed in a cell membrane of a cell using a suitable nucleic acid (e.g., a cDNA) that encodes an otopetrin polypeptide. In some implementations, a suitable cell is transfected with a cDNA encoding an otopetrin polypeptide. Non-limiting examples of a cDNA that encodes a Drosophila otopetrin polypeptide are provided in NCBI reference numbers NM_001272325. <NUM> (CG42492), NM_134914. <NUM> (CG332) and NM_001144688. <NUM> (CG42265).

In certain implementations an otopetrin polypeptide comprises one or more amino acid additions, deletions or substitutions. An otopetrin polypeptide can be at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or <NUM>% identical to an otopetrin polypeptide described herein. In certain implementations, an otopetrin polypeptide comprises or consists of an otopetrin polypeptide that is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or <NUM>% identical to an otopetrin polypeptide sequence of any one of SEQ ID Nos: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In some implementations, an otopetrin polypeptide comprises one or more amino acid analogs or one or more modified amino acids. Modified otopetrin polypeptides that comprise amino acid substitutions, amino acid deletions, amino acid additions, or amino acid analogs can be prepared by a suitable method for altering peptide sequences, non-limiting examples of which are described in <NPL>, or <NPL>. An otopetrin polypeptide can also be modified and made using suitable recombinant nucleic acid technology.

The term "percent identical" or "percent identity" refers to sequence identity between two amino acid sequences. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. When the equivalent site is occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, WI), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD. In one implementation, the percent identity of two sequences can be determined by the GCG program with a gap weight of <NUM>, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

In certain implementations, an otopetrin polypeptide described herein provides any amount of detectable proton translocation activity. In certain implementations, an otopetrin polypeptide for use in a method described herein is naturally occurring, truncated, mutated, or genetically altered while retaining any amount of detectable proton translocation activity. In some implementations, an otopetrin polypeptide described herein retains at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or more of the proton translocation activity of a wild-type otopetrin polypeptide peptide sequence. In some implementations, an otopetrin polypeptide described herein retains at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or more of the proton translocation activity of an otopetrin polypeptide of any one of SEQ ID Nos: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

In certain implementations, an otopetrin polypeptide comprises a functional portion of an otopetrin polypeptide described herein. A function portion of an otopetrin polypeptide is any portion of an otopetrin polypeptide that, when integrated into a membrane, displays proton translocation activity as measured by a method described herein. A functional portion of an otopetrin polypeptide can be made and/or expressed using a suitable recombinant method known in the arts and can be tested for proton translocation activity by a method described herein. Accordingly, one of skill in the art can readily identify a functional portion of an otopetrin polypeptide using the methods described herein. A functional portion of an otopetrin polypeptide, in certain implementations, retains at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or <NUM>% of the proton translocation activity of a wild type otopetrin polypeptide (e.g., an otopetrin polypeptide of any one of SEQ ID Nos: <NUM> to <NUM>).

In some implementations, an otopetrin polypeptide, or functional portion thereof, is linked, covalently or otherwise to another polypeptide, nucleic acid, carbohydrate, fatty acid or detectable reagent. In certain implementations, an otopetrin polypeptide, or functional portion thereof, comprises another polypeptide. In certain implementations, an otopetrin polypeptide or functional portion thereof, may be linked covalently to another polypeptide, or portion thereof, while retaining proton translocation activity. In some implementations, an otopetrin polypeptide is linked to another transmembrane protein, or portion thereof.

In some implementations, an otopetrin polypeptide is linked (covalently or non-covalently) to a distinguishable identifier. In some implementations an otopetrin polypeptide comprises one or more distinguishable identifiers. Any suitable distinguishable identifier can be linked to or associated with an otopetrin polypeptide. In some implementations, a distinguishable identifier is a detectable label. Non-limiting examples of a distinguishable identifier include a metallic label, a fluorescent label, a fluorescent protein (e.g., green fluorescent protein (GFP)), a pH sensitive protein or pH sensitive GFP (e.g., a pHluorin, or the like), any suitable fluorophore (e.g., mCherry), a chromophore, a chemiluminescent label, an electro-chemiluminescent label (e.g., Origen™), a phosphorescent label, a quencher (e.g., a fluorophore quencher), a fluorescence resonance energy transfer (FRET) pair (e.g., donor and acceptor), a protein (e.g., an enzyme (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase and the like)), an antigen or part thereof, a linker, a member of a binding pair), an enzyme substrate, a small molecule (e.g., biotin, avidin), a mass tag, quantum dots, nanoparticles, the like or combinations thereof. Any suitable fluorophore or light emitting material can be used as a distinguishable identifier. A light emitting distinguishable identifier can be detected and/or quantitated by a variety of suitable techniques such as, for example, flow cytometry, gel electrophoresis, protein-chip analysis (e.g., any chip methodology), microarray, mass spectrometry, cytofluorimetric analysis, fluorescence microscopy, confocal laser scanning microscopy, laser scanning cytometry, the like and combinations thereof. In some implementations, an otopetrin polypeptide is fused to GFP. In some implementations an otopetrin polypeptide is fused to a pHluorin.

In certain implementations a distinguishable identifier is indirectly associated with (e.g., bound to) an otopetrin polypeptide. In some implementations a distinguishable identifier is reversibly associated with an otopetrin polypeptide. In certain implementations a distinguishable identifier that is reversibly associated with an otopetrin polypeptide can be removed from an otopetrin polypeptide using a suitable method (e.g., by increasing salt concentration, denaturing, washing, adding a suitable solvent and/or salt, adding a suitable competitor, and/or by heating).

As described herein (e.g., see Examples), an otopetrin polypeptide is a multipass transmembrane protein that when integrated into a lipid bilayer, forms an ion channel or pore that selectively allows translocation of protons from one side of the lipid bilayer to the other side. Accordingly, the phrase "proton translocation activity" refers to translocation of protons through an ion channel or pore formed by an otopetrin polypeptide, or functional portion thereof, from one side of a lipid bilayer to the other side of the lipid bilayer. Proton translocation activity can be an active or passive process. In some implementations proton translocation activity by an otopetrin polypeptide is a passive process. Proton translocation activity of a membrane-integrated otopetrin polypeptide can be assayed or measured using a suitable in vitro or in vivo method. The translocation of protons across or through a channel or pore formed by an otopetrin polypeptide can be determined, measured, detected or assayed directly (e.g., by directly measuring or detecting changes (e.g., increases or decreases) in intracellular pH, current, resistance or voltage potential), or indirectly (e.g., by measuring or detecting changes (e.g., increases or decreases) in intracellular calcium or zinc (e.g., by imaging, e.g., calcium imaging)), by detecting otopetrin-mediated cell signaling or other otopetrin-mediated events, or by measuring or detecting (e.g., by imaging) light emitted from pH sensitive indicator. Non-limiting examples of a pH sensitive indicator includes pH sensitive fluorophores, pH sensitive dyes, pH sensitive proteins, the like and combinations thereof. In some implementations a pH sensitive indicator comprises a pHluorin. In some implementations, a pH sensitive indicator comprises pHrodo Red. The pH sensitive indicator pHrodo Red is weakly fluorescent at neutral pH but becomes increasingly fluorescent as pH decreases. This reagent can be used to quantify cellular cytosolic pH. Any suitable pH indicator can be used for a method described herein to detect changes in pH (intracellular or extracellular), non-limiting examples of which are listed in <NPL>.

In some implementations, proton translocation activity is determined, measured, detected or assayed by a method comprising voltage clamping (e.g., a two-electrode voltage clamping on Xenopus laevis oocytes or patch clamping transfected HEK-<NUM> cells), or imaging intracellular pH indicators. For example, in certain implementations, a nucleic acid encoding an otopetrin polypeptide protein, a functional portion thereof, or a homolog thereof is injected into Xenopus oocytes thereby mediating the expression and integration of an otopetrin polypeptide into the oocyte membrane. Proton translocation activity can then be assessed by measuring current or by measuring changes in membrane polarization, (e.g., changes in membrane potential). In certain implementations, proton translocation activity is determined by measuring changes in intracellular pH by use of a pH indicator such as a pHluorin. In certain implementations, proton translocation activity is determined by measuring changes in intracellular Ca<NUM>+ levels. For example, calcium flux can be measured by assessment of the uptake of Ca<NUM>+ or by using fluorescent dyes such as Fura-<NUM>. In a typical microfluorimetry assay, a dye such as Fura-<NUM>, which undergoes a change in fluorescence upon binding a single Ca<NUM>+ ion, is loaded into the cytosol of Otop-expressing cells. Upon exposure to a test compound, an increase in cytosolic calcium is reflected by a change in fluorescence of Fura-<NUM> that occurs when calcium is bound. Other suitable fluorescent calcium indicators can also be used for a method disclosed herein.

In some implementations, the proton translocation activity of an otopetrin polypeptide is assessed indirectly using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring the binding of an otopetrin to other molecules (e.g., peptide, signaling molecules, including peptides, small organic molecules, and lipids); and/or measuring increases or decreases in protein expression (e.g., transcription, protein levels, etc.) in response to intracellular pH changes that result from otopetrin-dependent proton translocation. In some implementations, proton translocation activity is determined by assessing changes in cell growth or viability where such events are mediated by otopetrin-dependent proton translocation. In certain implementations, proton translocation activity is determined or measured indirectly by detecting changes in amounts, or detecting modifications (e.g., phosphorylation) of intracellular second messengers (e.g., IP3, cGMP or cAMP), where such amounts or modifications are regulated or induced by otopetrin-dependent proton translocation.

In some implementations, proton translocation activity of an otopetrin polypeptide, or functional portion thereof, is determined by measuring or detecting a change in current, resistance or voltage potential across a lipid bilayer. A lipid bilayer can be lipid bilayer of a cell or may be synthetic. In some implementations, proton translocation activity is determined in response to a change in pH (i.e. proton concentration) on one side of a lipid bilayer. In some implementations, proton translocation activity is induced by a change in pH (i.e. proton concentration) on one side of a lipid bilayer. A change in pH can be provided by the addition of protons (e.g., an acid) to the fluid on one side of a membrane. A change in pH can be an increase or decrease in pH. In some implementations, a change in pH is a change of at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> pH unit. In some implementations a change is pH is a change of at least <NUM> pH units. A change in pH can be provided on either side of a membrane. In certain implementations, protons are added to the extracellular side of a membrane thereby providing an extracellular change in pH. Non-limiting examples of methods that can be used to determine, assay, or measure proton translocation activity include any suitable version of a patch clamp technique (e.g., cell-attached patch, inside-out patch, whole cell recording, outside-out patch, perforated patch, loose patch, a two-electrode voltage claim and a mammalian cell patch clamp (e.g., using transfected HEK-<NUM> cells)), mammalian cell - pH imaging with a dye (e.g., DFFDA BCECF, pHrodo red or Fura <NUM>-AM (Sigma Aldrich, CA no. <NUM>-<NUM>-<NUM>)) or by co-transfection with a pHluorin, for example, and imaging (e.g., calcium imaging), the like and combinations thereof. Additional non-limiting examples of methods that can be used to determine, assay, or measure proton translocation activity can found in <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; and <NPL>.

In certain implementation, proton translocation activity is determined using a voltage clamp technique on a cell or oocytes comprising a membrane integrated otopetrin polypeptide, or functional portion thereof. In some implementations, the presence of otopetrin-dependent proton translocation activity is determined as a net change in current of at least <NUM> pA, at least <NUM> pA, at least <NUM> pA, at least <NUM> pA, at least <NUM> nA, at least <NUM> nA, at least <NUM> nA, at least <NUM>µA, at least <NUM>µA, at least <NUM>µA, at least <NUM>µA, or at least <NUM>µA in response to pH change of at least <NUM> pH unit.

In some implementations, proton translocation activity is determined using a fluorescent-imaging plate reader (FLIPR). The FLIPR was a first-in-class instrument that utilized charge-coupled device imaging of a whole plate to capture fluorescent readouts (<NPL>; also see <NPL>). In certain implementations, a system comprises a FLIPR. In some implementations, a system is a high throughput system comprising a FLIPR.

In some implementations, a change in current is determined, measured or detected directly. In some implementations, a change in current is determined, measured or detected by measuring or detecting a corresponding change in resistance or voltage potential, which parameters are readily converted using a suitable mathematical algorithm. In some implementations, a modulation of otopetrin-dependent proton translocation activity is a net change (e.g., an increase or decrease of proton translocation activity) in otopetrin-dependent proton translocation activity of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>-fold, at least <NUM>-fold or at least <NUM>-fold. A net change can be determined by comparing an otopetrin-mediated proton translocation activity determined in the absence of a test compound to an otopetrin-mediated proton translocation activity determined in the presence of a test compound. In some implementations, an amount of otopetrin-mediated proton translocation activity determined in the absence of a test compound is referred to as a reference level or control. For example, a decrease in proton translocation activity, in some implementations, is determined by comparing an amount of proton translocation activity of an otopetrin polypeptide in the presence of a test compound to the proton translocation activity of an otopetrin polypeptide determined in the absence of a test compound (e.g., a reference level). In certain implementations, an increase in proton translocation activity is determined by comparing an amount of proton translocation activity of an otopetrin polypeptide in the presence of a test compound to the proton translocation activity of an otopetrin polypeptide determined in the absence of a test compound (e.g., a reference level).

The term "decrease" or "reduced", and grammatical variations thereof, as used herein, means a decrease of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>-fold, at least <NUM>-fold or at least <NUM>-fold as compared to a reference level or control. In certain implementations, a test compound that induces a decrease in the proton translocation activity of an otopetrin polypeptide is identified by a method described herein as a modulator of otopetrin-mediated proton translocation activity. A test compound or modulator that decreases or reduces the otopetrin-mediated proton translocation activity of an otopetrin polypeptide is referred to herein as an antagonist (e.g., an antagonist of an otopetrin polypeptide, an antagonist of otopetrin-mediated proton translocation activity).

The term "increase" or "enhance", and grammatical variations thereof, as used herein, means an increase of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>-fold, at least <NUM>-fold or at least <NUM>-fold as compared to a reference level or control. In certain implementations, a test compound that induces an increase in the proton translocation activity of an otopetrin polypeptide is identified by a method described herein as a modulator of otopetrin-mediated proton translocation activity. A test compound or modulator that increases or enhances the otopetrin-mediated proton translocation activity of an otopetrin polypeptide is referred to herein as an agonist (e.g., an agonist of an otopetrin polypeptide, an agonist of otopetrin-mediated proton translocation activity).

In some implementations, otopetrin-binding compounds are screened for agonistic or antagonist action in a functional assay that monitors a biological activity associated with otopetrin function such as effects upon intracellular levels of protons in a otopetrin-expressing host cell (e.g., protons, calcium, zinc), pH-activated conductance, cell death (i.e.; receptor-mediated cell death which can be monitored using, e.g., morphological assays, chemical assays, or immunological assays), depolarization of the otopetrin-expressing cells (e.g., using fluorescent voltage-sensitive dyes), second messenger production which can be detected by radioimmunoassay or ELISA), calcium-induced reporter gene expression, or other readily assayable biological activity associated with otopetrin activity or inhibition of otopetrin activity. In certain implementations, a functional assay is based upon detection of a biological activity of otopetrin that can be assayed using high-throughput screening of multiple samples simultaneously, e.g., a functional assay based upon detection of a change in fluorescence which in turn is associated with a change in otopetrin activity. Such functional assays can be used to screen candidate agents for activity as either otopetrin agonists or antagonists.

In some implementations, otopetrin-expressing cells (e.g., recombinant otopetrin-expressing cells) are pre-loaded with fluorescently-labeled pH indicators (e.g., pHrod Red). The Otopetrin-expressing cells are then exposed to a candidate otopetrin-binding compound and the effect of exposure to the compound is monitored. Candidate compounds that have otopetrin agonist activity are often those that, when contacted with the otopetrin-expressing cells, elicit an otopetrin-mediated change in intracellular pH or intracellular calcium relative to control cells (e.g., otopetrin-expressing cells in the absence of the candidate compound, host cells without otopetrin-encoding nucleic added). Similarly, functional otopetrin assays can be used to identify candidate compounds that block activity of otopetrins (e.g., block the change in intracellular pH induced by a change in extracellular pH).

In some implementations, a method of identifying a modulator of otopetrin-mediated proton translocation activity comprises a high-throughput screening process. High-throughput screening (HTS) is a method for scientific discovery that uses robotics, data processing and control software, liquid handling devices, and sensitive detectors to screen thousands to millions of test compounds in a relatively short period of time. Any suitable High-Throughput Screening process can be used for a method described herein, non-limiting examples of which are described in <CIT>;<CIT>; <CIT>; <CIT>; and<CIT>.

In some implementations, a difference in proton translocation activity is determined. A difference in proton translocation activity can be determined by comparing a first proton translocation activity determined in the absence of a test compound, and a second proton translocation activity determined in the presence of a test compound. In certain implementations, identifying a test compound as a modulator of an otopetrin proton translocation activity comprises determining a difference in proton translocation activity of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>-fold, at least <NUM>-fold or at least <NUM>-fold.

Any suitable test compound can be tested by a method described herein. In some implementations, a test compound is soluble in an aqueous medium (e.g., a cell culture medium) at a concentration tested. In some implementations, a test compound is completely soluble in an aqueous medium at a concentration tested. In some implementations, a test compound is partially soluble in an aqueous medium at a concentration tested. In certain implementations, a test compound is not toxic to a mammalian cell at a concentration tested. For example, in certain implementations, a test compound does not (i) inhibit growth, (ii) reduce viability, (iii) induce necrosis or apoptosis, (iv) induce damage of DNA or RNA, (v) damage a cell membrane, or (vi) induce proteolytic cleavage of cellular proteins, at a concentration tested. In some implementations, a test compound is not a carcinogen or co-carcinogen. In some implementations, a test compound is not a teratogen.

As used herein, the phrase "test compound" refers to any suitable compound that can be screened for the ability to specifically modulate the proton translocation activity of an otopetrin polypeptide. Non-limiting examples of a test compound include small compounds (e.g., small organic or inorganic molecules), large compounds (e.g., greater than <NUM> Da), polysaccharides, carbohydrates, sugars, fatty acids, lipids, biological macromolecules, (e.g., peptides, polypeptides, proteins, peptide analogs and derivatives, peptidomimetics, nucleic acids, nucleotides, nucleotide analogs), naturally occurring or synthetic compounds, binding agents (e.g., antibodies, or binding fragments thereof, including non-naturally occurring and synthetic binding agents (e.g., TandAbs, nanobodies, aptamers, BiTEs, SMIPs, DARPins, DNLs, affibodies, Duocalins, adnectins, fynomers, Kunitz Domains Albu-dabs, DARTs, DVD-IG, Covx-bodies, peptibodies, scFv-Igs, SVD-Igs, dAb-Igs, Knob-in-Holes, triomAbs, and the like), derivatives thereof, polymers thereof, salts thereof, isomers thereof, polymorphs thereof, and combinations thereof. In some implementations, a test compound is contained within an extract made from biological materials such as extracts of bacteria, plants, fungi, animal cells, or animal tissues. In some implementations, a test compound is contained within a biological fluid. Accordingly, in some implementations, a test compound comprises an extract or biological fluid. Small compounds may include molecules having a molecular weight greater than about <NUM> daltons (Da), but less than <NUM> Da, less than <NUM> Da, or less than <NUM> Da. Small compounds may comprise any suitable chemical moiety or group, non-limiting examples of which include alkanes, alkenes, alkynes, alcohols, halogens, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, saturated, partially saturated or unsaturated ring structures, nucleotides, nucleosides, polyatomic nonmetals (e.g., P, S, Se), transition metals, post-transition metals, metalloids, the like, salts thereof, and combinations thereof.

In certain implementations, test compounds include synthetic or naturally occurring compounds of a suitable library. A multitude of small molecule libraries are known in the art, some of which are commercially available. Commercially available compound libraries can be obtained from, for example, ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, Biomol International and Oxford. Methods for developing small molecule, polymeric and genome based libraries are described, for example, in <NPL>) and <NPL>). Chemical compound libraries from, for example, NIH Roadmap, Molecular Libraries Screening Centers Network (MLSCN) can also be used. Any suitable method can be used to make a small compound library. A compound library can be screened using a suitable HTS screening method and/or a method described herein to identified test compounds within the library that modulate an otopetrin-mediated proton translocation activity.

In certain implementations, a test compound comprises a molecular weight of <NUM> to <NUM>,<NUM> Da, <NUM> to <NUM>,<NUM> Da, <NUM> to <NUM>,<NUM> Da, <NUM> to <NUM>,<NUM> Da, <NUM> to <NUM>,<NUM> Da, <NUM> to <NUM>,<NUM> Da, <NUM> to <NUM> Da, or <NUM> to <NUM> Da. In certain implementations, a test compound comprises a molecular weight of <NUM> to <NUM>,<NUM> Da, <NUM>,<NUM> to <NUM>,<NUM> Da, <NUM>,<NUM> to <NUM>,<NUM> Da, or <NUM> to <NUM>,<NUM> Da.

A test compound can be tested at any suitable concentration. In some implementations, a test compound is tested at a concentration of at least <NUM> pM, at least <NUM> pM, at least <NUM> pM, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM> or at least <NUM>. In some implementations, a test compound is tested at a concentration in a range of <NUM> pM to <NUM>, <NUM> pM to <NUM>, <NUM> pM to <NUM>, <NUM> pM to <NUM>, <NUM> pM to <NUM>, <NUM> pM to <NUM>, <NUM> pM to <NUM>, <NUM> pM to <NUM>, or <NUM> pM to <NUM>. In some implementations, a test compound is tested at a concentration of less than <NUM>, less than <NUM>, less than <NUM> or less than <NUM>. In some implementations, a test compound is tested or assayed at one or more different concentrations.

In certain implementations, a test compound comprises or consists of an electrophile. An electrophile is a compound capable of forming a covalent bound with an otopetrin polypeptide. In certain implementations, a test compound comprises an enzyme (e.g., a protease).

In certain implementations an otopetrin polypeptide, or functional portion thereof, is associated with a cell membrane or a synthetic membrane. The phrase "associated with a cell membrane or a synthetic membrane" means that the otopetrin polypeptide, or functional portion thereof, is integrated into the membrane, thereby forming a functional proton channel comprising proton translocation activity.

An otopetrin polypeptide can be integrated into a lipid bilayer using a suitable method. An otopetrin polypeptide can be integrated into a lipid bilayer of a suitable cell, non-limiting examples of which include an animal cell, plant cell, a protist cell, a cell of an aquatic freshwater or marine animals, an arthropod cell, a fungi cell, a bacteria cell, an annelid cell, an echinoderm cell, a chordate cell, a mollusk cell, and the like. In certain implementations, an otopetrin polypeptide is integrated into a lipid bilayer of a mammalian cell, a primate cell, a non-human primate cell or a human cell. In certain implementations, a cell is a primary cell or an immortal cell (e.g., a tissue culture cell line), non-limiting examples of which include HeLa, HEK293, DU145, H295R, HT29, KBM-<NUM>, MCF-<NUM>, MDA-MB-<NUM>, PC3, THP-<NUM>, PC12, A549, CHO, COS, Caco-<NUM>, EL4, HEP G2, HL-<NUM>, the like, and derivatives thereof. In some implementations, a cell is a taste receptor cell (e.g., a sour taste cell). In certain implementations, a cell is an egg cell (e.g., an oocyte, e.g., a Xenopus oocyte). In certain implementations, a cell is a red blood cell, or derivative thereof. In certain implementations, a cell is a ghost cell, or an anucleated cell. An otopetrin polypeptide can be integrated into a lipid bilayer of a cell using any suitable method. In certain implementations, an otopetrin polypeptide is integrated into a lipid bilayer of a cell by introducing a nucleic acid into the cell that directs the expression of an otopetrin polypeptide. Accordingly, in some implementations a method described herein comprises expressing an otopetrin polypeptide, or functional portion thereof, in a cell. In some implementations, therefore, a cell is a transfected cell, for example a cell transfected with a nucleic acid directing the expression of an otopetrin polypeptide or functional portion thereof. In some implementations, a cell is a transduced cell, for example a cell transduced with a recombinant virus comprising a nucleic acid that directs the expression of an otopetrin polypeptide or functional portion thereof inside the transduced cell. In certain implementations, a cell comprises an otopetrin polypeptide or functional portion thereof, integrated into the cell membrane of the cell. In certain implementations, a cell comprises a heterologous otopetrin polypeptide, or functional portion thereof. The phrase "heterologous otopetrin polypeptide" indicates that the otopetrin polypeptide is derived from a different species than the cell that it is expressed in. In certain implementations, a cell is Xenopus oocyte comprising a heterologous otopetrin polypeptide. In certain implementations, a cell is taste receptor cell comprising a heterologous otopetrin polypeptide.

In certain implementations, an otopetrin polypeptide is integrated into a suitable liposome, micelle, nanodisc, bilayer sheet, or bicelle for use in a method described herein. In certain implementations, an otopetrin polypeptide is integrated into a suitable synthetic membrane for use in a method described herein. In some implementations, an otopetrin is integrated into a suitable synthetic membrane formed in the aperture of a suitable nanopore device for use in a method described herein.

In certain implementations, a nanopore device comprises a first fluid filled chamber and a second fluid filled chamber separated by an aperture comprising a synthetic membrane, wherein the membrane comprises one or more otopetrin polypeptides. In certain implementations, the first fluid filled chamber comprises a first electrode and the second fluid filled chamber comprises a second electrode, where the first and second electrode are operatively linked to a means for measurement or detection of at least voltage potential, current, and/or resistance. In certain implementations, a means for measurement or detection of voltage potential, current, and/or resistance is a device comprising an ammeter, voltmeter, ohmmeter, oscilloscope, the like or a combination thereof. In certain implementations, the first and or second chamber comprises one or more photocells for detection of light emitting molecular probes.

In certain implementations, provided herein, is a system for determining proton translocation activity of an otopetrin polypeptide. In certain implementations, provided herein, is a system for identifying a modulator of proton translocation activity of an otopetrin polypeptide (i.e., a modulator or otopetrin-mediated proton translocation activity). In certain implementations, a system comprises one or more otopetrin polypeptides integrated into a membrane, a first fluid-filled chamber and second fluid filled chamber, wherein the first fluid-filled chamber and the second fluid-filled chamber are located on opposing sides of the membrane. In certain implementations, an aperture comprises the membrane. For example, in some implementations, the system comprises an aperture and the membrane is housed within the aperture. In some implementations, the membrane is a synthetic membrane. In some implementations, the system comprises a nanopore device.

To characterize the function properties of Otop1, we varied pHo and measured the evoked currents in Xenopus oocytes expressing Otop1. Unless otherwise noted, these and all other experiments were conducted with the large cation NMDG+, which is generally impermeable through ion channels, substituting for Na+ in the extracellular solution. Otop1 currents in Xenopus oocytes increased monotonically in magnitude as pHo was lowered over a range of pH <NUM>-<NUM> (<FIG>) and the reversal potential (Erev) of the currents shifted toward more positive voltages. Note that because in these experiments endogenous and leak currents were not subtracted, the Otop1 currents deviate from Nernstian behavior<NUM>,<NUM>.

To determine if otop1 can generate the pore-forming subunit of an ion channel, irrespective of cellular context, we expressed it in HEK-<NUM> cells (<FIG>). An N-terminal YFP-tagged channel confirmed expression at the cell surface (<FIG>). Large inward currents were elicited in response to lowering pHo in Otop1-transfected cells (I = <NUM>,<NUM> ± <NUM> pA for pHo = <NUM>, compared with <NUM> ± <NUM> pA for untransfected cells) and the current magnitude increased monotonically with pHo (<FIG>), as it did in oocytes. Otop1 currents in HEK-<NUM> cells decayed within seconds, with faster kinetics observed in response to more acidic stimuli (<FIG>). The decay of the currents may be due to accumulation (or depletion) of protons near the channel, which could the affect the driving force for proton movement or gating of the channels<NUM>,<NUM>. For example, an H+ current of <NUM> pA, such as we observe, flowing for <NUM> second in a cell of <NUM> diameter (<NUM> fL volume) can be calculated to increase the total (bound + free) intracellular concentration of H+ by up to <NUM><NUM>. Moreover, removal of the excess H+ can take <NUM>-<NUM> seconds due to the slow diffusion of H+, which is bound to bulky pH buffers<NUM>. Indeed, when we imaged intracellular pH using the membrane permeant dye pHrodo Red, we observed a large change in pHi upon lowering pHo from <NUM> to <NUM> in Otop1-transfected cells, a response not observed in mock-transfected cells (<FIG>).

We carried out a detailed biophysical characterization of Otop1 to determine whether it is proton selective. First, we determined whether Otop1 is permeable to other cations by measuring responses to lowering pH while varying ionic conditions. Replacing NMDG+ in the extracellular solution with Na+ (<FIG>) did not change the amplitude of the current elicited in response to pH <NUM> (p> <NUM> by paired t-test), indicating that the channel is not measurably permeable to Na+. Similarly, substitution of NMDG+ with Cs+, Li+ or Ca<NUM>+ caused a less than <NUM>% increase in the current magnitude, indicating that Otop1 is also not appreciably permeable to these monovalent and divalent cations (<FIG>).

To determine the relative permeability of H+ to Na+ and Cl-, we measured Erev under conditions where <NUM> Na+ replaced <NUM> NMDG and where [Cl-] was increased, either in the presence or absence of Na+. In no case did we observe any change in Erev (<FIG>). Using the Goldman-Hodgkin-Katz (GHK) equation<NUM>, we calculate that a change of <NUM> mV, which would have been easily detectable, translates into a selectivity for H+ over Na+ of > 2x10<NUM>-fold and a selectivity of H+ over Cl- of >1x10<NUM>; as we observed no change in Erev, it is possible that the channel is perfectly selective for H+ over Cl- and Na+, like Hv1<NUM>.

The transition metal Zn<NUM>+ is a potent inhibitor of Hv1, binding to two external histidine residues that regulate gating<NUM>,<NUM>,<NUM>. It also inhibits other molecules involved in proton transport<NUM>, including the proton channel in taste cells<NUM>. To gain insights into structural elements involved in ion permeation by Otop1, we measured its sensitivity to inhibition by Zn<NUM>+. Otop1 currents in HEK-<NUM> cells evoked in response to pH <NUM> were inhibited by Zn<NUM>+ in a dose-dependent manner with an IC<NUM> of <NUM> ± <NUM> (<FIG>). The Hill coefficient of close to one (<NUM> ± <NUM>) indicates that a single Zn<NUM>+ ion binds to inhibit the channel.

Otop1 was tested to determine if it might be voltage-dependent. The response of Otop1 currents, evoked by pH <NUM> solution, to voltage steps between -<NUM> to +<NUM> mV (from a holding potential of <NUM> mV) was measured. The currents showed little evidence of a time-dependent change in amplitude, indicating that gating of Otop1 is not appreciably voltage-dependent over a range of physiologically relevant voltages (<FIG>).

Together these data establish that Otop1 encodes a proton-selective ion channel with unique structural and biophysical properties.

Otopetrins are evolutionarily conserved from nematodes to humans<NUM>,<NUM> (<FIG>). To determine if this family functions generally as proton channels, we examined some of the most distantly related members.

Otop1 has two murine homologs- mOtop2 and mOtop3<NUM> (<FIG>) which share <NUM>%-<NUM>% amino acid identity with Otop1 (<FIG>). When expressed in Xenopus oocytes, both, Otop2 and Otop3 generated large currents upon lowering pHo in an NMDG-based solution (<FIG>). Otop3 showed evidence of high selectivity for H+; the magnitude of Otop3 currents increased linearly as a function of pHo over the entire pH range tested (pH <NUM>-<NUM>; <FIG>) and Erev shifted <NUM> mV/log[H+](<FIG>. In contrast, Otop2 currents behaved anomalously; they saturated at ~pH <NUM> (<FIG>) and Erev shifted little over a range of pH <NUM>-<NUM> (<FIG>. These unexpected features of Otop2 are intrinsic to the channel protein as Otop2 currents measured in HEK-<NUM> cells showed very similar properties (<FIG>). Both Otop2 and Otop3 currents showed little evidence of voltage-dependence, like Otop1 (<FIG>. When expressed in HEK-<NUM> cells and measured with microfluorimetry, both Otop2 and Otop3 conducted protons into the cell cytosol in response to lowering pHo (<FIG>). Interestingly, pHi in Otop3 transfected cells failed to recover following return to neutral pHo, while Otop2 exhibited faster recovery as compared with Otop1, pointing to differences in H+ conduction by the channels. Thus although Otop2 and Otop3 have distinct properties, both permeate protons.

There are three genes in the drosophila genome related to mOtop1; they encode the predicted proteins dmOTOPLa, dmOTOPLb, and dmOTOPLc (<FIG>), none of which has previously been characterized. dmOtopLc, a protein of <NUM> amino acids, shows <NUM>% amino acid identity with Otop1 over the region of homology and ><NUM>% amino acid identity in several of its transmembrane domains (<FIG>). When expressed in Xenopus oocytes dmOTOPLc produced large currents in response to lowering the extracellular pH (<FIG>). Like Otop1 and Otop3, dmOTOPLc currents increased as pHo was lowered and Erev shifted to more positive voltages, indicative of proton selectivity (<FIG>). Interestingly, dmOTOPLc conducted relatively more current at pH6 than Otop1 or Otop3 and the relationship between the current amplitude and pH (<FIG>) was shallower; this may endow the channel with a broader dynamic range.

Together these data show that highly divergent and evolutionarily distant members of the Otopetrin family form channels with distinct functional properties but a common capacity to permeate protons.

To determine if proton influx through otopetrin channels, mOtop1, mOtop2, mOtop3 and hOtop1, could lead to a change in intracellular pH that could be detected with an intracellular pH-sensitive indicator dye, we transiently expressed each channel in HEK-<NUM> cells. Indeed, when we imaged intracellular pH from mOtop1-transfected cells using the membrane permeant pH-sensitive dye pHrodo Red and lowered the extracellular pH from <NUM> to <NUM>, we observed a large change in fluorescence indicative of a large change in intracellular pH (<FIG>). Similar results were observed (<FIG>) when monitoring changes in intracellular pH of HEK <NUM> cells transfected with hOtop1 using the pH indicator pHrodo red (mean +/- SEM), in response to a pH <NUM> solution. In both cases, a similar response was not observed in mock-transfected cells (<FIG>), indicating that the change in intracellular pH is a result of proton entry through Otop1 channels. <FIG> shows that HEK <NUM> cells transfected with Otop2 or Otop3 and loaded with pHrodo Red showed a large change in fluorescence upon lowering extracellular pH, indicating that both channels conduct protons into the cell cytosol in response to lowering extracellular pH (<FIG>). Interestingly, the intracellular pH in Otop3 transfected cells failed to recover following return to neutral pHo, while Otop2 exhibited faster recovery as compared with Otop1, pointing to differences in H+ conduction by the channels.

We also tested whether proton transport through Otop1 (<FIG>), Otop2 (<FIG>), and Otop3 (<FIG>) could be monitored with a genetically encoded pH indicator. For this we use pHluorin, which was co-transfected with each of the channels, or transfected alone (as a control). In HEK <NUM> cells transfected with Otop channels, large changes in fluorescent emission were detected from pHluorin when external pH was lowered (Otop1, Otop2 and Otop3) or external pH was raised (Otop <NUM> and Otop2). These responses were only observed in cells transfected with one of the three Otops and were not observed in untransfected cells.

We also tested whether entry of protons through Otop channels could cause a change in intracellular calcium that could be detected by micofluorometry. Transfected cells were loaded with the calcium indicator Fura2 AM and exposed to a change in extracellular pH from <NUM> to <NUM>. <FIG> (left panel) shows that only the Otop1 transfected cells responded with an elevation of intracellular calcium (n=<NUM> cells). The difference between Otop1 transfected and sham-transfected cells is significant (student's t-test. *** p<<NUM>). As a control, we showed that both transfected and untransfected cells responded to HOAc, pH <NUM>, which penetrates cell membranes and causes intracellular acidification, thereby elevating intracellular calcium by liberating it from intracellular buffers.

All experimental procedures were approved by IACUC of University of Southern California. Mice in which expression of eGFP was driven by TRPM5 promoter <NUM>,<NUM> and YFP was driven by the Pkd2l1 promoter(Pkd2l1-YFP)<NUM> were bred to generate mice that were positive for both GFP and YFP (Trpm5-GFP/Pkd2l1-YFP).

For expression in HEK-<NUM> cells, cDNAs were cloned into pcDNA3 vector with InFusion® HD Cloning Kit (Clontech). N-terminal YFP-tagged mOtop1 was generated by eliminating the start codon and subcloning Otop1 in frame to a <NUM>' YFP in pcDNA3. All sequences were verified by Sanger sequencing (Genewiz). In vitro transcription was performed with T7 mMESSAGE mMACHINE kit (Thermo Fisher Scientific). The mRNA were treated with TURBO DNase (<NUM> for <NUM>), purified with RNA Clean & Concentrator kit (Zymo), and checked for the integrity and concentration with gel electrophoresis and Nanodrop (Thermo Scientific).

Xenopus laevis oocytes were provided by Ecocyte Bioscience. mRNA (<NUM>-<NUM> ng, <NUM> nL) were injected into the oocytes with Nanoject II Auto-Nanoliter Injector (Drummond) and incubated in Standard Barth's solution (SBS, Ecocyte Bioscience) at <NUM> for <NUM>-<NUM> days before recording.

Two-electrode voltage clamp (TEVC) was performed as previously described <NUM>. The borosilicate glass pipette was pulled with P-<NUM> Flaming/Brown type micropipette puller and its resistance was within the range of <NUM>-<NUM> MΩ. The current was measured with GeneClamp <NUM> amplifier (Axon). Solution exchange was executed by gravity driven perfusion. For most experiments, the membrane potential was held at -<NUM> mV, and voltage ramps were applied every second from -<NUM> mV to +<NUM> mV (1V/s). The oocytes were incubated in the Na+-free extracellular solution with <NUM> DIDS for <NUM>-<NUM> to inhibit Ca<NUM>+-activated Cl- channels<NUM>,<NUM> before the application of acids. Significance was determined by ANOVA.

Xenopus oocytes were incubated in ND96 solution containing (in mM): <NUM> NaCl, <NUM> KCl, <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, <NUM> HEPES, pH adjusted to <NUM> with HCl. For measuring the current change in response to a change in pHo, sodium-free solutions were used, containing <NUM> N-Methyl-D-glucamine (NMDG), <NUM> KCl, <NUM> CaCl<NUM>, <NUM> MgCl<NUM>, buffered with either <NUM> HEPES pH <NUM>-<NUM> or with <NUM>-(N-morpholino)ethanesulfonic acid (MES, <NUM>) for pH <NUM>-<NUM>. pH was adjusted with HCl. The following chemicals were added to the sodium-free solutions as indicated in figures: <NUM> <NUM>,<NUM>'-Diisothiocyano-<NUM>,<NUM>'-stilbenedisulfonic acid (DIDS), <NUM>-<NUM> ZnCl<NUM>, <NUM> Amantadine.

Otop and GFP (<NUM>:<NUM>) were co-transfected into HEK-<NUM> cells (CRL-<NUM>, ATCC) using TransIT-LT1 Transfection Reagent (Mirus Bio Corporation, Madison, WI). Patch clamp recording and imaging experiments on GFP-positive cells were performed at room temperature ~<NUM>-<NUM> after transfection.

Whole-cell patch clamp recording was performed as previously described<NUM>. In brief, recordings were made with an Axopatch 200A or Axopatch 200B amplifier, digitized with a Digidata 1322a <NUM>-bit data acquisition system, acquired with pClamp <NUM>, and analyzed with Clampfit <NUM> (Molecular Devices, Palo Alto, CA). Records were sampled at <NUM> and filtered at <NUM>. Patch pipettes with resistance of <NUM>-<NUM> MΩ were fabricated from borosilicate glass, and only recordings in which a gigaohm seal was achieved were used in the analysis. For most experiments, after the whole cell configuration was achieved, the membrane potential was held at -80mV, or ramped from -80mV to +80mV (1V/s) once per second. For experiments to determine proton selectivity of Otop1 the membrane potential was held at EH for the extracellular solution bathing the cell and ramped from -80mV to +80mV (1V/s) once per second. Solutions were changed using a linear array of microperfusion pipes (Warner Instruments, Hamden, CT).

Tyrode's solution contained <NUM> NaCl, <NUM> KCl, <NUM> MgCl<NUM>, <NUM> CaCl<NUM>, <NUM> dextrose, <NUM> HEPES (pH <NUM> with NaOH). Pipette solutions contained <NUM> Cs-aspartate, <NUM> CsCl, <NUM> Mg-ATP, <NUM> EGTA, <NUM> CaCl<NUM> (<NUM> free Ca<NUM>+), and <NUM> HEPES (pH <NUM> with CsOH). For experiments in which pHo was varied (<FIG>), extracellular solutions contained <NUM> NMDG, <NUM> CaCl<NUM>, and either <NUM> HEPES (for pH <NUM>), <NUM> MES (for pH <NUM>-<NUM>), or <NUM> HomoPIPES (for pH <NUM>-<NUM>), pH adjusted with HCl. For ion substitution experiment (<FIG>), solutions contained <NUM> NMDG-Cl was replaced by equimolar concentrations of NaCl, LiCl, or CsCl and <NUM> amiloride was added to block endogenous ENaC channels. For high calcium solutions, <NUM> NMDG was replaced by <NUM> CaCl<NUM> to maintain consistent osmolality. In the experiments shown in <FIG>, 3f-g, and <FIG>, <NUM> DIDS was added to block endogenous Cl- currents<NUM>. DIDS was omitted in all other experiments.

For measurement of proton selectivity (<FIG>), the pipette solution contained: <NUM> TMA- methane sulfonate, <NUM> TEA-Cl, <NUM> MgATP, <NUM> EGTA, <NUM> CaCl<NUM> and <NUM> MES titrated to (<NUM>) with ~<NUM> TMA-OH and adjusted to <NUM> mOsm with dH<NUM><NUM>. NMDG based (Na-free) external solution contained: <NUM> NMDG-methane sulfonate, <NUM> CaCl<NUM>, <NUM> HEPES (pH7. <NUM> - <NUM>), or <NUM> MES (pH5. <NUM> - <NUM>), or <NUM> HomoPIPES (pH4. <NUM> - <NUM>), pH adjusted with ~<NUM> - <NUM> NMDG-OH and adjusted to <NUM> mOsm with dH<NUM><NUM>. Na-containing solution contained: <NUM> Na-methane sulfonate, <NUM> CaCl<NUM>, <NUM> HEPES (pH7. <NUM>), or <NUM> MES (pH5. <NUM>), pH adjusted with NaOH. Where indicated, ZnCl<NUM> (<NUM>, final concentration), NMDG-Cl (<NUM>), NaCl (<NUM>) or MgCl<NUM> (<NUM>) were added to the Na-free or Na-containing solution and osmolarity was adjusted with dH<NUM>O. The pH of each solution was measured before use and adjusted if necessary. Note that the measurements were limited in accuracy by small variations in pH on the order of +/- <NUM> pH units or ~+/-<NUM>% change in [H+] at pH <NUM>; this could cause small changes in current magnitude when measuring response in the presences of different ions such as those shown observed in <FIG>. Liquid junction potentials of <NUM> mV were measured between the pipette solution and the bath solution (Tyrode's) and were corrected posthoc. To minimize junction potentials between solutions delivered by sewer pipes and the bath solution, a <NUM> KCl agar bridge was utilized and the bath solution was adjusted to contain a similar composition of ions as the sewer pipe in use<NUM>. Junction potentials of <<NUM> mV were not corrected.

HEK-<NUM> cells co-transfected with Otop and GFP were mixed with HEK-<NUM> cells transfected with CFP and plated on protamine coated coverslips at <NUM>. After at least one hour, cells were loaded with the intracellular pH indicator pHrodo Red AM, using PowerLoad concentrate according to the manufacturer's instructions (Molecular Probes). Cells co-transfected with Otop and GFP were identified using a U-MNIBA2 GFP filter cube (Olympus) while "sham" transfected cells expressing CFP were identified using a UN31044v2 CFP filter cube (Olympus) -or by absence of fluorescence. pH imaging optics and image acquisition were the same as previously described <NUM><NUM>. pHrodo Red fluorescence intensity for each cell was measured in response to pH <NUM> solutions buffered with MES (<NUM> NaCl, <NUM> MES, <NUM> CaCl<NUM>) or with acetic acid (<NUM> NaCl, <NUM> acetic acid, <NUM> CaCl<NUM>). The fluorophore was excited with <NUM> light, and emission at <NUM> was detected by a Hamamatsu digital CCD camera attached to an Olympus IX71 microscope using a U-N31004 Texas Red/Cy3. <NUM> filter cube (Chroma Technologies). The pHrodo Red fluorescence intensity of each cell was normalized to its baseline fluorescence in pH <NUM> solution (<NUM> NaCl, <NUM> HEPES, <NUM> CaCl<NUM>) before the first acid application.

All data are presented as mean ± SEM. unless otherwise noted. Statistical analyses (ANOVA or student's t-test) were performed using Graphpad Prism (Graphpad Software Inc). Sample sizes in the present study are similar to those reported in the literature for similar studies. Representative data shown in the figures was in some cases decimated <NUM>-fold before exporting into the graphics programs Origin (Microcal) and Coreldraw (Corel).

For <FIG>, The maximum-likelihood phylogenetic tree was created from the multi-sequence alignment of <NUM> otopetrin family proteins using Align-X (Invitrogen) and NJplot<NUM>. Accession numbers are as follows: Human, NP_819056; Cow, NP_001193713; Mouse, NP_766297; Dog, XP_545943; Chicken, XP_015141351; Frog, XP_012811170; Zebrafish, NP_942098; m_OTOP2, NP_766389; m_OTOP3, NP_081408; dm_OTOPLa, AAF46050; dm_OTOPLb, AAN10385; dm_OTOPLc, ACL82893; ce_OTOPL1, CCD61337. See <NPL>).

Claim 1:
A method of identifying a modulator of otopetrin-mediated proton translocation activity comprising:
(a) contacting an otopetrin polypeptide, or a portion thereof having proton translocation activity, or host cell expressing said polypeptide with a test compound, wherein the otopetrin polypeptide or the portion thereof is integrated into a cell membrane, the host cell membrane, or a synthetic membrane; and
(b) determining a proton translocation activity mediated by the otopetrin polypeptide, or the portion thereof;
wherein an increase or decrease of at least <NUM>% of the proton translocation activity, compared to an amount of proton translocation activity determined in an absence of the test compound, identifies the test compound as a modulator of the proton translocation activity mediated by the otopetrin polypeptide, or the portion thereof.