WO2013138948A1 - Redox-sensitive calcium-sensor proteins and methods of use thereof - Google Patents

Redox-sensitive calcium-sensor proteins and methods of use thereof Download PDF

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Publication number
WO2013138948A1
WO2013138948A1 PCT/CN2012/000335 CN2012000335W WO2013138948A1 WO 2013138948 A1 WO2013138948 A1 WO 2013138948A1 CN 2012000335 W CN2012000335 W CN 2012000335W WO 2013138948 A1 WO2013138948 A1 WO 2013138948A1
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Prior art keywords
cell
protein
calcium
receptors
seq
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PCT/CN2012/000335
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French (fr)
Inventor
Jie Liu
Bin Cai
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Rugen Therapeutics R & D (Shanghai) Co., Ltd.
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Application filed by Rugen Therapeutics R & D (Shanghai) Co., Ltd. filed Critical Rugen Therapeutics R & D (Shanghai) Co., Ltd.
Priority to PCT/CN2012/000335 priority Critical patent/WO2013138948A1/en
Priority to CN201280051540.1A priority patent/CN103958684A/en
Publication of WO2013138948A1 publication Critical patent/WO2013138948A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • compositions directed to genetically-encoded calcium indicator proteins that are capable of exhibiting significantly decreased fluorescence under oxidative conditions and that, upon exposure to intracellular calcium ions, produce a fluorescent light signal having increased signal-to-noise ratios as well as methods for using and producing the same.
  • Calcium ions serve as a second messenger in several important signal transduction pathways, is involved in the release of neurotransmitters in neurons to facilitate nerve transduction, mediates the contraction of all muscle cell types, and plays a vital role in fertility, enabling both sperm capacitation and motility as well as the oocyte's mechanism to prevent polyspermy.
  • many enzymes require calcium ions as a cofactor in order to function properly. Therefore, methods to monitor the dynamic changes in intracellular calcium concentration accompanying these diverse cellular and physiological phenomena have broad utility in biomedical research and drug discovery.
  • GECI genetically encoded calcium indicators
  • the small molecule fluorescent dyes such as fura-2 and fluo-3, change their fluorescence properties upon binding calcium. Their wide dynamic range, high sensitivity and fast kinetics have made them very popular tools. However, the dyes cannot be targeted to specific cells and dye loading can cause cytotoxicity.
  • GECIs provide an alternative to dyes that can be easily directed to any of subcellular compartments by simply fusing a signaling peptide to the GECI protein. Additionally, GECIs can be targeted to specific cell-types or tissues of a living organism and their expression level in target cells is stable from days to months, enabling extended time-lapse experiments.
  • Single-fluorescent protein GECIs possess a circularly permuted fluorescent protein whose fluorescence is modulated by calcium binding-dependent changes in the chromophore environment.
  • One type of single-fluorescent protein GECI is GCamP, which is composed of a circularly permutated enhanced GFP (cpEGFP) moiety, a calcium-binding protein calmodulin (CaM), and a Ca 2+ -CaM-binding peptide referred to as M13 (Nakai et al., Nature Biotech, 2001, 19: 137-41 ; Akerboom et al., 2009, J Biol. Chem., 284:6455-64).
  • cpEGFP circularly permutated enhanced GFP
  • CaM calcium-binding protein calmodulin
  • M13 Ca 2+ -CaM-binding peptide
  • the native EGFP has its chromophore located in the center of an 1 1 -strand ⁇ -barrel-like structure, which protects the chromophore from bulk solvent.
  • the native N- and C- terminus of the EGFP are jointed together by a linker, and the new N- and C- terminus are generated by opening one of the ⁇ -sheets at the side of the barrel, leading to exposure of the chromophore to solvent.
  • Ml 3 and the CaM domain are fused to the N- and C- terminus of the cpEGFP, respectively.
  • the artificial opening on the barrel of the cpEGFP domain is partly occluded by the Ca -CaM/M13 complex, so that the chromophore is protected from solvent and is stabilized in the fluorescent, deprotonated form.
  • the apo-CaM domain will not associate with Ml 3 in the absence of calcium, leading to solvent-access mediated darkening of the cpEGFP chromophore.
  • GECIs such as GCamP
  • existing GECIs have serious drawbacks that limit their practical use, such as low signal-to-noise ratio, temperature sensitivity, suboptimal kinetics, nonlinearity and low photostability.
  • low signal-to-noise ratio is a major drawback of GCamP and other GECIs.
  • Dead or damaged cells interfere with the measurement of calcium-specific fluorescence signals. Since these cells frequently exhibit increased intracellular calcium levels as a consequence of necrosis or programmed cell death, their fluorescence can increase background fluorescence detection and lower the overall sensitivity of calcium-based assays utilizing GECIs.
  • the invention provided herein discloses, inter alia, GCamP calcium sensor proteins which exhibit decreased fluorescence under the oxidative conditions typically observed in dead, dying, or damaged cells and that possess overall higher signal-to-noise ratios in comparison to unmodified GCamP calcium sensor proteins.
  • the invention additionally provides methods for using GCamP calcium sensor proteins in assays whose goal is to screen for a compound capable of modulating (for example, increasing or decreasing) intracellular calcium concentrations.
  • isolated nucleic acids comprising a nucleotide sequence encoding a calcium sensor protein comprising, from N terminus to C terminus, an Ml 3 domain, a circularly permuted green fluorescent protein domain, and a calmodulin (CaM) domain, wherein at least one pair of amino acid residues located at N77 and Y95, L134 and T201, T331 and T365, and/or T332 and E348 in the calcium sensor protein is/are replaced with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: l, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
  • CaM calmodulin
  • vectors comprising any of the nucleic acids described herein.
  • isolated cells comprising any of the nucleic acids or vectors described herein.
  • non-human animals comprising any of the cells described herein.
  • tissue slices comprising any of the cells described herein.
  • isolated calcium sensor proteins comprising an amino acid sequence comprising, from N terminus to C terminus, an Ml 3 domain, a circularly permuted green fluorescent protein domain, and a calmodulin (CaM) domain, wherein at least one pair of amino acid residues located at N77 and Y95, LI 34 and T201, T331 and T365, and/or T332 and E348 is/are replaced with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: 1, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
  • CaM calmodulin
  • kits for screening for an agent that is capable of increasing or decreasing intracellular calcium concentrations in a cell comprising: (i) contacting the agent with a cell expressing a calcium sensor protein encoded by any of the nucleic acids described herein; and (ii) determining a level of fluorescence, wherein an increase in fluorescence indicates that the agent is capable of increasing intracellular calcium concentrations and a decrease in fluorescence indicates that the agent is capable of decreasing intracellular calcium concentrations.
  • kits comprising one or more of (i) one or more of the nucleic acids, such as a vector, described herein; (ii) one or more of the calcium sensor proteins described herein; (iii) one or more of the cells described herein; (iv) one or more of the non-human animals described herein; (v) and/or one or more of the tissue slices described herein.
  • FIG. 1 depicts a schematic of the primary amino acid sequence of GCaMP2 illustrating the domain organization. Carets below the schematic show the positions of inter- domain linkers.
  • FIG. 2 depicts in vitro screening of redoxGCamP calcium sensor proteins.
  • A 10 ⁇ ionomycin or
  • B 100 ⁇ acetylcholine were added and the fluorescent change of the transfected HEK293 cells recorded.
  • F ma x represents the maximum fluorescent signal;
  • F 0 represents the background fluorescent signal.
  • C The fluorescent intensity of the crude lysates of the transfected HEK293 cells in MOPS buffer in the presence of 100 ⁇ DTT or 100 ⁇ H 2 0 2 , respectively.
  • D The fluorescent change of the transfected HEK293 cells after 30 minutes' treatment of the cell death assay buffer in the presence or absence of ⁇ H 2 0 2 .
  • FIG. 3 depicts fluorescence excitation and emission spectra of the crude lysates of the transfected HEK293 cells (A-F: untransfected, GCamP, N77C/Y95C, L134C/T E201C, T331C/T365C, and T332C/E348C) in the presence of 2 mM CaCl 2 or 10 mM EGTA,
  • the excitation spectra 300-500 nm were detected at 520 ran, and the emission spectra (500-600 nm) were excited at 470 nm.
  • FIG. 4 depicts the results of in vitro calcium titration experiments for
  • FIG. 5 depicts the results of experiments measuring the fluorescent spectra of isolated GCamP proteins under variable calcium, reducing, and oxidative conditions.
  • FIG. 6 depicts the results of experiments measuring the fluorescent spectra of isolated N77C/Y95C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions.
  • A Represents the excitation spectrum detected at 525 nm while (B) represents the emission spectrum excited at 470 nm.
  • FIG. 7 depicts the results of experiments measuring the fluorescent spectra of isolated L134C/T201 C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions.
  • A Represents the excitation spectrum detected at 525 nm while
  • B represents the emission spectrum excited at 470 nm.
  • FIG. 8 depicts the results of experiments measuring the fluorescent spectra of isolated T331C/T365C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions.
  • A Represents the excitation spectrum detected at 525 nm while
  • B represents the emission spectrum excited at 470 nm.
  • FIG. 9 depicts the results of experiments measuring the fluorescent spectra of isolated T332C/E348C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions.
  • A Represents the excitation spectrum detected at 525 nm while
  • B represents the emission spectrum excited at 470 nm.
  • FIG. 10 depicts observed fluorescent intensity of HEK293 cells co-transfected with GCamP or redoxGCamP calcium sensor proteins and 5HT2a receptor.
  • This invention provides, inter alia, genetically-encoded calcium indicator (GECI) proteins capable of exhibiting significantly decreased fluorescence under oxidative conditions as well as methods for using and producing the same.
  • GECI genetically-encoded calcium indicator
  • the inventors have constructed a series of GCamP calcium sensor proteins possessing overall improved signal-to-noise ratios in comparison to GCamP calcium sensor proteins which are not sensitive to oxidative conditions.
  • the inventors identified several sites for the substitution of cysteine residue pairs into the primary amino acid sequence of the GCamP chimeric protein.
  • thiol groups on those cysteine resides are susceptible to disulfide bond formation under oxidizing conditions and the formation of those disulfide bonds result in major conformational changes to the tertiary and/or quaternary structure of GCamP, thereby decreasing the protein's ability to fluoresce.
  • the thiol groups of the substituted cysteines remain protonated, thus preventing any structural disruption of GCamP which could negatively impact fluorescence emission or excitation upon exposure to intracellular calcium.
  • These unique calcium sensor proteins can be targeted to specific cells or tissues in vitro or in a live organism for measurement of intracellular calcium levels. Furthermore, the calcium sensor proteins expressed in cell types that utilize intracellular calcium as a second messenger can be used to screen for chemical compounds capable of selectively altering intracellular calcium levels via interaction with, for example, a plasma membrane-bound protein receptor (such as, but not limited to, a G-protein coupled receptor).
  • a plasma membrane-bound protein receptor such as, but not limited to, a G-protein coupled receptor
  • protein includes polypeptides, peptides, fragments of proteins, and fusion proteins.
  • an "isolated” molecule or cell is one which has been identified and separated and/or recovered from a component of its natural environment.
  • isolated protein means a protein derived from a different organism, species, or strain than the host cell. In some embodiments, a heterologous protein is not identical to a wild-type protein that is found in the same host cell in nature.
  • nucleic acid refers to two or more deoxyribonucleotides and/or ribonucleotides covalently joined together in either single or double-stranded form.
  • nucleic acid means a nucleic acid of interest that is free of one or more nucleic acids (e.g. , genes) which, in the genome occurring in nature of the organism from which the nucleic acid of interest is derived, flank the nucleic acid of interest.
  • nucleic acids e.g. , genes
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • heterologous nucleic acid is meant a nucleic acid sequence derived from a different organism, species or strain than the host cell.
  • the heterologous nucleic acid is not identical to a wild-type nucleic acid that is found in the same host cell in nature.
  • a nucleic acid encoding a calcium sensor protein which is transformed in or integrated into the chromosome of a host cell is a heterologous nucleic acid.
  • an "expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid of interest.
  • An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer.
  • An expression control sequence can be "native” or heterologous.
  • a native expression control sequence is derived from the same organism, species, or strain as the gene being expressed.
  • a heterologous expression control sequence is derived from a different organism, species, or strain as the gene being expressed.
  • An "inducible promoter” is a promoter that is active under environmental or developmental regulation.
  • operably linked is meant a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • a “mutation” includes an amino acid deletion, an amino acid insertion, and an amino acid substitution of at least one amino acid into a defined primary amino acid sequence.
  • mutation of one or more amino acids in a primary amino acid sequence can result in the protein encoded by that amino acid sequence having altered activity or expression levels within a cell.
  • mutation of one or more amino acids (such as a conservative mutation) in a primary amino acid sequence may not result in the protein encoded by that amino acid sequence having substantial changes in activity or expression levels within a cell.
  • substitution means that at least one amino acid component of a defined primary amino acid sequence is replaced with another amino acid (for example, a cysteine residue).
  • substitution of one or more amino acids in a primary amino acid sequence can result in the protein encoded by that amino acid sequence having altered activity or expression levels within a cell.
  • substitution of one or more amino acids (such as a conservative substitution) in a primary amino acid sequence may not result in the protein encoded by that amino acid sequence having substantial changes in activity or expression levels within a cell.
  • percent (%) amino acid sequence identity refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific protein or amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • percent (%) nucleotide sequence identity refers to the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the specific DNA or RNA sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • a "chimeric protein” is a protein comprising one or more portions derived from one or more different proteins, for example, a GCamP chimeric protein. Chimeric proteins may be produced by culturing a recombinant cell transfected with a nucleic acid that encodes the chimeric protein.
  • GPCR G-protein coupled receptor
  • Diacylglycerol activates certain protein kinase Cs (PKCs) while inositol phosphates (such as, but not limited to, IP3) stimulate the mobilization of calcium from intracellular stores such as the endoplasmic reticulum, the sarcoplasmic reticulum (for muscle cells), and/or the mitochondria.
  • PKCs protein kinase Cs
  • IP3 inositol phosphates
  • compositions of the Invention A Proteins
  • isolated calcium sensor proteins comprising an Ml 3 domain, a circularly permuted green fluorescent protein (cpGFP) domain, and a calmodulin (CaM) domain, wherein at least one pair of cysteine residues are added to the primary amino acid sequence of the calcium sensor protein, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
  • cpGFP circularly permuted green fluorescent protein
  • CaM calmodulin
  • the term “circularly permuted” means that the native GFP's N- and C-terminal ends are joined together (such as joined together by an amino acid linker sequence) and new N- and C-terminal ends are generated by cleavage of two peptide bonds in the GFP amino acid sequence.
  • the cpGFP domain of the calcium sensor protein comprises a first cpGFP domain (the native GFP's former C-terminal end) and a second cpGFP domain (the native GFP's former N-terminal end).
  • the cpGFP domain comprises the first cpGFP domain comprising amino acid residues 149-238 of the amino acid sequence shown in SEQ ID NO: l 1 and the second cpGFP domain comprising amino acid residues 1 -144 of the amino acid sequence shown in SEQ ID NO: 1 1.
  • the cpGFP domain comprises the first cpGFP domain comprising amino acid residues 148-237 of the amino acid sequence shown in SEQ ID NO: 12 and the second cpGFP domain comprising amino acid residues 1 -143 of the amino acid sequence shown in SEQ ID NO: 12. See amino acid sequence shown in Tsien, Annu. Rev.
  • the cpGFP domain comprises the first cpGFP domain comprising amino acid residues 148-237 of the amino acid sequence shown in SEQ ID NO: 13 and the second cpGFP domain comprising amino acid residues 1 -143 of the amino acid sequence shown in SEQ ID NO: 13 (Tsien, Annu. Rev. Biochem., 1998, 67:509-44 at pg. 513).
  • the calcium sensor protein domains are arranged so that the Ml 3 domain is located C-terminal to the CaM domain and the cpGFP domain.
  • the domains of the calcium sensor protein can be arranged so that the Ml 3 domain is located N-terminal to the cpGFP domain and the CaM domain.
  • the GFP of the calcium sensor protein is circularly permuted at any location between amino acid residues 135-155 of the amino acid sequence shown in SEQ ID NO: l 1 , 12, or 13. In a particular embodiment, the GFP is circularly permuted at amino acid residue position 144 of the amino acid sequence shown in SEQ ID NO: 11.
  • the isolated calcium sensor protein further comprises an optional tag (such as a purification tag).
  • a purification tag for isolation and purification of expressed proteins are numerous and commonly used in the art. Non-limiting examples include a His tag, a maltose binding protein tag, or a glutathione tag.
  • the protein purification tag is a histidine tag comprising at least six histidine residues. In another
  • the protein tag contains a protease recognition site so that the protein purification tag can be removed from the rest of the calcium sensor protein.
  • a linker can be inserted into the primary amino acid sequence of the cpGFP domain at the site of circular permutation.
  • the linker at the site of circular permutation is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.
  • the linker at the site of circular permutation is 6 amino acids in length.
  • the linker at the site of circular permutation comprises the amino acid sequence: G G T G G S (SEQ ID NO: 14).
  • a linker can be inserted between the optional protein purification tag and the Ml 3 domain.
  • the linker between the optional protein purification tag and the Ml 3 domain is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In one embodiment, the linker between the optional protein purification tag and the Ml 3 domain is 3 amino acids in length. In yet another embodiment, the linker between the optional protein purification tag and the Ml 3 domain comprises the amino acid sequence: M V D. In yet another embodiment a linker can be inserted between the Ml 3 domain and the first cpGFP domain. In some embodiments, the linker between the Ml 3 domain and the first cpGFP domain is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.
  • the linker between the Ml 3 domain and the first cpGFP domain is 2 amino acids in length. In yet another embodiment, the linker between the Ml 3 domain and the first cpGFP domain comprises the amino acid sequence: L E. In another embodiment, a linker can be inserted between the second cpGFP domain and the CaM domain. In some embodiments, the linker between the second cpGFP domain and the CaM domain is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In other embodiments, the linker between the second cpGFP domain and the CaM domain comprises the amino acid sequence: TR.
  • the addition of at least one pair of cysteine residues to the primary amino acid sequence of the calcium sensor protein can take the form of either inserting a pair of cysteine residues into the primary amino acid sequence or substituting a pair of residues in the primary amino acid sequence with cysteine residues.
  • any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pairs of cysteine residues are inserted into or replace at least two amino acids within the primary amino acid sequence of the calcium sensor protein.
  • the one or more inserted or replaced pairs of cysteine residues are located in the cpGFP domain of the calcium sensor protein.
  • the one or more inserted or replaced pairs of cysteine residues are located in the CaM domain of the calcium sensor protein. In another embodiment, the one or more inserted or replaced pairs of cysteine residues are located in the Ml 3 domain of the calcium sensor protein. In other embodiments, one member of the at least one or more inserted or replaced pairs of cysteine residues is located in the cpGFP domain while the other member is located in the CaM domain. In another embodiment, one member of the at least one or more inserted or replaced pairs of cysteine residues is located in the cpGFP domain while the other member is located in the Ml 3 domain. In yet another embodiment, one member of the at least one or more inserted or replaced pairs of cysteine residues is located in the CaM domain while the other member is located in the Ml 3 domain.
  • At least two residues in the primary amino acid sequence of the calcium sensor protein are substituted with at least two cysteine residues.
  • L134/T201, T365/T332, and T332/E348, are substituted with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: 1.
  • the calcium sensor protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: l, 2, 3, 4, or 5.
  • the calcium sensor protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO:l, 2, 3, 4, or 5.
  • the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:2. In another embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 3. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:3. In yet another embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 4. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:4. In still another embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 5. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:5.
  • the sensor protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent amino acid sequence identity to any of the calcium sensor proteins disclosed in Souslova et al., BMC Biotechnology, 2007, 7:37, the contents of which are incorporated by reference herein in its entirety.
  • the calcium sensor protein comprises an insertion of one or more cysteine residue pairs within about 5 amino acid residues of N77/Y95, L134/T201,
  • Modifications of proteins are made by methods that are well known in the art. For example, modifications can be made by site-specific mutagenesis of nucleotides in the polynucleotides encoding the calcium sensor protein, thereby producing DNA encoding the modification, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitutions at predetermined sites in DNA having a known sequence are also well known in the art, for example primer mutagenesis and PCR mutagenesis.
  • Calcium sensor proteins described herein may include conservative substitutions.
  • the calcium sensor proteins disclosed herein exhibit reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
  • the amount of fluorescence under oxidative conditions is reduced by at least any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%>, 60%, 65%, 70%, or 75%, inclusive, including any percentages in between these values in comparison to the amount of fluorescence produced under reducing conditions.
  • the calcium sensor proteins disclosed herein exhibit a higher signal-to-noise ratio in comparison to calcium sensor proteins (for example, unmodified GCaMP calcium sensor proteins) which do not comprise at least one or more pairs of cysteine residues added to the primary amino acid sequence of the calcium sensor protein.
  • the signal-to- noise ratio is improved by at least any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%), 65%, 60%, 65%, 70%, or 75%, inclusive, including any percentages in between these values in comparison to the signal-to-noise ratio of unmodified calcium sensor proteins (for example, unmodified GCaMP calcium sensor proteins).
  • the oxidative conditions are those conditions within the cytoplasm of damaged, dead, or dying cells.
  • the reducing conditions are those conditions within the cytoplasm of living cells or cells that are not substantially damaged.
  • any of the calcium sensor protein described above further comprise one or more protein tags (for example, a signal peptide) which is capable of targeting the protein to a particular cellular compartment.
  • the calcium sensor protein further comprises a mitochondrial import signal peptide.
  • the mitochondrial import signal peptide can target the protein to either the mitochondrial intermembrane space (for example, the tag can be any peptide sequence capable of binding to a Translocase of Inner Membrane (TIM) pore and/or a Translocase of Outer Membrane (TOM) pore in the mitochondrial membrane) or the mitochondrial matrix (for example, the tag can comprise one or more positively charged amino acids and/or one or more hydroxylated amino acids).
  • TIM Translocase of Inner Membrane
  • TOM Translocase of Outer Membrane
  • the calcium sensor protein further comprises an endoplasmic reticulum (ER) retention signal.
  • the ER retention signal is a non-cleavable K D E L amino acid sequence.
  • the calcium sensor protein further comprises a peroxisome targeting signal.
  • the peroxisome targeting signal can be Peroxisome targeting signal 1 (PTS1) in some
  • the peroxisome targeting signal can be Peroxisome targeting signal 2 (PTS2), comprising a nonapeptide located near the N-terminus with a consensus sequence of (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F) (where X can be any amino acid) (SEQ ID NO: 15).
  • PTS2 Peroxisome targeting signal 2
  • isolated nucleic acids that encode any of the proteins disclosed herein.
  • the disclosure provides isolated, synthetic, or recombinant polynucleotides comprising a nucleic acid sequence having at least about 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequence identity to the nucleic acid of SEQ ID NOs: 6, 7, 8, 9, or 10.
  • the disclosure provides isolated, synthetic, or recombinant polynucleotides comprising a nucleic acid sequence having at least about 70%, e.g., at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequence identity to nucleotides 106-1356, of SEQ ID NOs: 6, 7, 8, 9, or 10.
  • the disclosure specifically provides a nucleic acid encoding a calcium sensor protein.
  • the disclosure provides an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes: (1) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:l ; (2) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO: 1 ; (3) a protein comprising an amino acid sequence with at least 85%, 86%, 87%>, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9
  • the disclosure also provides cassettes and/or vectors comprising the above- described nucleic acids.
  • the nucleic acid encoding a calcium sensor protein of the disclosure is operably linked to a promoter.
  • Promoters are well known in the art and any promoter that functions in the host cell can be used for expression of the calcium sensor proteins of the present disclosure.
  • Initiation control regions or promoters, which are useful to drive expression of a calcium sensor protein in a specific cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these nucleic acids can be used.
  • vectors comprising the polynucleotides disclosed herein encoding a calcium sensor protein.
  • Suitable vectors are those which are compatible with the host cell employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Suitable vectors can be maintained in low, medium, or high copy number in the host cell. In some embodiments, the vector is an expression vector.
  • the vectors that can used according to the present invention also include vectors comprising a polynucleotide which encodes an RNA ⁇ e.g., an mRNA) that when transcribed from the polynucleotides of the vector will result in the
  • Vectors which may be used include, without limitation, lentiviral, HSV, and adenoviral vectors.
  • Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV and EIAV.
  • Lentiviruses may be pseudotyped with the envelope proteins of other viruses, including, but not limited to VSV, rabies, Mo-MLV, baculovirus and Ebola.
  • Such vectors may be prepared using standard methods in the art.
  • the vector is a recombinant AAV vector.
  • AAV vectors are
  • DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • the AAV genome has been cloned, sequenced and
  • ITR inverted terminal repeat
  • AAV AAV as a vector for gene therapy
  • Wild-type AAV could infect, with a comparatively high titer, dividing or non- dividing cells, or tissues of mammal, including human, and also can integrate into in human cells at specific site (on the long arm of chromosome 19)
  • Samulski, R. J, et al, EMBO J. 10: 3941-3950, 1991 the disclosures of which are hereby incorporated by reference herein in their entireties a tin, R. M., et al, Proc. Natl. Acad. Sci. USA 87: 2211-2215, 1990
  • Samulski, R. J, et al, EMBO J. 10: 3941-3950 1991 the disclosures of which are hereby incorporated by reference herein in their entireties.
  • AAV vector without the rep and cap genes loses specificity of site-specific integration, but may still mediate long-term stable expression of exogenous genes.
  • AAV vector exists in cells in two forms, wherein one is episomic outside of the chromosome; another is integrated into the chromosome, with the former as the major form.
  • AAV has not hitherto been found to be associated with any human disease, nor any change of biological characteristics arising from the integration has been observed.
  • AAV1 AAV 12, AAV13, AAV 14, AAV 15, and AAV 16
  • AAV5 is originally isolated from humans
  • AAVl-4 and AAV6 are all found in the study of adenovirus (Ursula Bantel-Schaal, Hajo Delius and Harald Kunststoff Hausen. J. Virol. 1999, 73: 939-947).
  • AAV vectors may be prepared using standard methods in the art.
  • Adeno- associated viruses of any serotype are suitable (See, e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease” J. R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P. Tattersall "The Evolution of Parvovirus Taxonomy” In Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski "The Genus Dependovirus” (JR Kerr, SF Cotmore.
  • the replication defective recombinant AAVs according to the invention can be prepared by co- transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes ⁇ rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus).
  • ITR inverted terminal repeat
  • a human helper virus for example an adenovirus
  • the vector(s) for use in the methods of the invention are encapsidated into a virus particle (e.g. AAV virus particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV 12, AAV13, AAV14, AAV15, and AAV16).
  • a virus particle e.g. AAV virus particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV 12, AAV13, AAV14, AAV15, and AAV16.
  • the invention includes a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Patent No. 6,596,
  • one or more vectors may be administered to any of the host cells described herein. If more than one vector is used, it is understood that they may be administered at the same or at different times to the host cell.
  • isolated cells are provided herein comprising any of the proteins, nucleic acids, and/or vectors described above.
  • the isolated cell is a prokaryotic cell or a eukaryotic cell.
  • the isolated cell is an invertebrate cell or a vertebrate cell.
  • the cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a nematode cell, an insect cell, a fish cell, a plant cell, an avian cell, an animal cell, and a mammalian cell.
  • Examples of cells capable of expressing a calcium sensor protein include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as those in the genera Synechocystis,
  • the cell is a yeast cell selected from the group consisting of a Saccharomyces, a Pichia, and a Candida.
  • the cell is a Caenorhabdus elegans nematode cell.
  • the cell is an insect cell, such as a Drosophila cell.
  • the cell is a zebrafish cell.
  • the cell is a sea urchin cell.
  • the cell is an amphibian cell, such as, but not limited to, aXenopus cell, for example, aXenopus oocyte.
  • Examples of mammalian cells capable of expressing a calcium sensor protein can be selected from the group consisting of a hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a porcine cell, an equine cell, a sheep cell, a monkey cell, a chimpanzee cell, and a human cell.
  • the animal cell is a neural cell (such as, but not limited to, a peripheral nervous system cell or a central nervous system cell), a muscle cell (such as a cardiac, skeletal, or smooth muscle cell), a gamete (such as a sperm cell or an oocyte), a cancer cell, an immune cell (such as, but not limited to, a macrophage, a T-cell, or a B-cell), a stem cell (such as, but not limited to, an embryonic stem cell or an adult stem cell), or an endocrine cell (such as, but not limited to, a thyroid cell, a hypothalamic cell, a pituitary cell, an adrenal cell, a testicular cell, an ovarian cell, a pancreatic cell (such as a ⁇ cell), a stomach cell, or an intestinal cell).
  • the cell is a human cell in cell culture.
  • the cell is a non-human cell in cell culture.
  • the cell is a human cell in cell culture.
  • the cell
  • the cell is a Hela cell, a KEK293 cell, a Chinese hamster ovary (CHO) cell, a Jurkat T cell, a neuroblastoma cell, or a human embryonic kidney cell (HEK) cell (such as a HEK293 cell or a HEK293T cell).
  • a Hela cell a Hela cell
  • a KEK293 cell a Chinese hamster ovary (CHO) cell
  • a Jurkat T cell a neuroblastoma cell
  • HEK human embryonic kidney cell
  • Suitable cell culture media and conditions vary with the specific cell type to be cultured but generally consist of an isotonic, buffered, basal nutrient medium which provides an energy source, coupled with inorganic salts, amino acids, vitamins and various supplements.
  • supplements may include serum (e.g., fetal calf serum, or the like) various antibiotics to prevent contamination or to provide selective conditions, attachment and growth factors, or the like.
  • serum e.g., fetal calf serum, or the like
  • a number of media formulations are known in the art, such as, but not limited to, minimal essential medium (MEM), Rosewell Park Memorial Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM).
  • Suitable culture conditions for many cell types are also known in the art (See, e.g., Morgan et al. 1993, Animal Cell Culture, BIOS
  • Transformation of cells is done using standard techniques which are known in the art and which are appropriate to the particular cell type. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector), by transfecting a plasmid into a cell via the use of an anionic lipid or by electroporation, or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,912,040, 4,740,461 , and 4,959,455 (which are hereby incorporated herein by reference in their entireties).
  • the transformation procedure used may depend upon the host to be transformed.
  • Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei.
  • the cells described herein can further comprise one or more membrane proteins whose function is responsible for either increasing or decreasing intracellular calcium as a second messenger.
  • Calcium ions are one of the most widespread second messengers used in cellular signal transduction. Calcium is transported into the intracellular environment either from outside the cell via calcium channels or from internal calcium storage sites such as the endoplasmic reticulum and the mitochondria. Levels of intracellular calcium are regulated by transport proteins that remove it from the cell. For example, the plasma membrane Ca ATPase (PMCA) pumps calcium out of the cell by in an ATP-dependent process.
  • voltage- dependent, calcium-selective ion channels are important for synaptic transmission through the release of neurotransmitters into the synaptic cleft by vesicle fusion of synaptic vesicles.
  • membrane proteins either the cell plasma membrane or the membranes of intracellular organelles
  • intracellular calcium's role as a second messenger is mediated by facilitating increases or decreases in the ion's concentration in the cytoplasm at any given time.
  • cells expressing a calcium sensor protein can further comprise one or more G protein-coupled receptors (GPCRs).
  • GPCRs also known as seven- transmembrane domain receptors
  • GPCRs comprise a large family of transmembrane receptor proteins (representing about 5% of the total genome of humans) that bind to molecules present in the extracellular environment and are capable of triggering signal transduction cascades within the cell and, ultimately, cellular responses.
  • GPCRs are found only in eukaryotes, including yeast, choanoflagellates, and animals.
  • the molecules that bind and activate these receptors include, but are not limited to, light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins.
  • GPCRs include, but are not limited to, G q protein or G q/ n, alpha- 1 adrenegic receptors (al-AR), urotensin (UT) receptors, 5-HT2 and 5-HT6 serotonin receptors, hypocretic (orexin) receptors, histamine HI receptors, bradykinin Bl and B2 receptors, bombesin BB2 receptors, P2Y purinergic receptors, acetycholine receptors (e.g., Ml, M3 and M5), mGluR5 glutamate receptors, vasopressin V2 and VI receptors, angiotensin AGTR1 receptors,
  • alpha- 1 adrenegic receptors alpha- 1 adrenegic receptors (al-AR), urotensin (UT) receptors, 5-HT2 and 5-HT6 serotonin receptors, hypocretic (orexin) receptors, histamine HI receptors, bradykin
  • the GPCR is endogenously expressed in the cell expressing the calcium sensor protein. In other embodiments, the GPCR is heterologously expressed in the cell expressing the calcium sensor protein.
  • PLC phospholipase C
  • PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers: Inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG).
  • IP 3 binds to a receptor protein present in the membrane of the smooth endoplasmic reticulum (SER) and mitochondria, which results in the release of the calcium ions stored within these organelles into the cytoplasm, thus dramatically increasing intracellular calcium levels.
  • cells expressing a calcium sensor protein can further comprise one or more calcium channel proteins.
  • a calcium channel is an ion channel which displays selective permeability to calcium ions.
  • the calcium channel protein is a voltage-dependent calcium channel.
  • excitable cells non-limiting examples include: muscle, glial, and neuronal cells
  • these channels are slightly permeable to sodium ions, leading to them also being known as Ca 2+ -Na + channels.
  • their permeability to calcium is around 1000-fold greater compared to sodium ions under normal physiological conditions (Hall, Guyton and Hall Textbook of Medical Physiology with Student Consult Online Access, 2011, (12th ed; Philadelphia: Elsevier Saunders), p. 64). At physiologic or resting membrane potential, these channels are normally closed.
  • Voltage- dependent calcium channels are formed as a complex of several different subunits: a ls ⁇ 2 ⁇ , ⁇ -4 , and ⁇ .
  • the ] subunit forms the ion conducting pore while the associated subunits have several functions including modulation of gating.
  • Voltage-dependent calcium channels include, but are not limited to, L-type calcium channels (having, for example, a Ca v l .1, Ca v l .2, Ca v l .3,or Ca v l .4 cti subunit and associated ⁇ 2 ⁇ , ⁇ , and/or ⁇ subunits), P-type calcium channels (having, for example, a Ca v 2.1 aj subunit and associated ⁇ 2 ⁇ , ⁇ , ⁇ subunits), N-type calcium channels (having, for example, a Ca v 2.1 ⁇ subunit and associated ⁇ 2 ⁇ / ⁇ , ⁇ 3 , ⁇ 4> ⁇ subunits), R-type calcium channels (having, for example, a Ca v 2.3 aj subunit and associated ⁇ 2 ⁇ , ⁇ , ⁇ subunits) or T-type calcium channels (having, for example, a Ca v 3.1, Ca v 3.2 ,or Ca v 3.3 j sub
  • the channel sensor protein is a ligand-gated calcium channel.
  • Ligand-gated calcium channels include, but are not limited to, the P2X receptor, the inositol 1,4,5-trisphosphate (IP 3 ) receptor, one or more Ryanodine receptors, one or more two pore channels, one or more cation channels of sperm (Catsper channels), or one or more plasma membrane store-operated channels.
  • the ligand-gated calcium channel is endogenously expressed in the cell expressing the calcium sensor protein. In other embodiments, the ligand-gated calcium channel is heterologously expressed in the cell expressing the calcium sensor protein.
  • cells expressing a calcium sensor protein can further comprise one or more calcium pump proteins.
  • intracellular calcium ordinarily in most cells, intracellular calcium
  • Calcium pump proteins are membrane proteins which pump intracellular calcium against its concentration gradient and into organelles such as mitochondria or the endoplasmic reticulum.
  • Calcium pump proteins include, but are not limited to, a plasma membrane Ca ATPase (PMCA) pump (including any of PMC A 1 , PMC A2, PMC A3, or PMCA4 isoforms) and the sodium-calcium antiporter protein (a.k.a. the Na + /Ca 2+ exchanger or NCX) which removes a single calcium ion in exchange for the import of three sodium ions.
  • the calcium pump protein is endogenously expressed in the cell expressing the calcium sensor protein.
  • the calcium pump protein is heterologously expressed in the cell expressing the calcium sensor protein.
  • non-human animals comprising any of the cells described herein or any of the calcium sensor proteins described herein.
  • Non-human animals are useful for studying the effects of and identifying compounds and agents capable of altering intracellular calcium concentrations in a live organism.
  • a "non-human animal" can include a mammal, a bird, a fly, a fish, a nematode worm, an amphibian, or a yeast.
  • the non-human animal is a mammal selected from the group consisting of a rodent (such as a rat or mouse in which one or more of the cells include a transgene encoding any of the calcium sensor proteins described herein), a non-human primate, a sheep, a dog, a cow, or a goat.
  • the non-human animal is a transgenic animal.
  • a transgene is exogenous DNA (such as a DNA encoding any of the calcium sensor proteins described herein) that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal.
  • Transgenes preferably direct the expression of an encoded gene product in one or more specific cell types or tissues of the transgenic animal.
  • Methods for creating transgenic animals are routine and well known in the art (see, e.g., U.S. Pat. Nos. 4,870,009, 4,736,866, and 4,873,191, the contents of which are incorporated by reference).
  • tissue slices obtained from non-human animals expressing any of the calcium sensor proteins described herein in one or more cell types or tissues.
  • the tissue slices are selected from the group consisting of central nervous system tissue slices (including brain and spinal cord slices), peripheral nervous system slices, muscle tissue slices (such as cardiac, skeletal, or smooth muscle tissue), endocrine tissue slices (such as tissue containing endocrine hormone-producing cells or glands), ovarian tissue slices, testicular tissue slices, bone marrow tissue slices, and tissue slices from cancerous tumors.
  • kits for screening for an agent that is capable of increasing or decreasing intracellular calcium concentrations in a cell comprising contacting the agent with a cell expressing a calcium sensor protein encoded by any of the nucleic acids described herein and determining a level of fluorescence, wherein an increase in fluorescence indicates that the agent is capable of increasing intracellular calcium concentrations and a decrease in fluorescence indicates that the agent is capable of decreasing intracellular calcium concentrations.
  • the cell expresses a calcium sensor protein such as those disclosed herein.
  • the cell can be selected from the group consisting of an animal cell, a bacterial cell, an insect cell, a nematode cell, and a yeast cell.
  • the animal cell is a human cell or a non-human cell.
  • the human cell is in a cell culture.
  • the non-human cell is selected from the group consisting of a rodent cell, a mouse cell, a rat cell, and a non-human primate cell.
  • the non-human cell is in a cell culture or in a non-human animal.
  • the animal cell is a muscle cell, a gamete, a neural cell, a cancer cell, or an endocrine cell.
  • the muscle cell is selected from the group consisting of smooth muscle, skeletal muscle, and cardiac muscle.
  • the cell is in a non- human animal, such as any of the non-human animals described herein.
  • the cell is in a tissue slice, such as any of the tissue slices described herein.
  • the cells further comprise one or more membrane proteins selected from the group consisting of a GPCR (such as any of the GPCRs described herein), a receptor tyrosine kinase, an ion channel protein (such as any of the calcium ion channel proteins described herein), or an ion pump protein (such as any of the calcium ion pump proteins or antiporters described herein).
  • a GPCR such as any of the GPCRs described herein
  • a receptor tyrosine kinase such as any of the calcium ion channel proteins described herein
  • an ion pump protein such as any of the calcium ion pump proteins or antiporters described herein.
  • the one or more membrane proteins are endogenously expressed in the cell expressing the calcium sensor protein.
  • the one or more membrane proteins are heterologously expressed in the cell expressing the calcium sensor protein.
  • cells in culture expressing any of the calcium sensor proteins described herein or non-human animals and/or tissue slices comprising cells expressing any of the calcium sensor proteins described herein can be used to screen for agents (for example, small molecules) that regulate the function of cell plasma membrane proteins (such as, but not limited to, a GPCR, an ion channel, or an ion pump) which directly or indirectly modulate intracellular calcium levels within living cells.
  • agents for example, small molecules
  • cell plasma membrane proteins such as, but not limited to, a GPCR, an ion channel, or an ion pump
  • Any agent known in the art can be tested for its ability to modulate (for example, increase or decrease) intracellular calcium concentrations.
  • candidate compounds can be directly provided to a cell expressing any of the calcium sensor proteins described herein and/or one or more receptor (such as a GPCR), calcium channel, and/or calcium pump proteins disclosed herein.
  • the methods described herein can be used to screen chemical libraries for molecules which modulate, e.g., increase or decrease, the intracellular concentration of calcium ions.
  • the chemical libraries can be peptide libraries, peptidomimetic libraries, chemically synthesized libraries, recombinant (e.g., phage display) libraries, in vitro translation- based libraries, and other non-peptide synthetic organic libraries.
  • the methods described herein may also be used to screen endogenous candidate compounds comprising biological materials (including, but not limited to, plasma and tissue extracts) and to screen libraries of endogenous compounds known to have biological activity.
  • the methods described herein are high-throughput methods for screening agents that are capable of increasing or decreasing intracellular calcium concentration.
  • direct identification of candidate agents is conducted in conjunction with agents generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis.
  • the candidate agent may be a member of a chemical library. This may comprise any convenient number of individual members, for example tens to hundreds to thousands to millions of suitable compounds, for example peptides, peptoids and other oligomeric compounds (cyclic or linear), and template-based smaller molecules (e.g.,
  • benzodiazepines hydantoins, biaryls
  • carbocyclic and polycyclic compounds e.g., naphthalenes, phenothiazines, acridines, steroids
  • carbohydrate and amino acid derivatives dihydropyridines, benzhydryls, heterocycles (e.g., trizines, indoles, thiazolidines).
  • the numbers quoted and the types of compounds listed are illustrative, but not intended to be limiting.
  • Preferred chemical libraries comprise chemical compounds of low molecular weight and potential therapeutic agents.
  • combinatorial chemistry can be used to identify modulators of the intracellular calcium concentration using the methods disclosed herein.
  • Combinatorial chemistry is capable of creating libraries containing hundreds of thousands of compounds, many of which may be structurally similar. While high throughput screening programs are capable of screening these vast libraries for affinity for known targets, newer approaches have been developed that achieve libraries of smaller dimension but which provide maximum chemical diversity. ⁇ See e.g., Matter, 1997 ', Journal of ' Medicinal Chemistry, 40: 1219-1229).
  • the agent is an antibody, a protein (for example, a polypeptide hormone) or any combination thereof.
  • the agent is an inhibitory nucleic acid selected from the group consisting of a triplex forming oligonucleotide, an aptamer, a ribozyme, a short interfering RNA (siRNA), an antisense oligonucleotide, and a micro-RNA (miRNA).
  • cells in culture expressing any of the calcium sensor proteins described herein or non-human animals and/or tissue slices comprising cells expressing any of the calcium sensor proteins described herein can be used to investigate the effects of intracellular calcium levels on protein function.
  • These protein functions can include, for example, the response of other calcium sensor proteins to changes in cytosolic calcium, the behavior of calcium dependent enzymes (for example, CaM Kinase enzymes) when bound to calcium, or the behavior of calcium pumps, and/or calcium ion channels to regulate intracellular calcium ion concentrations.
  • Additional aspects include using the cells, non-human animals, and/or tissue slices expressing any of the calcium sensor proteins described herein to investigate the effects of cytosolic calcium concentration on signaling pathways (such as, but not limited to, the phospholipase C pathway, the protein kinase C pathway, and/or the phosphoinositide 3-kinase pathway) or cell status (such as, but not limited to, cell growth, proliferation, transcription, metabolism, exocytosis, motility, nerve depolarization, muscle contraction, fertilization, apoptosis, and/or necrosis).
  • signaling pathways such as, but not limited to, the phospholipase C pathway, the protein kinase C pathway, and/or the phosphoinositide 3-kinase pathway
  • cell status such as, but not limited to, cell growth, proliferation, transcription, metabolism, exocytosis, motility, nerve depolarization, muscle contraction, fertilization, apoptosis, and/or necrosis).
  • neural cells in culture expressing any of the calcium sensor proteins described herein or neural tissue slices comprising cells expressing any of the calcium sensor proteins described herein can be used to detect the effects of a neuromodulator (for example, an agent that depolarizes a neuron) on a connection between a first neuron and a second neuron or a plurality of neurons forming a circuit.
  • a neuromodulator for example, an agent that depolarizes a neuron
  • Administering a neuromodulator for example, an agent that depolarizes a neuron
  • a neuromodulator for example, an agent that depolarizes a neuron
  • Administering a neuromodulator to the first neuron in the neural tissue slice to modulate (for example, increase or decrease) the eliciting of one or more action potential(s) in the second neuron or plurality of neurons forming a circuit can be optically detecting by monitoring the change in intensity of the fluorescence produced by the calcium sensor proteins in the second neuron or plurality of neurons forming a circuit.
  • the fluorescence of cells transformed or transfected with a DNA construct encoding any of the calcium sensor proteins disclosed herein may suitably be measured by optical means by, for example, a
  • spectrophotometer a fluorimeter, a fluorescence microscope, a cooled charge-coupled device (CCD) imager (such as a scanning imager or an area imager), a fluorescence activated cell sorter, a confocal microscope, or a scanning confocal device, wherein the spectral properties of the cells may be determined as scans of light excitation and emission.
  • CCD charge-coupled device
  • kits comprising one or more of any of the vectors disclosed herein, one or more of the calcium sensor proteins disclosed herein, any of the cells described herein, one or more of the non-human animals disclosed herein, and/or any of the tissue slices disclosed herein.
  • the kit further comprises instructions for screening for an agent that is capable of increasing or decreasing intracellular calcium
  • kit further comprises instructions for screening for an agent that is capable of increasing or decreasing intracellular calcium
  • Example 1 Design and construction of GCamP mutant calcium sensor proteins
  • the GCamP gene was further sub-cloned to the pcDNA3.1 vector in order to express in mammalian cells.
  • This pcDNA3.1 -GCamP plasmid was used as the template for generating the mutants by site-directed mutagenesis. For the mutants which have two separate mutation sites, the mutants containing the single mutation site were generated first and were sub-cloned together.
  • the forward and reverse primers of the N77CY95C mutant are: 5'-CGT CCT CGA TGT TGT GGC GGA TCT TGA AGC ACG CCT TGA TGC CGT TCT TC-3' (SEQ ID NO: 16) and 5'- GCG GCG TGC AGC TGG CCT ACC ACT GCC AGC AGA AC A CCC CCA TCG G-3' (SEQ ID NO: 17);
  • the forward and reverse primers of the L134C mutant are: 5'-AAC TCG CAC AGG ACC ATG TGA TCG CGC TTC-3' (SEQ ID NO: 18) and 5'-CGT GAC AGC TGC CGG GAT CAC TCT CG-3' (SEQ ID NO: 19);
  • the forward and reverse primers of the T201C mutant are: 5'-TTC AGG CAA AGC TTG CCG TTG GTG GCA TC-3' (SEQ ID NO: 20) and 5'-GTT CAT CTG CAC CAC CGG
  • a total of twelve GCamP mutant calcium sensor proteins were constructed with each having a pair of cysteine residues inserted into a domain of the GCamP chimeric protein.
  • Example 2 In vitro screening of potential redoxGCamP mutants for fluorescent emission
  • HEK293 cells were transfected with the calcium sensor proteins constructed in
  • Example 1 10 ⁇ ionomycin or 100 ⁇ acetylcholin were added and the fluorescent change of the transfected HEK293 cells were recorded by the FlexStation (excited at 470nm, detected at 520 nm).
  • Fmax represents the maximum fluorescent signal
  • F 0 represents the background fluorescent signal.
  • the fluorescent intensity of the crude lysates of the transfected HEK293 cells was measured in MOPS buffer (2 mM CaCl 2 , 100 mM KC1, 100 ⁇ PMSF, 20 mM MOPS, pH 7.5), in the presence of 100 ⁇ DTT or 100 ⁇ H 2 0 2 , respectively or after 30 minutes' treatment of the cell death assay buffer (HBSS, 2 mM CaCl 2 , 10 mM HEPES, 0.2% NaN 3 , 0.1% Triton X- 100, pH 7.4), in the presence or absence of 100 ⁇ ⁇ 2 0 2 .
  • the HEK293 cells were obtained from Cellbank in Shanghai, and were cultured in
  • DMEM containing 10% FBS fetal bovine serum
  • plasmids were transfected by PEL Briefly, plasmids were mixed with lmg/ml PEI solution in Opti-MEM at a ratio of 1 :2. After incubating for 10 minutes at room temperature, the DNA/PEI mixture was added to the culture plate. The cells were used for the following experiments 48 hours after transfection.
  • HEK293 cells were re-plated to the Costar black with clear bottom 96-well plate 12 hours before the experiments.
  • Cells were washed with the assay buffer (137 mM sodium chloride, 5.4 mM potassium chloride, 0.44 mM potassium phosphate monobasic, 5.56 mM glucose, 0.25 mM sodium phosphate dibasic, 4.2 mM sodium bicarbonate, 2mM calcium chloride, 10 mM HEPES, pH 7.4), and were incubated at room temperature for 30 minutes. Ionomycin and acetylcholine solution were prepared in the Costar V-bottom 96-well compound plate.
  • Both the compound plate and the assay plate were loaded to FlexStation III (Molecular Devices) under the flex mode.
  • the baseline (F 0 ) was recorded for 20 seconds before the compound solution was automatically transferred to the assay plate by the instrument.
  • the fluorescent signal was continuously recorded for 100 seconds, and the maximum signal is referred to as F max .
  • the excitation wavelength is at 470 nm, and the fluorescent signal was detected at 520 nm.
  • transfected HEK293 cells were washed and then were re-suspended with the MOPS buffer (2 mM CaCl 2 , 100 mM KC1, 100 ⁇ PMSF, 20 mM MOPS, pH 7.5).
  • the cells were lysed by sonication. After centrifugation, the supernatant was collected as the crude lysate.
  • the crude lysates were loaded to a Costar black with clear bottom 96-well plate, and were incubated with either 100 ⁇ H202 or 100 ⁇ DTT for 30 minutes at room temperature before measuring by FlexStation III under the spectrum mode.
  • the excitation wavelength is at 470 nm, and the emission record is from 500 to 600 nm.
  • Example 3 Characterization of the fluorescent spectra of redoxGCamP proteins in cellular crude lysates
  • MOPS buffer 100 mM KC1, 100 ⁇ PMSF, 20 mM MOPS, pH 7.5.
  • the cells were lysed by sonication. After centrifugation, the supernatant was collected as the crude lysate.
  • the crude lysates were loaded to a Costar black with clear bottom 96-well plate, and were incubated with either 2 mM CaCl 2 or 10 mM EGTA for 5 minutes at room temperature before measuring by FlexStation III under the spectrum mode.
  • the excitation wavelength is at 470 nm, and the emission record is from 500 to 600 nm (1 nm step).
  • Example 4 Characterization of the fluorescent spectra of purified redoxGCamP proteins by [0106] This study characterized the fluorescent spectra of purified redoxGCamP proteins in a calcium titration assay.
  • the purified proteins were diluted in either the calcium free buffer (10 mM EGTA, lOOmM KC1, 30mM MOPS, pH 7.2) or the high calcium buffer (10 mM Ca 2+ -EGTA, lOOmM KC1, 30mM MOPS, pH 7.2) at the final concentration of 1 ⁇ .
  • the calcium free buffer and the high calcium buffer containing the purified protein were mixed at different ratios to provide different concentrations of free calcium ions.
  • the fluorescent signal was monitored by FlexStation III (Molecular Devices) under the end-point mode. The samples were excited at 485 ran, and were detected at 520 nm.
  • F max was defined as the signal intensity at maximum concentration of free calcium
  • F 0 was defined as the signal intensity in the calcium free buffer.
  • the calcium titration curve was fitted by the four-parameter logistic dose-response model by GraphPad to obtain the EC 50 and Hill coefficient of each sample.
  • Example 5 Characterization of the fluorescent spectra of purified redoxGCamP proteins under reductive or oxidative conditions and in the presence or absence of calcium
  • This study measured emitted fluorescent light from purified unmodified and redox-sensitive GCamP proteins in the presence or absence of calcium and in the presence of either an oxidizing (H 2 0 2 ) or a reducing agent (dithiothreitol).
  • cell pellet was resuspended by the His60 Ni xTractor Buffer with ⁇ iL of DNase (Takara). After centrifugation at 4°C, the supernatant was collected and flow through the His60 Ni Gravity Column which have been equilibrated. The column was washed with 10 column volumes of Equilibration Buffer followed by 10 column volumes of Wash Buffer. The target protein was eluted by 10 column volumes of Elution Buffer and was collected as 1 ml fractions. The protein was desalted by the PD-10 column (GE Healthcare). The concentration of the protein was determined by the BCA Protein Assay Kit (Tiangen).
  • the samples were diluted in the MOPS buffer (lOOmM KC1, 30mM MOPS, pH 7.2) to a final concentration of 1 ⁇ , and were scanned by FlexStation (spectrum mode) reader (from Molecular Devices, Inc.) in a Costar black with clear bottom plate to obtain the excitation and emission spectra.
  • MOPS buffer lOOmM KC1, 30mM MOPS, pH 7.2
  • FlexStation (spectrum mode) reader from Molecular Devices, Inc.
  • N77C/Y95C redoxGCamP protein exhibited higher fluorescence in the presence of calcium and a reducing agent in comparison to the amount of light emitted in the presence of calcium in an oxidative environment ( Figure 6). Similar results were observed for the L134C/T E201C
  • Example 6 Use of redoxGCamP mutant proteins to measure changes in intracellular calcium concentration mediated by activation of a G protein-coupled receptor
  • This study tests the ability of the redoxGCamP mutant proteins to detect increases in intracellular calcium concentration in HEK293 cells expressing the 5-HT 2 A receptor, which is a subtype of the 5-HT 2 receptor belonging to the serotonin receptor family of G protein-coupled receptors (GPCRs).
  • GPCRs G protein-coupled receptors
  • the 5-HT2A gene was cloned from the cDNA library by PCR, and was inserted to the pcDNA3.1 vector.
  • the forward and reverse primers used were 5'-ACG AAG CTT ATG GAA ATT CTC TGT GAA GAC AAT ATC TCC CTG AGC TCA ATT CC-3' (SEQ ID NO: 48) and 5'-TA GGA TCC TCA CAC ACA GCT AAC CTT TTC ATT CAC GGT TTCAAT ATT GTC TGT AC-3' (SEQ ID NO: 49).
  • the HEK 293 cells were co-transfected with 5-HT2A and the redoxGCamP mutants.
  • the assay buffer (137 mM sodium chloride, 5.4 mM potassium chloride, 0.44 mM potassium phosphate monobasic, 5.56 mM glucose, 0.25 mM sodium phosphate dibasic, 4.2 mM sodium bicarbonate, 2mM calcium chloride, 10 mM HEPES, pH 7-4), and were incubated at room temperature for 30 minutes.
  • Serotonin (Sigma) solution was freshly prepared in the Costar V-bottom 96-well compound plate. Both the compound plate and the assay plate were loaded to FlexStation III (Molecular Devices) under the flex mode.
  • the baseline (F 0 ) was recorded for 20 seconds before the compound solution was automatically transferred to the assay plate by the instrument.
  • the fluorescent signal was further continuously recorded for 130 seconds, and the maximum signal is referred to as F max .
  • the excitation wavelength is at 470nm, and the fluorescent signal was detected at 525 nm.
  • VDEMIREADIDGDGQV YEEFVQMMTAK SEQ ID NO: l
  • VDEMIREADIDGDGQVNYEEFVQMMTAK SEQ ID NO:4 T332C/E348C REDOX-SENSITIVE PROTEIN
  • NUCLEOTIDE SEQUENCE FOR N77C/Y95C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgtgcttcaagatccgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacgg
  • NUCLEOTIDE SEQUENCE FOR T331C/T365C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgaacttcaagatccgccacaacatcgaggacggcggcgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcg
  • NUCLEOTIDE SEQUENCE FOR T332C/E348C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgaacttcaagatccgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggacggcggcgtgccacaacatcgaggac

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Abstract

Provided herein are compositions directed to the production and use of genetically-encoded calcium indicator proteins which exhibit significantly decreased fluorescence under oxidative conditions and that, upon exposure to intercellular calcium ions, produce a fluorescent light signal having increased signal to noise ratios as well as methods for using the same.

Description

REDOX-SENSITIVE CALCIUM-SENSOR PROTEINS AND METHODS OF USE
THEREOF
FIELD OF THE INVENTION
[0001] This disclosure relates to compositions directed to genetically-encoded calcium indicator proteins that are capable of exhibiting significantly decreased fluorescence under oxidative conditions and that, upon exposure to intracellular calcium ions, produce a fluorescent light signal having increased signal-to-noise ratios as well as methods for using and producing the same.
BACKGROUND OF THE INVENTION
[0002] Calcium (Ca2+) plays a vital role in the physiology and biochemistry of cells.
Calcium ions serve as a second messenger in several important signal transduction pathways, is involved in the release of neurotransmitters in neurons to facilitate nerve transduction, mediates the contraction of all muscle cell types, and plays a vital role in fertility, enabling both sperm capacitation and motility as well as the oocyte's mechanism to prevent polyspermy. In addition, many enzymes require calcium ions as a cofactor in order to function properly. Therefore, methods to monitor the dynamic changes in intracellular calcium concentration accompanying these diverse cellular and physiological phenomena have broad utility in biomedical research and drug discovery.
[0003] Two classes of Ca2+ indicators have been developed: small molecule fluorescent dyes and genetically encoded calcium indicators (GECI). The small molecule fluorescent dyes, such as fura-2 and fluo-3, change their fluorescence properties upon binding calcium. Their wide dynamic range, high sensitivity and fast kinetics have made them very popular tools. However, the dyes cannot be targeted to specific cells and dye loading can cause cytotoxicity. GECIs, on the other hand, provide an alternative to dyes that can be easily directed to any of subcellular compartments by simply fusing a signaling peptide to the GECI protein. Additionally, GECIs can be targeted to specific cell-types or tissues of a living organism and their expression level in target cells is stable from days to months, enabling extended time-lapse experiments.
[0004] Single-fluorescent protein GECIs possess a circularly permuted fluorescent protein whose fluorescence is modulated by calcium binding-dependent changes in the chromophore environment. One type of single-fluorescent protein GECI is GCamP, which is composed of a circularly permutated enhanced GFP (cpEGFP) moiety, a calcium-binding protein calmodulin (CaM), and a Ca2+-CaM-binding peptide referred to as M13 (Nakai et al., Nature Biotech, 2001, 19: 137-41 ; Akerboom et al., 2009, J Biol. Chem., 284:6455-64). The native EGFP has its chromophore located in the center of an 1 1 -strand β-barrel-like structure, which protects the chromophore from bulk solvent. In the cpEGFP domain of the GCamP sensor protein, the native N- and C- terminus of the EGFP are jointed together by a linker, and the new N- and C- terminus are generated by opening one of the β-sheets at the side of the barrel, leading to exposure of the chromophore to solvent. Ml 3 and the CaM domain are fused to the N- and C- terminus of the cpEGFP, respectively. In the Ca2+-binding form, the artificial opening on the barrel of the cpEGFP domain is partly occluded by the Ca -CaM/M13 complex, so that the chromophore is protected from solvent and is stabilized in the fluorescent, deprotonated form. In contrast, the apo-CaM domain will not associate with Ml 3 in the absence of calcium, leading to solvent-access mediated darkening of the cpEGFP chromophore.
[0005] While the development and use of GECIs such as GCamP have provided researchers with valuable tools to examine fluctuations in intracellular calcium ion concentrations, existing GECIs have serious drawbacks that limit their practical use, such as low signal-to-noise ratio, temperature sensitivity, suboptimal kinetics, nonlinearity and low photostability. In particular, low signal-to-noise ratio is a major drawback of GCamP and other GECIs. Dead or damaged cells interfere with the measurement of calcium-specific fluorescence signals. Since these cells frequently exhibit increased intracellular calcium levels as a consequence of necrosis or programmed cell death, their fluorescence can increase background fluorescence detection and lower the overall sensitivity of calcium-based assays utilizing GECIs.
[0006] What is needed, therefore, is a GECI protein that exhibits increased signal-to-noise ratios during measurement of intracellular calcium levels, particularly when an assay includes cells which are dead, dying, or damaged.
[0007] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.
BRIEF SUMMARY OF THE INVENTION
[0008] The invention provided herein discloses, inter alia, GCamP calcium sensor proteins which exhibit decreased fluorescence under the oxidative conditions typically observed in dead, dying, or damaged cells and that possess overall higher signal-to-noise ratios in comparison to unmodified GCamP calcium sensor proteins. The invention additionally provides methods for using GCamP calcium sensor proteins in assays whose goal is to screen for a compound capable of modulating (for example, increasing or decreasing) intracellular calcium concentrations.
[0009] Accordingly, provided herein are isolated nucleic acids comprising a nucleotide sequence encoding a calcium sensor protein comprising, from N terminus to C terminus, an Ml 3 domain, a circularly permuted green fluorescent protein domain, and a calmodulin (CaM) domain, wherein at least one pair of amino acid residues located at N77 and Y95, L134 and T201, T331 and T365, and/or T332 and E348 in the calcium sensor protein is/are replaced with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: l, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions. In another aspect, provided herein are vectors comprising any of the nucleic acids described herein. In still another aspect are isolated cells comprising any of the nucleic acids or vectors described herein. In one aspect, provided herein are non-human animals comprising any of the cells described herein. In another aspect, provided herein are tissue slices comprising any of the cells described herein.
[0010] In another aspect, provided herein are isolated calcium sensor proteins comprising an amino acid sequence comprising, from N terminus to C terminus, an Ml 3 domain, a circularly permuted green fluorescent protein domain, and a calmodulin (CaM) domain, wherein at least one pair of amino acid residues located at N77 and Y95, LI 34 and T201, T331 and T365, and/or T332 and E348 is/are replaced with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: 1, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
[0011] In yet another aspect, provided herein are methods for screening for an agent that is capable of increasing or decreasing intracellular calcium concentrations in a cell comprising: (i) contacting the agent with a cell expressing a calcium sensor protein encoded by any of the nucleic acids described herein; and (ii) determining a level of fluorescence, wherein an increase in fluorescence indicates that the agent is capable of increasing intracellular calcium concentrations and a decrease in fluorescence indicates that the agent is capable of decreasing intracellular calcium concentrations. [0012] In another aspect, provided herein is a kit comprising one or more of (i) one or more of the nucleic acids, such as a vector, described herein; (ii) one or more of the calcium sensor proteins described herein; (iii) one or more of the cells described herein; (iv) one or more of the non-human animals described herein; (v) and/or one or more of the tissue slices described herein.
[0013] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 depicts a schematic of the primary amino acid sequence of GCaMP2 illustrating the domain organization. Carets below the schematic show the positions of inter- domain linkers.
[0015] FIG. 2 depicts in vitro screening of redoxGCamP calcium sensor proteins. (A) 10 μΜ ionomycin or (B) 100 μΜ acetylcholine were added and the fluorescent change of the transfected HEK293 cells recorded. Fmax represents the maximum fluorescent signal; F0 represents the background fluorescent signal. (C) The fluorescent intensity of the crude lysates of the transfected HEK293 cells in MOPS buffer in the presence of 100 μΜ DTT or 100 μΜ H202, respectively. (D) The fluorescent change of the transfected HEK293 cells after 30 minutes' treatment of the cell death assay buffer in the presence or absence of ΙΟΟμΜ H202.
[0016] FIG. 3 depicts fluorescence excitation and emission spectra of the crude lysates of the transfected HEK293 cells (A-F: untransfected, GCamP, N77C/Y95C, L134C/T E201C, T331C/T365C, and T332C/E348C) in the presence of 2 mM CaCl2 or 10 mM EGTA,
respectively. The excitation spectra (300-500 nm) were detected at 520 ran, and the emission spectra (500-600 nm) were excited at 470 nm.
[0017] FIG. 4 depicts the results of in vitro calcium titration experiments for
characterization of redoxGCamP calcium sensor proteins.
[0018] FIG. 5 depicts the results of experiments measuring the fluorescent spectra of isolated GCamP proteins under variable calcium, reducing, and oxidative conditions. (A)
Represents the excitation spectrum detected at 525 nm while (B) represents the emission spectrum excited at 470 nm. [0019] FIG. 6 depicts the results of experiments measuring the fluorescent spectra of isolated N77C/Y95C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions. (A) Represents the excitation spectrum detected at 525 nm while (B) represents the emission spectrum excited at 470 nm.
[0020] FIG. 7 depicts the results of experiments measuring the fluorescent spectra of isolated L134C/T201 C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions. (A) Represents the excitation spectrum detected at 525 nm while (B) represents the emission spectrum excited at 470 nm.
[0021] FIG. 8 depicts the results of experiments measuring the fluorescent spectra of isolated T331C/T365C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions. (A) Represents the excitation spectrum detected at 525 nm while (B) represents the emission spectrum excited at 470 nm.
[0022] FIG. 9 depicts the results of experiments measuring the fluorescent spectra of isolated T332C/E348C redoxGCamP calcium sensor proteins under variable calcium, reducing, and oxidative conditions. (A) Represents the excitation spectrum detected at 525 nm while (B) represents the emission spectrum excited at 470 nm.
[0023] FIG. 10 depicts observed fluorescent intensity of HEK293 cells co-transfected with GCamP or redoxGCamP calcium sensor proteins and 5HT2a receptor.
DETAILED DESCRIPTION
[0024] This invention provides, inter alia, genetically-encoded calcium indicator (GECI) proteins capable of exhibiting significantly decreased fluorescence under oxidative conditions as well as methods for using and producing the same. The inventors have constructed a series of GCamP calcium sensor proteins possessing overall improved signal-to-noise ratios in comparison to GCamP calcium sensor proteins which are not sensitive to oxidative conditions. The inventors identified several sites for the substitution of cysteine residue pairs into the primary amino acid sequence of the GCamP chimeric protein. Without being bound to theory, it is believed that the thiol groups on those cysteine resides are susceptible to disulfide bond formation under oxidizing conditions and the formation of those disulfide bonds result in major conformational changes to the tertiary and/or quaternary structure of GCamP, thereby decreasing the protein's ability to fluoresce. However, under the reducing conditions typically observed within living cells, it is believed that the thiol groups of the substituted cysteines remain protonated, thus preventing any structural disruption of GCamP which could negatively impact fluorescence emission or excitation upon exposure to intracellular calcium.
[0025] These unique calcium sensor proteins can be targeted to specific cells or tissues in vitro or in a live organism for measurement of intracellular calcium levels. Furthermore, the calcium sensor proteins expressed in cell types that utilize intracellular calcium as a second messenger can be used to screen for chemical compounds capable of selectively altering intracellular calcium levels via interaction with, for example, a plasma membrane-bound protein receptor (such as, but not limited to, a G-protein coupled receptor).
I. General Techniques
[0026] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1 87); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994). Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, "Advanced Organic Chemistry Reactions, Mechanisms and Structure " 4th ed., John Wiley & Sons (New York, N.Y. 1992), and "Neuronal Calcium Sensor Proteins, " NOVA Publishers, (Philippov & Koch, eds., 2006) provide one skilled in the art with a general guide to many of the terms used in the present application.
II. Definitions
[0027] As used herein, the term "protein" includes polypeptides, peptides, fragments of proteins, and fusion proteins.
[0028] As used herein, an "isolated" molecule or cell (such as an isolated protein, an isolated nucleic acid or an isolated cell) is one which has been identified and separated and/or recovered from a component of its natural environment. [0029] The term "heterologous protein" means a protein derived from a different organism, species, or strain than the host cell. In some embodiments, a heterologous protein is not identical to a wild-type protein that is found in the same host cell in nature.
[0030] As used herein, a "nucleic acid" refers to two or more deoxyribonucleotides and/or ribonucleotides covalently joined together in either single or double-stranded form.
[0031] The term "recombinant nucleic acid" means a nucleic acid of interest that is free of one or more nucleic acids (e.g. , genes) which, in the genome occurring in nature of the organism from which the nucleic acid of interest is derived, flank the nucleic acid of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
[0032] By "heterologous nucleic acid" is meant a nucleic acid sequence derived from a different organism, species or strain than the host cell. In some embodiments, the heterologous nucleic acid is not identical to a wild-type nucleic acid that is found in the same host cell in nature. For example, a nucleic acid encoding a calcium sensor protein which is transformed in or integrated into the chromosome of a host cell is a heterologous nucleic acid.
[0033] As used herein, an "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid of interest. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. An expression control sequence can be "native" or heterologous. A native expression control sequence is derived from the same organism, species, or strain as the gene being expressed. A heterologous expression control sequence is derived from a different organism, species, or strain as the gene being expressed. An "inducible promoter" is a promoter that is active under environmental or developmental regulation.
[0034] By "operably linked" is meant a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. [0035] A "mutation" includes an amino acid deletion, an amino acid insertion, and an amino acid substitution of at least one amino acid into a defined primary amino acid sequence. In some aspects, mutation of one or more amino acids in a primary amino acid sequence can result in the protein encoded by that amino acid sequence having altered activity or expression levels within a cell. In other aspects, mutation of one or more amino acids (such as a conservative mutation) in a primary amino acid sequence may not result in the protein encoded by that amino acid sequence having substantial changes in activity or expression levels within a cell.
[0036] An amino acid "substitution" means that at least one amino acid component of a defined primary amino acid sequence is replaced with another amino acid (for example, a cysteine residue). In some aspects, substitution of one or more amino acids in a primary amino acid sequence can result in the protein encoded by that amino acid sequence having altered activity or expression levels within a cell. In other aspects, substitution of one or more amino acids (such as a conservative substitution) in a primary amino acid sequence may not result in the protein encoded by that amino acid sequence having substantial changes in activity or expression levels within a cell.
[0037] As used herein, "percent (%) amino acid sequence identity" with respect to a protein or amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific protein or amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
[0038] As used herein, "percent (%) nucleotide sequence identity" with respect to a DNA or RNA sequence refers to the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the specific DNA or RNA sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
[0039] A "chimeric protein" is a protein comprising one or more portions derived from one or more different proteins, for example, a GCamP chimeric protein. Chimeric proteins may be produced by culturing a recombinant cell transfected with a nucleic acid that encodes the chimeric protein.
[0040] A "G-protein coupled receptor (GPCR)" refers to any member of a superfamily of receptors that mediates signal transduction by coupling with a G protein. One example of a class of GPCR which influences cytosolic calcium levels works through the Gq type of G proteins, which activate a phospholipase C (PLC) pathway, resulting in the hydrolysis of
phosphoinositides to generate two classes of different second messengers, namely, diacylglycerol and inositol phosphates. Diacylglycerol, in turn, activates certain protein kinase Cs (PKCs) while inositol phosphates (such as, but not limited to, IP3) stimulate the mobilization of calcium from intracellular stores such as the endoplasmic reticulum, the sarcoplasmic reticulum (for muscle cells), and/or the mitochondria.
[0041] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0042] As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.
[0043] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
III. Compositions of the Invention A Proteins
[0044] In some aspects, provided herein are isolated calcium sensor proteins comprising an Ml 3 domain, a circularly permuted green fluorescent protein (cpGFP) domain, and a calmodulin (CaM) domain, wherein at least one pair of cysteine residues are added to the primary amino acid sequence of the calcium sensor protein, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions. In this context, the term "circularly permuted" ("cp") means that the native GFP's N- and C-terminal ends are joined together (such as joined together by an amino acid linker sequence) and new N- and C-terminal ends are generated by cleavage of two peptide bonds in the GFP amino acid sequence. As can be observed in the schematic shown in Figure 1 , the cpGFP domain of the calcium sensor protein comprises a first cpGFP domain (the native GFP's former C-terminal end) and a second cpGFP domain (the native GFP's former N-terminal end).
[0045] Accordingly, in one aspect, the cpGFP domain comprises the first cpGFP domain comprising amino acid residues 149-238 of the amino acid sequence shown in SEQ ID NO: l 1 and the second cpGFP domain comprising amino acid residues 1 -144 of the amino acid sequence shown in SEQ ID NO: 1 1. In one embodiment, the cpGFP domain comprises the first cpGFP domain comprising amino acid residues 148-237 of the amino acid sequence shown in SEQ ID NO: 12 and the second cpGFP domain comprising amino acid residues 1 -143 of the amino acid sequence shown in SEQ ID NO: 12. See amino acid sequence shown in Tsien, Annu. Rev.
Biochem., 1998, 67:509-44 at pg. 513, the disclosure of which is incorporated by reference herein in its entirety. In one embodiment, the cpGFP domain comprises the first cpGFP domain comprising amino acid residues 148-237 of the amino acid sequence shown in SEQ ID NO: 13 and the second cpGFP domain comprising amino acid residues 1 -143 of the amino acid sequence shown in SEQ ID NO: 13 (Tsien, Annu. Rev. Biochem., 1998, 67:509-44 at pg. 513).
[0046] In another embodiment, the calcium sensor protein domains are arranged so that the Ml 3 domain is located C-terminal to the CaM domain and the cpGFP domain. In some embodiments,, the domains of the calcium sensor protein can be arranged so that the Ml 3 domain is located N-terminal to the cpGFP domain and the CaM domain. In other embodiments, the GFP of the calcium sensor protein is circularly permuted at any location between amino acid residues 135-155 of the amino acid sequence shown in SEQ ID NO: l 1 , 12, or 13. In a particular embodiment, the GFP is circularly permuted at amino acid residue position 144 of the amino acid sequence shown in SEQ ID NO: 11.
[0047] In some embodiments, the isolated calcium sensor protein further comprises an optional tag (such as a purification tag). Protein purification tags for isolation and purification of expressed proteins are numerous and commonly used in the art. Non-limiting examples include a His tag, a maltose binding protein tag, or a glutathione tag. In one embodiment, the protein purification tag is a histidine tag comprising at least six histidine residues. In another
embodiment, the protein tag contains a protease recognition site so that the protein purification tag can be removed from the rest of the calcium sensor protein.
[0048] In some embodiments, a linker can be inserted into the primary amino acid sequence of the cpGFP domain at the site of circular permutation. In some embodiments, the linker at the site of circular permutation is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In one embodiment, the linker at the site of circular permutation is 6 amino acids in length. In yet another embodiment, the linker at the site of circular permutation comprises the amino acid sequence: G G T G G S (SEQ ID NO: 14). In another embodiment, a linker can be inserted between the optional protein purification tag and the Ml 3 domain. In some
embodiments, the linker between the optional protein purification tag and the Ml 3 domain is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In one embodiment, the linker between the optional protein purification tag and the Ml 3 domain is 3 amino acids in length. In yet another embodiment, the linker between the optional protein purification tag and the Ml 3 domain comprises the amino acid sequence: M V D. In yet another embodiment a linker can be inserted between the Ml 3 domain and the first cpGFP domain. In some embodiments, the linker between the Ml 3 domain and the first cpGFP domain is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In other embodiments, the linker between the Ml 3 domain and the first cpGFP domain is 2 amino acids in length. In yet another embodiment, the linker between the Ml 3 domain and the first cpGFP domain comprises the amino acid sequence: L E. In another embodiment, a linker can be inserted between the second cpGFP domain and the CaM domain. In some embodiments, the linker between the second cpGFP domain and the CaM domain is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In other embodiments, the linker between the second cpGFP domain and the CaM domain comprises the amino acid sequence: TR.
[0049] In some aspects, the addition of at least one pair of cysteine residues to the primary amino acid sequence of the calcium sensor protein can take the form of either inserting a pair of cysteine residues into the primary amino acid sequence or substituting a pair of residues in the primary amino acid sequence with cysteine residues. In some embodiments, any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pairs of cysteine residues are inserted into or replace at least two amino acids within the primary amino acid sequence of the calcium sensor protein. In another embodiment, the one or more inserted or replaced pairs of cysteine residues are located in the cpGFP domain of the calcium sensor protein. In still another embodiment, the one or more inserted or replaced pairs of cysteine residues are located in the CaM domain of the calcium sensor protein. In another embodiment, the one or more inserted or replaced pairs of cysteine residues are located in the Ml 3 domain of the calcium sensor protein. In other embodiments, one member of the at least one or more inserted or replaced pairs of cysteine residues is located in the cpGFP domain while the other member is located in the CaM domain. In another embodiment, one member of the at least one or more inserted or replaced pairs of cysteine residues is located in the cpGFP domain while the other member is located in the Ml 3 domain. In yet another embodiment, one member of the at least one or more inserted or replaced pairs of cysteine residues is located in the CaM domain while the other member is located in the Ml 3 domain.
[0050] In some aspects, at least two residues in the primary amino acid sequence of the calcium sensor protein are substituted with at least two cysteine residues. In one embodiment, any combination of one or more pairs of amino acid residues from the primary amino acid sequence of the calcium sensor protein selected from the group consisting of N77/Y95,
L134/T201, T365/T332, and T332/E348, are substituted with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: 1.
[0051] In some aspects, the calcium sensor protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: l, 2, 3, 4, or 5. In another aspect, the calcium sensor protein comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO:l, 2, 3, 4, or 5. In one embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:2. In another embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 3. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:3. In yet another embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 4. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:4. In still another embodiment, the calcium sensor protein comprises the amino acid sequence of SEQ ID NO: 5. In another embodiment, the calcium sensor protein comprises amino acid residues 36-451 of SEQ ID NO:5. In another embodiment, the sensor protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent amino acid sequence identity to any of the calcium sensor proteins disclosed in Souslova et al., BMC Biotechnology, 2007, 7:37, the contents of which are incorporated by reference herein in its entirety.
[0052] In some embodiments, the calcium sensor protein comprises an insertion of one or more cysteine residue pairs within about 5 amino acid residues of N77/Y95, L134/T201,
T365/T332, and/or T332/E348, wherein the amino acid residue position corresponds to the position in SEQ ID NO: 1
[0053] Modifications of proteins, including specific amino acid substitutions or insertions, are made by methods that are well known in the art. For example, modifications can be made by site-specific mutagenesis of nucleotides in the polynucleotides encoding the calcium sensor protein, thereby producing DNA encoding the modification, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitutions at predetermined sites in DNA having a known sequence are also well known in the art, for example primer mutagenesis and PCR mutagenesis.
[0054] Calcium sensor proteins described herein may include conservative substitutions.
Conservative substitutions are shown in the "Table of Amino Acid Substitutions" below under the heading of "preferred substitutions."
Amino Acid Substitutions
Figure imgf000014_0001
Figure imgf000015_0001
[0055] In some aspects, the calcium sensor proteins disclosed herein exhibit reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions. In some embodiments, the amount of fluorescence under oxidative conditions is reduced by at least any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%>, 60%, 65%, 70%, or 75%, inclusive, including any percentages in between these values in comparison to the amount of fluorescence produced under reducing conditions. In other embodiments, the calcium sensor proteins disclosed herein exhibit a higher signal-to-noise ratio in comparison to calcium sensor proteins (for example, unmodified GCaMP calcium sensor proteins) which do not comprise at least one or more pairs of cysteine residues added to the primary amino acid sequence of the calcium sensor protein. In some embodiments, the signal-to- noise ratio is improved by at least any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%), 65%, 60%, 65%, 70%, or 75%, inclusive, including any percentages in between these values in comparison to the signal-to-noise ratio of unmodified calcium sensor proteins (for example, unmodified GCaMP calcium sensor proteins). In some embodiments the oxidative conditions are those conditions within the cytoplasm of damaged, dead, or dying cells. In other embodiments, the reducing conditions are those conditions within the cytoplasm of living cells or cells that are not substantially damaged.
[0056] In some aspects, any of the calcium sensor protein described above further comprise one or more protein tags (for example, a signal peptide) which is capable of targeting the protein to a particular cellular compartment. In some embodiments, the calcium sensor protein further comprises a mitochondrial import signal peptide. The mitochondrial import signal peptide can target the protein to either the mitochondrial intermembrane space (for example, the tag can be any peptide sequence capable of binding to a Translocase of Inner Membrane (TIM) pore and/or a Translocase of Outer Membrane (TOM) pore in the mitochondrial membrane) or the mitochondrial matrix (for example, the tag can comprise one or more positively charged amino acids and/or one or more hydroxylated amino acids). In another embodiment, the calcium sensor protein further comprises an endoplasmic reticulum (ER) retention signal. In one embodiment, the ER retention signal is a non-cleavable K D E L amino acid sequence. In another embodiment, the calcium sensor protein further comprises a peroxisome targeting signal. The peroxisome targeting signal can be Peroxisome targeting signal 1 (PTS1) in some
embodiments, comprising a C-terminal tripeptide with a consensus sequence of (S/A/C)-(K/R/H)- (L/A). In another embodiment, the PTS1 signal is S K L. In other embodiments, the peroxisome targeting signal can be Peroxisome targeting signal 2 (PTS2), comprising a nonapeptide located near the N-terminus with a consensus sequence of (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F) (where X can be any amino acid) (SEQ ID NO: 15).
B. Nucleic acids and vectors
[0057] Provided herein are isolated nucleic acids that encode any of the proteins disclosed herein. The disclosure provides isolated, synthetic, or recombinant polynucleotides comprising a nucleic acid sequence having at least about 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequence identity to the nucleic acid of SEQ ID NOs: 6, 7, 8, 9, or 10. In another embodiment, the disclosure provides isolated, synthetic, or recombinant polynucleotides comprising a nucleic acid sequence having at least about 70%, e.g., at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequence identity to nucleotides 106-1356, of SEQ ID NOs: 6, 7, 8, 9, or 10.
[0058] The disclosure specifically provides a nucleic acid encoding a calcium sensor protein. For example, the disclosure provides an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes: (1) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:l ; (2) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO: 1 ; (3) a protein comprising an amino acid sequence with at least 85%, 86%, 87%>, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2; (4) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO: 2; (5) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3; (6) a protein comprising an amino acid sequence with at least 85%, 86%>, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO: 3; (7) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%), 99% or 100%) sequence identity to the amino acid sequence represented by SEQ ID NO:4; (8) a protein comprising an amino acid sequence with at least 85%), 86%, 87%), 88%), 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO: 4; (9) a protein comprising an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100% sequence identity to the amino acid sequence represented by SEQ ID NO:5; or (10) a protein comprising an amino acid sequence with at least 85%), 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to amino acid residues 36-451 of SEQ ID NO: 5.
[0059] The disclosure also provides cassettes and/or vectors comprising the above- described nucleic acids. Suitably, the nucleic acid encoding a calcium sensor protein of the disclosure is operably linked to a promoter. Promoters are well known in the art and any promoter that functions in the host cell can be used for expression of the calcium sensor proteins of the present disclosure. Initiation control regions or promoters, which are useful to drive expression of a calcium sensor protein in a specific cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these nucleic acids can be used.
[0060] Also provided herein are vectors comprising the polynucleotides disclosed herein encoding a calcium sensor protein. Suitable vectors are those which are compatible with the host cell employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Suitable vectors can be maintained in low, medium, or high copy number in the host cell. In some embodiments, the vector is an expression vector. Protocols for obtaining, maintaining, and using such vectors are known to those in the art {see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989). The vectors that can used according to the present invention also include vectors comprising a polynucleotide which encodes an RNA {e.g., an mRNA) that when transcribed from the polynucleotides of the vector will result in the
accumulation of calcium sensor proteins in the cytoplasm of host cells. Vectors which may be used include, without limitation, lentiviral, HSV, and adenoviral vectors. Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV and EIAV. Lentiviruses may be pseudotyped with the envelope proteins of other viruses, including, but not limited to VSV, rabies, Mo-MLV, baculovirus and Ebola. Such vectors may be prepared using standard methods in the art.
[0061] In some embodiments, the vector is a recombinant AAV vector. AAV vectors are
DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and
characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.
[0062] The application of AAV as a vector for gene therapy has been rapidly developed in recent years. Wild-type AAV could infect, with a comparatively high titer, dividing or non- dividing cells, or tissues of mammal, including human, and also can integrate into in human cells at specific site (on the long arm of chromosome 19) (Kotin, R. M., et al, Proc. Natl. Acad. Sci. USA 87: 2211-2215, 1990) (Samulski, R. J, et al, EMBO J. 10: 3941-3950, 1991 the disclosures of which are hereby incorporated by reference herein in their entireties). AAV vector without the rep and cap genes loses specificity of site-specific integration, but may still mediate long-term stable expression of exogenous genes. AAV vector exists in cells in two forms, wherein one is episomic outside of the chromosome; another is integrated into the chromosome, with the former as the major form. Moreover, AAV has not hitherto been found to be associated with any human disease, nor any change of biological characteristics arising from the integration has been observed. There are sixteen serotypes of AAV reported in literature, respectively named AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVIO, AAV1 1, AAV 12, AAV13, AAV 14, AAV 15, and AAV 16, wherein AAV5 is originally isolated from humans (Bantel-Schaal, and H. zur Hausen. 1984. Virology 134: 52-63), while AAVl-4 and AAV6 are all found in the study of adenovirus (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Virol. 1999, 73: 939-947).
[0063] AAV vectors may be prepared using standard methods in the art. Adeno- associated viruses of any serotype are suitable (See, e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J. R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P. Tattersall "The Evolution of Parvovirus Taxonomy" In Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski "The Genus Dependovirus" (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) pi 5-23, Hudder Arnold, London, UK (2006), the disclosures of which are hereby incorporated by reference herein in their entireties). Methods for purifying for vectors may be found in, for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 and WO/1999/01 1764 titled "Methods for Generating High Titer Helper-free
Preparation of Recombinant AAV Vectors", the disclosures of which are herein incorporated by reference in their entirety. Preparation of hybrid vectors is described in, for example, PCT
Application No. PCT/US2005/027091, the disclosure of which is herein incorporated by reference in its entirety. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., International Patent Application Publication Nos.: WO 91/18088 and WO 93/09239; U.S. Patent Nos: 4,797,368, 6,596,535, and 5,139,941 ; and European Patent No: 0488528, all of which are herein incorporated by reference in their entireties). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co- transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes {rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques.
[0064] In some embodiments, the vector(s) for use in the methods of the invention are encapsidated into a virus particle (e.g. AAV virus particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV 12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the invention includes a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.
[0065] For the animal cells described herein, it is understood that one or more vectors may be administered to any of the host cells described herein. If more than one vector is used, it is understood that they may be administered at the same or at different times to the host cell.
C. Host cells
[0066] In some aspects, isolated cells are provided herein comprising any of the proteins, nucleic acids, and/or vectors described above. In one embodiment, the isolated cell is a prokaryotic cell or a eukaryotic cell. In another embodiment, the isolated cell is an invertebrate cell or a vertebrate cell. In some embodiments, the cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a nematode cell, an insect cell, a fish cell, a plant cell, an avian cell, an animal cell, and a mammalian cell.
[0067] Examples of cells capable of expressing a calcium sensor protein include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as those in the genera Synechocystis,
Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena,
Thiobacillus, Methanobacterium and Klebsiella. In one embodiment, the cell is a yeast cell selected from the group consisting of a Saccharomyces, a Pichia, and a Candida. In another embodiment, the cell is a Caenorhabdus elegans nematode cell. In another embodiment, the cell is an insect cell, such as a Drosophila cell. In still another embodiment, the cell is a zebrafish cell. In another embodiment, the cell is a sea urchin cell. In another embodiment, the cell is an amphibian cell, such as, but not limited to, aXenopus cell, for example, aXenopus oocyte.
[0068] Examples of mammalian cells capable of expressing a calcium sensor protein can be selected from the group consisting of a hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a porcine cell, an equine cell, a sheep cell, a monkey cell, a chimpanzee cell, and a human cell. In another embodiment, the animal cell is a neural cell (such as, but not limited to, a peripheral nervous system cell or a central nervous system cell), a muscle cell (such as a cardiac, skeletal, or smooth muscle cell), a gamete (such as a sperm cell or an oocyte), a cancer cell, an immune cell (such as, but not limited to, a macrophage, a T-cell, or a B-cell), a stem cell (such as, but not limited to, an embryonic stem cell or an adult stem cell), or an endocrine cell (such as, but not limited to, a thyroid cell, a hypothalamic cell, a pituitary cell, an adrenal cell, a testicular cell, an ovarian cell, a pancreatic cell (such as a β cell), a stomach cell, or an intestinal cell). In some embodiments, the cell is a human cell in cell culture. In other embodiments, the cell is a non-human cell in cell culture. In still other embodiments, the cell is a cancer cell.
[0069] In some embodiments, the cell is a Hela cell, a KEK293 cell, a Chinese hamster ovary (CHO) cell, a Jurkat T cell, a neuroblastoma cell, or a human embryonic kidney cell (HEK) cell (such as a HEK293 cell or a HEK293T cell).
[0070] Suitable cell culture media and conditions vary with the specific cell type to be cultured but generally consist of an isotonic, buffered, basal nutrient medium which provides an energy source, coupled with inorganic salts, amino acids, vitamins and various supplements. For culturing mammalian cells, supplements may include serum (e.g., fetal calf serum, or the like) various antibiotics to prevent contamination or to provide selective conditions, attachment and growth factors, or the like. A number of media formulations are known in the art, such as, but not limited to, minimal essential medium (MEM), Rosewell Park Memorial Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM). Suitable culture conditions for many cell typesare also known in the art (See, e.g., Morgan et al. 1993, Animal Cell Culture, BIOS
Scientific Publishers Ltd., Oxford, UK, and Adams, 1990, Cell Culture for Biochemists, 2nd ed, Elsevier.
[0071] Transformation of cells is done using standard techniques which are known in the art and which are appropriate to the particular cell type. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector), by transfecting a plasmid into a cell via the use of an anionic lipid or by electroporation, or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,912,040, 4,740,461 , and 4,959,455 (which are hereby incorporated herein by reference in their entireties). In certain embodiments, the transformation procedure used may depend upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei.
D. Additional host cell plasma membrane proteins
[0072] In some aspects, the cells described herein can further comprise one or more membrane proteins whose function is responsible for either increasing or decreasing intracellular calcium as a second messenger. Calcium ions are one of the most widespread second messengers used in cellular signal transduction. Calcium is transported into the intracellular environment either from outside the cell via calcium channels or from internal calcium storage sites such as the endoplasmic reticulum and the mitochondria. Levels of intracellular calcium are regulated by transport proteins that remove it from the cell. For example, the plasma membrane Ca ATPase (PMCA) pumps calcium out of the cell by in an ATP-dependent process. In neurons, voltage- dependent, calcium-selective ion channels are important for synaptic transmission through the release of neurotransmitters into the synaptic cleft by vesicle fusion of synaptic vesicles.
Therefore, it is through one or more membrane proteins (either the cell plasma membrane or the membranes of intracellular organelles) that intracellular calcium's role as a second messenger is mediated by facilitating increases or decreases in the ion's concentration in the cytoplasm at any given time.
[0073] In some aspects, therefore, cells expressing a calcium sensor protein can further comprise one or more G protein-coupled receptors (GPCRs). GPCRs (also known as seven- transmembrane domain receptors) comprise a large family of transmembrane receptor proteins (representing about 5% of the total genome of humans) that bind to molecules present in the extracellular environment and are capable of triggering signal transduction cascades within the cell and, ultimately, cellular responses. GPCRs are found only in eukaryotes, including yeast, choanoflagellates, and animals. The molecules that bind and activate these receptors include, but are not limited to, light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins.
[0074] GPCRs include, but are not limited to, Gq protein or Gq/n, alpha- 1 adrenegic receptors (al-AR), urotensin (UT) receptors, 5-HT2 and 5-HT6 serotonin receptors, hypocretic (orexin) receptors, histamine HI receptors, bradykinin Bl and B2 receptors, bombesin BB2 receptors, P2Y purinergic receptors, acetycholine receptors (e.g., Ml, M3 and M5), mGluR5 glutamate receptors, vasopressin V2 and VI receptors, angiotensin AGTR1 receptors,
cholecystokinin CCKAR and CCKBR receptors, endothelin ENDRA receptors, ghrelin GHSRla receptors, melatonin MTNRl A receptors, neurotensin NTSRl receptors, platelet-activating factor PTAFR receptors, luteinizing hormone receptors (LHRs), follicle stimulating hormone receptors (FSHRs), gonadotrophic releasing hormone receptors (GnRHRs), and prolactin releasing peptide receptor PRLHR receptors. In some embodiments, the GPCR is endogenously expressed in the cell expressing the calcium sensor protein. In other embodiments, the GPCR is heterologously expressed in the cell expressing the calcium sensor protein.
[0075] With particular relevance to intracellular calcium levels in many cells is the phosphatidylinositol signaling pathway. In this case, an extracellular signal molecule binds to a Gq GPCR on a cell's surface which leads to the activation of a phospholipase C (PLC; such as, but not limited to, PLCp, PLCy, PLC6, PLCE, PLCC, and PLCr]) located on the plasma membrane and that is associated with Gq. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers: Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). In turn, IP3 binds to a receptor protein present in the membrane of the smooth endoplasmic reticulum (SER) and mitochondria, which results in the release of the calcium ions stored within these organelles into the cytoplasm, thus dramatically increasing intracellular calcium levels.
[0076] In some aspects, cells expressing a calcium sensor protein can further comprise one or more calcium channel proteins. A calcium channel is an ion channel which displays selective permeability to calcium ions. In mammalian biology, there are two types of calcium channel proteins: voltage-dependent calcium channels and ligand-gated calcium channels.
[0077] In some embodiments, the calcium channel protein is a voltage-dependent calcium channel. These types of channels are found in excitable cells (non-limiting examples include: muscle, glial, and neuronal cells) and are permeable to calcium ions. Additionally, these channels are slightly permeable to sodium ions, leading to them also being known as Ca2+-Na+ channels. However, their permeability to calcium is around 1000-fold greater compared to sodium ions under normal physiological conditions (Hall, Guyton and Hall Textbook of Medical Physiology with Student Consult Online Access, 2011, (12th ed; Philadelphia: Elsevier Saunders), p. 64). At physiologic or resting membrane potential, these channels are normally closed. They are activated in response to a depolarized membrane potential causing calcium entry into the cell resulting, depending on the cell type, in muscular contraction, excitation of neurons, up- regulation of gene expression, release of hormones or neurotransmitters, or cell motility. Voltage- dependent calcium channels are formed as a complex of several different subunits: als α2δ, βι-4, and γ. The ] subunit forms the ion conducting pore while the associated subunits have several functions including modulation of gating. Voltage-dependent calcium channels include, but are not limited to, L-type calcium channels (having, for example, a Cavl .1, Cavl .2, Cavl .3,or Cavl .4 cti subunit and associated α2δ, β, and/or γ subunits), P-type calcium channels (having, for example, a Cav2.1 aj subunit and associated α2δ, β, γ subunits), N-type calcium channels (having, for example, a Cav2.1 \ subunit and associated α2δ/βι, β3, β4> γ subunits), R-type calcium channels (having, for example, a Cav2.3 aj subunit and associated α2δ, β, γ subunits) or T-type calcium channels (having, for example, a Cav3.1, Cav3.2 ,or Cav3.3 j subunit). In some embodiments, the voltage-dependent calcium channel is endogenously expressed in the cell expressing the calcium sensor protein. In other embodiments, the voltage-dependent calcium channel is heterologously expressed in the cell expressing the calcium sensor protein.
[0078] In other embodiments, the channel sensor protein is a ligand-gated calcium channel. Ligand-gated calcium channels include, but are not limited to, the P2X receptor, the inositol 1,4,5-trisphosphate (IP3) receptor, one or more Ryanodine receptors, one or more two pore channels, one or more cation channels of sperm (Catsper channels), or one or more plasma membrane store-operated channels. In some embodiments, the ligand-gated calcium channel is endogenously expressed in the cell expressing the calcium sensor protein. In other embodiments, the ligand-gated calcium channel is heterologously expressed in the cell expressing the calcium sensor protein.
[0079] In some aspects, cells expressing a calcium sensor protein can further comprise one or more calcium pump proteins. Ordinarily in most cells, intracellular calcium
concentrations are maintained at relatively low levels in comparison to the concentration of calcium within intracellular organelles and in the extracellular environment. Calcium pump proteins are membrane proteins which pump intracellular calcium against its concentration gradient and into organelles such as mitochondria or the endoplasmic reticulum. Calcium pump proteins include, but are not limited to, a plasma membrane Ca ATPase (PMCA) pump (including any of PMC A 1 , PMC A2, PMC A3, or PMCA4 isoforms) and the sodium-calcium antiporter protein (a.k.a. the Na+/Ca2+ exchanger or NCX) which removes a single calcium ion in exchange for the import of three sodium ions. In some embodiments, the calcium pump protein is endogenously expressed in the cell expressing the calcium sensor protein. In other
embodiments, the calcium pump protein is heterologously expressed in the cell expressing the calcium sensor protein.
E. Non-human animals
[0080] Also provided herein, in one aspect, are non-human animals comprising any of the cells described herein or any of the calcium sensor proteins described herein. Non-human animals are useful for studying the effects of and identifying compounds and agents capable of altering intracellular calcium concentrations in a live organism. As used herein, a "non-human animal" can include a mammal, a bird, a fly, a fish, a nematode worm, an amphibian, or a yeast. In some embodiments, the non-human animal is a mammal selected from the group consisting of a rodent (such as a rat or mouse in which one or more of the cells include a transgene encoding any of the calcium sensor proteins described herein), a non-human primate, a sheep, a dog, a cow, or a goat. In some embodiments, the non-human animal is a transgenic animal. As used herein, a "transgene" is exogenous DNA (such as a DNA encoding any of the calcium sensor proteins described herein) that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal. Transgenes preferably direct the expression of an encoded gene product in one or more specific cell types or tissues of the transgenic animal. Methods for creating transgenic animals are routine and well known in the art (see, e.g., U.S. Pat. Nos. 4,870,009, 4,736,866, and 4,873,191, the contents of which are incorporated by reference).
[0081] Also provided herein are tissue slices obtained from non-human animals expressing any of the calcium sensor proteins described herein in one or more cell types or tissues. In some embodiments, the tissue slices are selected from the group consisting of central nervous system tissue slices (including brain and spinal cord slices), peripheral nervous system slices, muscle tissue slices (such as cardiac, skeletal, or smooth muscle tissue), endocrine tissue slices (such as tissue containing endocrine hormone-producing cells or glands), ovarian tissue slices, testicular tissue slices, bone marrow tissue slices, and tissue slices from cancerous tumors.
Methods of the Invention [0082] Because of the important role intracellular calcium plays in mammalian cellular physiology and because of the sheer number of membrane proteins capable of modulating cytosolic concentrations of this cation (for example, the myriad of GPCRs encoded in
mammalian genomes), tools permitting accurate and real time measurement of intracellular calcium in cells are of great use in the search for agents capable of modulating cytosolic Ca levels. Accordingly, provided herein are methods of screening for an agent that is capable of increasing or decreasing intracellular calcium concentrations in a cell comprising contacting the agent with a cell expressing a calcium sensor protein encoded by any of the nucleic acids described herein and determining a level of fluorescence, wherein an increase in fluorescence indicates that the agent is capable of increasing intracellular calcium concentrations and a decrease in fluorescence indicates that the agent is capable of decreasing intracellular calcium concentrations.
[0083] In some aspects of the method provided herein, the cell expresses a calcium sensor protein such as those disclosed herein. In some embodiments, the cell can be selected from the group consisting of an animal cell, a bacterial cell, an insect cell, a nematode cell, and a yeast cell. In other embodiments, the animal cell is a human cell or a non-human cell. In one embodiment, the human cell is in a cell culture. In yet another embodiment, the non-human cell is selected from the group consisting of a rodent cell, a mouse cell, a rat cell, and a non-human primate cell. In still another embodiment, the non-human cell is in a cell culture or in a non-human animal. In some embodiments, the animal cell is a muscle cell, a gamete, a neural cell, a cancer cell, or an endocrine cell. In further embodiments, the muscle cell is selected from the group consisting of smooth muscle, skeletal muscle, and cardiac muscle. In another embodiment, the cell is in a non- human animal, such as any of the non-human animals described herein. In yet another
embodiment, the cell is in a tissue slice, such as any of the tissue slices described herein.
[0084] In some aspects of the methods provided herein, the cells further comprise one or more membrane proteins selected from the group consisting of a GPCR (such as any of the GPCRs described herein), a receptor tyrosine kinase, an ion channel protein (such as any of the calcium ion channel proteins described herein), or an ion pump protein (such as any of the calcium ion pump proteins or antiporters described herein). In some embodiments, the one or more membrane proteins are endogenously expressed in the cell expressing the calcium sensor protein. In other embodiments, the one or more membrane proteins are heterologously expressed in the cell expressing the calcium sensor protein. A. Assays utilizins calcium sensor proteins
[0085] In another aspect, cells in culture expressing any of the calcium sensor proteins described herein or non-human animals and/or tissue slices comprising cells expressing any of the calcium sensor proteins described herein can be used to screen for agents (for example, small molecules) that regulate the function of cell plasma membrane proteins (such as, but not limited to, a GPCR, an ion channel, or an ion pump) which directly or indirectly modulate intracellular calcium levels within living cells. Any agent known in the art can be tested for its ability to modulate (for example, increase or decrease) intracellular calcium concentrations. For identifying an agent that modulates activity, candidate compounds can be directly provided to a cell expressing any of the calcium sensor proteins described herein and/or one or more receptor (such as a GPCR), calcium channel, and/or calcium pump proteins disclosed herein.
[0086] In some aspects, the methods described herein can be used to screen chemical libraries for molecules which modulate, e.g., increase or decrease, the intracellular concentration of calcium ions. The chemical libraries can be peptide libraries, peptidomimetic libraries, chemically synthesized libraries, recombinant (e.g., phage display) libraries, in vitro translation- based libraries, and other non-peptide synthetic organic libraries. The methods described herein may also be used to screen endogenous candidate compounds comprising biological materials (including, but not limited to, plasma and tissue extracts) and to screen libraries of endogenous compounds known to have biological activity.
[0087] In some aspects, the methods described herein are high-throughput methods for screening agents that are capable of increasing or decreasing intracellular calcium concentration. In some embodiments, direct identification of candidate agents is conducted in conjunction with agents generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. The candidate agent may be a member of a chemical library. This may comprise any convenient number of individual members, for example tens to hundreds to thousands to millions of suitable compounds, for example peptides, peptoids and other oligomeric compounds (cyclic or linear), and template-based smaller molecules (e.g.,
benzodiazepines, hydantoins, biaryls), carbocyclic and polycyclic compounds (e.g., naphthalenes, phenothiazines, acridines, steroids), carbohydrate and amino acid derivatives, dihydropyridines, benzhydryls, heterocycles (e.g., trizines, indoles, thiazolidines). The numbers quoted and the types of compounds listed are illustrative, but not intended to be limiting. Preferred chemical libraries comprise chemical compounds of low molecular weight and potential therapeutic agents. In another embodiment, combinatorial chemistry can be used to identify modulators of the intracellular calcium concentration using the methods disclosed herein. Combinatorial chemistry is capable of creating libraries containing hundreds of thousands of compounds, many of which may be structurally similar. While high throughput screening programs are capable of screening these vast libraries for affinity for known targets, newer approaches have been developed that achieve libraries of smaller dimension but which provide maximum chemical diversity. {See e.g., Matter, 1997 ', Journal of 'Medicinal Chemistry, 40: 1219-1229).
[0088] In other aspects, the agent is an antibody, a protein (for example, a polypeptide hormone) or any combination thereof. In another aspect, the agent is an inhibitory nucleic acid selected from the group consisting of a triplex forming oligonucleotide, an aptamer, a ribozyme, a short interfering RNA (siRNA), an antisense oligonucleotide, and a micro-RNA (miRNA).
[0089] In another aspect, cells in culture expressing any of the calcium sensor proteins described herein or non-human animals and/or tissue slices comprising cells expressing any of the calcium sensor proteins described herein can be used to investigate the effects of intracellular calcium levels on protein function. These protein functions can include, for example, the response of other calcium sensor proteins to changes in cytosolic calcium, the behavior of calcium dependent enzymes (for example, CaM Kinase enzymes) when bound to calcium, or the behavior of calcium pumps, and/or calcium ion channels to regulate intracellular calcium ion concentrations. Additional aspects include using the cells, non-human animals, and/or tissue slices expressing any of the calcium sensor proteins described herein to investigate the effects of cytosolic calcium concentration on signaling pathways (such as, but not limited to, the phospholipase C pathway, the protein kinase C pathway, and/or the phosphoinositide 3-kinase pathway) or cell status (such as, but not limited to, cell growth, proliferation, transcription, metabolism, exocytosis, motility, nerve depolarization, muscle contraction, fertilization, apoptosis, and/or necrosis).
[0090] In other aspects, neural cells in culture expressing any of the calcium sensor proteins described herein or neural tissue slices comprising cells expressing any of the calcium sensor proteins described herein can be used to detect the effects of a neuromodulator (for example, an agent that depolarizes a neuron) on a connection between a first neuron and a second neuron or a plurality of neurons forming a circuit. Administering a neuromodulator (for example, an agent that depolarizes a neuron) to the first neuron in the neural tissue slice to modulate (for example, increase or decrease) the eliciting of one or more action potential(s) in the second neuron or plurality of neurons forming a circuit can be optically detecting by monitoring the change in intensity of the fluorescence produced by the calcium sensor proteins in the second neuron or plurality of neurons forming a circuit.
B. Fluorescence detection assays
[0091] In some aspects of any of the methods provided herein, the fluorescence of cells transformed or transfected with a DNA construct encoding any of the calcium sensor proteins disclosed herein may suitably be measured by optical means by, for example, a
spectrophotometer, a fluorimeter, a fluorescence microscope, a cooled charge-coupled device (CCD) imager (such as a scanning imager or an area imager), a fluorescence activated cell sorter, a confocal microscope, or a scanning confocal device, wherein the spectral properties of the cells may be determined as scans of light excitation and emission.
V. Kits
[0092] Also provided herein are kits comprising one or more of any of the vectors disclosed herein, one or more of the calcium sensor proteins disclosed herein, any of the cells described herein, one or more of the non-human animals disclosed herein, and/or any of the tissue slices disclosed herein. In some embodiments, the kit further comprises instructions for screening for an agent that is capable of increasing or decreasing intracellular calcium
concentrations in a cell. In another embodiment, the kit further comprises instructions for screening for an agent that is capable of increasing or decreasing intracellular calcium
concentrations in a non-human animal.
EXAMPLES
Example 1 : Design and construction of GCamP mutant calcium sensor proteins
[0093] The solved crystal structure of GCamP was used to identify several potential sites for the introduction of cysteine residues into the GCamP chimeric protein {see Figure 1). It was hypothesized that the thiol groups on those cysteine resides would be susceptible to disulfide bond formation under oxidizing conditions and cause major conformational changes to the structure of GCamP, thereby decreasing the protein's ability to fluoresce. It was further hypothesized that formation of intramolecular or intermolecular disulfide bonds would 1) distort the barrel structure of the GCamP 's cpEGFP domain to increase the solvent accessibility to the chromophore, and consequently decrease the fluorescence intensity of the Ca -saturated form , 2) disrupt the interface between the cpEGFP domain and the Ca2+-CaM domain to destabilize the whole protein and thereby decrease the sensor performance, and/or 3) alter the intramolecular environment of the chromophore center, such as the hydrogen bond network, to change the fluorescent properties of the calcium sensor protein.
Materials and Methods
[0094] The GCamP gene was fully synthesized and inserted in the pUC57 vector by
GenScript. The GCamP gene was further sub-cloned to the pcDNA3.1 vector in order to express in mammalian cells. This pcDNA3.1 -GCamP plasmid was used as the template for generating the mutants by site-directed mutagenesis. For the mutants which have two separate mutation sites, the mutants containing the single mutation site were generated first and were sub-cloned together. The forward and reverse primers of the N77CY95C mutant are: 5'-CGT CCT CGA TGT TGT GGC GGA TCT TGA AGC ACG CCT TGA TGC CGT TCT TC-3' (SEQ ID NO: 16) and 5'- GCG GCG TGC AGC TGG CCT ACC ACT GCC AGC AGA AC A CCC CCA TCG G-3' (SEQ ID NO: 17); the forward and reverse primers of the L134C mutant are: 5'-AAC TCG CAC AGG ACC ATG TGA TCG CGC TTC-3' (SEQ ID NO: 18) and 5'-CGT GAC AGC TGC CGG GAT CAC TCT CG-3' (SEQ ID NO: 19); the forward and reverse primers of the T201C mutant are: 5'-TTC AGG CAA AGC TTG CCG TTG GTG GCA TC-3' (SEQ ID NO: 20) and 5'-GTT CAT CTG CAC CAC CGG CAA GCT G-3' (SEQ ID NO: 21); the forward and reverse primers of the D129C mutant are: 5'-GCT GCC ACA TGG TCC TGC TGG AGT TC-3' (SEQ ID NO: 22) and 5'-GCT TCT CGT TGG GGT CTT T.CG-3' (SEQ ID NO: 23); the forward and reverse primers of the K210C mutant are: 5'-GCT GCC TGC CCG TGC CCT GGC CCA C-3' (SEQ ID NO: 24) and 5'-CGG TGG TGC AGA TGA ACT TC-3' (SEQ ID NO: 25); the forward and reverse primers of the G209C mutant are: 5' -CCT GCA AGC TGC CCG TGC CCT GGC C-3' (SEQ ID NO: 26) and 5'-TGG TGC AGA TGA ACT TCA GG-3' (SEQ ID NO: 27); the forward and reverse primers of the P212C mutant are: 5'-TGT GCG TGC CCT GGC CCA CCC TCG TG-3' (SEQ ID NO: 28) and 5'-GCT TGC CGG TGG TGC AGA TG-3' (SEQ ID NO: 29); the forward and reverse primers of the L295C mutant are: 5' -TCT GCG GGC ACA AGC TGG AGT ACA AC-3' (SEQ ID NO: 30) and 5'-TGT TGC CGT CCT CCT TGA AG-3' (SEQ ID NO: 31); the forward and reverse primers of the T196C mutant are: 5'-CCT GCT ACG GCA AGC TGA CCC TGA AG-3' (SEQ ID NO: 32) and CAT CGC CCT CAC CCT CGC CG-3' (SEQ ID NO: 33); the forward and reverse primers of the R231C mutant are: 5'- GCT GCT ACC CCG ACC ACA TGA AGC AG-3' (SEQ ID NO: 34) and 5'-TGA AGC ACT GCA CGC CGT AG-3' (SEQ ID NO: 35); the forward and reverse primers of the T331C mutant are: 5'-TAT GCA CCA AGG AGC TGG GGA CGG TG-3' (SEQ ID NO: 36) and 5'-TTG TCC CAT CCC CGT CCT TG-3' (SEQ ID NO: 37); the forward and reverse primers of the T365C mutant are: 5'-GCT GCA TCG ACT TCC CTG AGT TCC TG-3' (SEQ ID NO: 38) and 5 '-CAT TAC CGT CGG CAT CTA C- 3' (SEQ ID NO: 39); the forward and reverse primers of the T329C mutant are: 5'-GGT GCA TAA CAA CCA AGG AGC TGG G-3' (SEQ ID NO: 40) and 5'-CAT CCC CGT CCT TGT CAA ATA G-3' (SEQ ID NO: 41); the forward and reverse primers of the T332C mutant are: 5'- CAT GCA AGG AGC TGG GGA CGG TGA TG-3' (SEQ ID NO: 42) and 5'-TTA TTG TCC CAT CCC CGT CC-3' (SEQ ID NO: 43); the forward and reverse primers of the E348C mutant are: 5'-CAT GCG CAG AGC TGC AGG ACA TGA TC-3' (SEQ ID NO: 44) and 5'-TGG GGT TCT GCC CCA GAG AC-3' (SEQ ID NO: 45); the forward and reverse primers of the L351C mutant are: 5'-AGT GCC AGG ACA TGA TCA ATG AAG-3' (SEQ ID NO: 46) and 5'-CTG CTT CTG TGG GGT TCT GC-3' (SEQ ID NO: 47).
Results
[0095] A total of twelve GCamP mutant calcium sensor proteins were constructed with each having a pair of cysteine residues inserted into a domain of the GCamP chimeric protein.
Example 2: In vitro screening of potential redoxGCamP mutants for fluorescent emission
[0096] This study screened the potential redoxGCamP mutants constructed in Example 1 for the ability to emit fluorescence upon exposure to increased intracellular calcium
concentrations in HEK293 cells. Additionally, cell lysates containing transfected redoxGCamP mutants were exposed to either reducing conditions or oxidizing conditions and the differential effect upon emitted fluorescence was measured.
Materials and Methods
[0097] HEK293 cells were transfected with the calcium sensor proteins constructed in
Example 1. 10 μΜ ionomycin or 100 μΜ acetylcholin were added and the fluorescent change of the transfected HEK293 cells were recorded by the FlexStation (excited at 470nm, detected at 520 nm). Fmax represents the maximum fluorescent signal, F0 represents the background fluorescent signal. The fluorescent intensity of the crude lysates of the transfected HEK293 cells was measured in MOPS buffer (2 mM CaCl2, 100 mM KC1, 100 μΜ PMSF, 20 mM MOPS, pH 7.5), in the presence of 100 μΜ DTT or 100 μΜ H202, respectively or after 30 minutes' treatment of the cell death assay buffer (HBSS, 2 mM CaCl2, 10 mM HEPES, 0.2% NaN3, 0.1% Triton X- 100, pH 7.4), in the presence or absence of 100μΜ Η202. [0098] The HEK293 cells were obtained from Cellbank in Shanghai, and were cultured in
DMEM containing 10% FBS. To express the proteins in HEK293 cells, the plasmids were transfected by PEL Briefly, plasmids were mixed with lmg/ml PEI solution in Opti-MEM at a ratio of 1 :2. After incubating for 10 minutes at room temperature, the DNA/PEI mixture was added to the culture plate. The cells were used for the following experiments 48 hours after transfection.
[0099] For the cell-based screening of the mutants (Figure 2A-B), the transfected
HEK293 cells were re-plated to the Costar black with clear bottom 96-well plate 12 hours before the experiments. Before the screening, Cells were washed with the assay buffer (137 mM sodium chloride, 5.4 mM potassium chloride, 0.44 mM potassium phosphate monobasic, 5.56 mM glucose, 0.25 mM sodium phosphate dibasic, 4.2 mM sodium bicarbonate, 2mM calcium chloride, 10 mM HEPES, pH 7.4), and were incubated at room temperature for 30 minutes. Ionomycin and acetylcholine solution were prepared in the Costar V-bottom 96-well compound plate. Both the compound plate and the assay plate were loaded to FlexStation III (Molecular Devices) under the flex mode. The baseline (F0) was recorded for 20 seconds before the compound solution was automatically transferred to the assay plate by the instrument. The fluorescent signal was continuously recorded for 100 seconds, and the maximum signal is referred to as Fmax. The excitation wavelength is at 470 nm, and the fluorescent signal was detected at 520 nm.
[0100] For the screening of the cell lysates (Figure 2C), transfected HEK293 cells were washed and then were re-suspended with the MOPS buffer (2 mM CaCl2, 100 mM KC1, 100 μΜ PMSF, 20 mM MOPS, pH 7.5). The cells were lysed by sonication. After centrifugation, the supernatant was collected as the crude lysate. The crude lysates were loaded to a Costar black with clear bottom 96-well plate, and were incubated with either 100 μΜ H202 or 100 μΜ DTT for 30 minutes at room temperature before measuring by FlexStation III under the spectrum mode. The excitation wavelength is at 470 nm, and the emission record is from 500 to 600 nm.
[0101] In the cell death assay (Figure 2D), the transfected HEK293 cells were incubated with the assay buffer containing 0.2% NaN3, 0.1% TritonX-100, with or without 100 μΜ H202 for 30 minutes at room temperature. The signal was measured by FlexStation III under the well- scan mode. F0 is defined as the fluorescent signal by cells incubating with assay buffer.
Results [0102] Seven of the GCamP mutants emitted light when expressed in cells exposed to molecules which are known in increase intracellular calcium concentrations (Figure 2A-B). Of these, four (N77C/Y95C, L134C/T201C, T331C/T365C, and T332C/E348C) exhibited decreased fluorescence when exposed to oxidative conditions in cell lysates in comparison to fluorescence emitted under reducing conditions (Figure 2C-D).
Example 3 : Characterization of the fluorescent spectra of redoxGCamP proteins in cellular crude lysates
[0103] This study characterized the fluorescent spectra of the redoxGCamP mutant proteins identified in Example 2 in the presence of calcium or in the presence of a divalent cation chealator.
Materials and Methods
[0104] Transfected HEK293 cells were washed and then were re-suspended with the
MOPS buffer (100 mM KC1, 100 μΜ PMSF, 20 mM MOPS, pH 7.5). The cells were lysed by sonication. After centrifugation, the supernatant was collected as the crude lysate. The crude lysates were loaded to a Costar black with clear bottom 96-well plate, and were incubated with either 2 mM CaCl2 or 10 mM EGTA for 5 minutes at room temperature before measuring by FlexStation III under the spectrum mode. The excitation wavelength is at 470 nm, and the emission record is from 500 to 600 nm (1 nm step).
Results
[0105] The results indicate that the GCamP calcium sensor protein (Figure 3B) and the
N77C/Y95C (Figure 3C), L134C/T E201C (Figure 3D), T331C/T365C (Figure 3E), and T332C/E348C (Figure 3F) redoxGCamP mutant calcium sensor proteins exhibit greater excitation and emission spectra fluorescence when exposed to calcium in comparison to fluorescence in the presence of the divalent cation chealator EGTA. In particular, the excitation and emission spectra of the L134C/T E201C (Figure 3D) redoxGCamP mutant was comparable to that of the GCamP protein (Figure 3B) when exposed to 2 mM CaCl2 or in the presence of EGTA.
Example 4: Characterization of the fluorescent spectra of purified redoxGCamP proteins by [0106] This study characterized the fluorescent spectra of purified redoxGCamP proteins in a calcium titration assay.
Materials and Methods
[0107] GCamP and the mutants were sub-cloned to the pEcoli-Cterm 6xHN vector
(Clontech), which is an inducible bacterial expression vector. The plasmids were transformed to BL21 strain under the selection of ampicillin on agar plate. The colonies were picked to innoculate 3 ml of LB/amp cultures and shake overnight at 37°C. In the morning, 1 ml of the overnight culture was inoculated to 50 ml of fresh LB/amp culture. When OD60o reaches 0.8, the culture was induced to express proteins by adding ImM IPTG, and were keep shaking for another 4 hours at 37°C. The cultures were then centrifuged to remove the supernatant and were stored at -80°C freezer for further analysis.
[0108] The proteins were purified by the His60 Ni Gravity Column Purification Kit
(Clontech). Briefly, cell pellet was resuspended.by the His60 Ni xTractor Buffer with \ μΙ, of DNase (Takara). After centrifugation at 4°C, the supernatant was collected and flow through the His60 Ni Gravity Column which have been equilibrated. The column was washed with 10 column volumes of Equilibration Buffer followed by 10 column volumes of Wash Buffer. The target protein was eluted by 10 column volumes of Elution Buffer and was collected as 1 ml fractions. The protein was desalted by the PD-10 column (GE Healthcare). The concentration of the protein was determined by the BCA Protein Assay Kit (Tiangen). For calcium titration, the purified proteins were diluted in either the calcium free buffer (10 mM EGTA, lOOmM KC1, 30mM MOPS, pH 7.2) or the high calcium buffer (10 mM Ca2+-EGTA, lOOmM KC1, 30mM MOPS, pH 7.2) at the final concentration of 1 μΜ. The calcium free buffer and the high calcium buffer containing the purified protein were mixed at different ratios to provide different concentrations of free calcium ions. The fluorescent signal was monitored by FlexStation III (Molecular Devices) under the end-point mode. The samples were excited at 485 ran, and were detected at 520 nm. Fmax was defined as the signal intensity at maximum concentration of free calcium, and F0 was defined as the signal intensity in the calcium free buffer. The calcium titration curve was fitted by the four-parameter logistic dose-response model by GraphPad to obtain the EC50 and Hill coefficient of each sample.
Results [0109] The calculated apparent Kd and Hill coefficients are shown in Table 1. As shown in Figure 4, fluorescence emission by the redoxGCamP was comparable to GCamP protein with the exception of the T331 C/365C redoxGCamP mutant.
Table 1: Apparent Kd and Hill coefficients for redoxGCamP
Figure imgf000035_0001
Example 5: Characterization of the fluorescent spectra of purified redoxGCamP proteins under reductive or oxidative conditions and in the presence or absence of calcium
[0110] This study measured emitted fluorescent light from purified unmodified and redox-sensitive GCamP proteins in the presence or absence of calcium and in the presence of either an oxidizing (H202) or a reducing agent (dithiothreitol).
Materials and Methods
[0111] GCamP and the mutants were sub-cloned to the pEcoli-Cterm 6xHN vector
(Clontech), which is an inducible bacterial expression vector. The plasmids were transformed to BL21 strain under the selection of ampicillin on agar plate. The colonies were picked to innoculate 3 ml of LB/amp cultures and shake overnight at 37°C. In the morning, 1 ml of the overnight culture was inoculated to 50 ml of fresh LB/amp culture. When OD600 reaches 0.8, the culture was induced to express proteins by adding ImM IPTG, and were keep shaking for another 4 hours at 37°C. The cultures were then centrifiiged to remove the supernatant and were stored at -80°C freezer for further analysis.
[0112] The proteins were purified by the His60 Ni Gravity Column Purification Kit
(Clontech). Briefly, cell pellet was resuspended by the His60 Ni xTractor Buffer with \ iL of DNase (Takara). After centrifugation at 4°C, the supernatant was collected and flow through the His60 Ni Gravity Column which have been equilibrated. The column was washed with 10 column volumes of Equilibration Buffer followed by 10 column volumes of Wash Buffer. The target protein was eluted by 10 column volumes of Elution Buffer and was collected as 1 ml fractions. The protein was desalted by the PD-10 column (GE Healthcare). The concentration of the protein was determined by the BCA Protein Assay Kit (Tiangen). The samples were diluted in the MOPS buffer (lOOmM KC1, 30mM MOPS, pH 7.2) to a final concentration of 1 μΜ, and were scanned by FlexStation (spectrum mode) reader (from Molecular Devices, Inc.) in a Costar black with clear bottom plate to obtain the excitation and emission spectra.
Results
[0113] Measurement of the unmodified GCamP protein demonstrated that the presence of a reducing or oxidizing agent had no effect on the excitation or emission spectra (Figure 5). Indeed, the only change in the amount of light emitted resulted from the decrease in fluorescence associated with the addition of a calcium chealtor. The N77C/Y95C, L 134C/T E201 C, and T332C/E348C redoxGCamP proteins exhibited overall decreased relative fluorescence in comparison to the emission and excitation spectra of the unmodified GCamP protein (Figures 5, 6, 7, and 9). In contrast, the T331C/T365C redox-sensitive mutant exhibited comparable, if not higher, relative fluorescence in both emission and excitation spectra compared to GCamP
(Figures 5 and 8). In contrast, however, for both excitation and emission spectra, the
N77C/Y95C redoxGCamP protein exhibited higher fluorescence in the presence of calcium and a reducing agent in comparison to the amount of light emitted in the presence of calcium in an oxidative environment (Figure 6). Similar results were observed for the L134C/T E201C
(Figure 7), T331C/T365C (Figure 8), and T332C/E348C (Figure 9) mutant GCamP proteins.
[0114] In conclusion, this study demonstrated that the redoxGCamP proteins emit less fluorescence in an oxidative environment in comparison to the amount of fluorescent light the same proteins emit under reducing conditions.
Example 6: Use of redoxGCamP mutant proteins to measure changes in intracellular calcium concentration mediated by activation of a G protein-coupled receptor
[0115] This study tests the ability of the redoxGCamP mutant proteins to detect increases in intracellular calcium concentration in HEK293 cells expressing the 5-HT2A receptor, which is a subtype of the 5-HT2 receptor belonging to the serotonin receptor family of G protein-coupled receptors (GPCRs).
Materials and Methods [0116] The 5-HT2A gene was cloned from the cDNA library by PCR, and was inserted to the pcDNA3.1 vector. The forward and reverse primers used were 5'-ACG AAG CTT ATG GAA ATT CTC TGT GAA GAC AAT ATC TCC CTG AGC TCA ATT CC-3' (SEQ ID NO: 48) and 5'-TA GGA TCC TCA CAC ACA GCT AAC CTT TTC ATT CAC GGT TTCAAT ATT GTC TGT AC-3' (SEQ ID NO: 49). The HEK 293 cells were co-transfected with 5-HT2A and the redoxGCamP mutants. Briefly, plasmids (5-HT2A:redoxGCamP = 1 : 1) were mixed with lmg/ml PEI solution in Opti-MEM at a ratio of 1 :2. After incubating for 10 minutes at room temperature, the DNA/PEI mixture was added to the culture plate. The cells were used for the following experiments 48 hours after transfection. The transfected HEK293 cells were re-plated to the Costar black with clear bottom 96-well plate 12 hours before the experiments.
[0117] Before the screening, cells were washed with the assay buffer (137 mM sodium chloride, 5.4 mM potassium chloride, 0.44 mM potassium phosphate monobasic, 5.56 mM glucose, 0.25 mM sodium phosphate dibasic, 4.2 mM sodium bicarbonate, 2mM calcium chloride, 10 mM HEPES, pH 7-4), and were incubated at room temperature for 30 minutes. Serotonin (Sigma) solution was freshly prepared in the Costar V-bottom 96-well compound plate. Both the compound plate and the assay plate were loaded to FlexStation III (Molecular Devices) under the flex mode. The baseline (F0) was recorded for 20 seconds before the compound solution was automatically transferred to the assay plate by the instrument. The fluorescent signal was further continuously recorded for 130 seconds, and the maximum signal is referred to as Fmax. The excitation wavelength is at 470nm, and the fluorescent signal was detected at 525 nm.
Results
[0118] Following activation of the 5-HT2A receptor with serotonin, the redoxGCamP proteins exhibited light at an intensity which was comparable, albeit slightly less, than the
GCamP protein (Figure 10). This study shows that the redoxGCamP mutants are able to detect changes in intracellular calcium concentration that result from an extracellular molecule acting on a plasma membrane cellular receptor protein.
[0119] The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. All publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
SEQUENCES
GCaMF PROTEIN
MMGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLAT^
DKQKNGIKANF IRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQCKLSKDPNEKRDHMVLLEFVT
AAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDV GHKFSVSGEGEGDATYGKLTLKFICTT
GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVN
RIELKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQ
DMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEE
VDEMIREADIDGDGQV YEEFVQMMTAK SEQ ID NO: l
N77C/Y95C REDOX-SENSITIVE PROTEIN
MMGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKTGHAVRAIGRLSSLENVYIMA DKQKNGI ACFKIRHNIEDGGVQLAYHCQQNTPIGDGPVLLPDNHYLSTQCKLSKDPNE RDHMVLLEFVT AAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDV GHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEF EAFSLFD DGDGTITTKELGTVMRSLGQNPTEAELQD MINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEV DEMIREADIDGDGQVNYEEFVQMMTAK SEQ ID NO:2
L134C/T201C REDOX-SENSITIVE PROTEIN
MMGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKW KTGHAVRAIGRLSSLENVYI A DKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQCKLSKDPNEKRDHMVLCEFVT AAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLCLKFICTTG KLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQD MINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEV DEMIREADIDGDGQVNYEEFVQMMTAK SEQ ID NO:3
T331C/T365C REDOX-SENSITIVE PROTEIN
MMGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKTGHAVRAIGRLSSLENVYIMA
DKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQCKLSKDPNEKRDHMVLLEFVT
AAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG
KLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNR
IELKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTICTKELGTVMRSLGQNPTEAELQD
MINEVDADGNGCIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEE
VDEMIREADIDGDGQVNYEEFVQMMTAK SEQ ID NO:4 T332C/E348C REDOX-SENSITIVE PROTEIN
MMGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKW KTGHAVRAIGPvLSSLENVYIMA
DKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQCKLSKDPNEKRDHMVLLEFVT
AAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDV GHKFSVSGEGEGDATYGKLTLKFICTTG
KLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNR
IELKGIDFKEDGNILGHKLEY TRDQLTEEQIAEFKEAFSLFDKDGDGTITCKELGTVMRSLGQNPTEAECQ
DMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEE
VDEMIREADIDGDGQVNYEEFVQMMTAK SEQ ID NO: 5
NUCLEOTIDE SEQUENCE FOR GCaMF PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgcagtcagagctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgaacttcaagatccgccacaacatcgaggacggcggcgtgcagctcgcctaccactaccagcagaacacccccatcggcgacggccccgtgctgctgccc gacaaccactacctgagcacccagtgcaaactttcgaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggc atggacgagctgtacaagggcggtaccggagggagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaa acggccacaagttcagcgtgtccggcgagggtgagggcgatgccacctaeggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacatccagg agcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttca aggaggacggcaacatcctggggcacaagctggagtacaacacgcgtgaccaactgactgaagagcagatcgcagaatttaaagaggctttctccctatttgacaagga cggggatgggacaataacaaccaaggagctggggacggtgatgcggtctctggggcagaaccccacagaagcagagctgcaggacatgatcaatgaagtagatgcc gacggtaatggcacaatcgacttccctgagttcctgacaatgatggcaagaaaaatgaaagacacagacagtgaagaagaaattagagaagcgttccgtgtgtttgataa ggatggcaatggctacatcagtgcagcagagcttcgccacgtgatgacaaaccttggagagaagttaacagatgaagaggttgatgaaatgatcagggaagcagacatc gatggggatggtcaggtaaactacgaagagtttgtacaaatgatgacagcgaagtga SEQ ID NO: 6
NUCLEOTIDE SEQUENCE FOR N77C/Y95C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgcagtcagagctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgtgcttcaagatccgccacaacatcgaggacggcggcgtgcagctggcctaccactgccagcagaacacccccatcggcgacggccccgtgctgctgccc gacaaccactacctgagcacccagtgcaaactttcgaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggc atggacgagctgtacaagggcggtaccggagggagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaa acggccacaagttcagcgtgtccggcgagggtgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacatccagg agcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttca aggaggacggcaacatcctggggcacaagctggagtacaacacgcgtgaccaactgactgaagagcagatcgcagaatttaaagaggctttctccctatttgacaagga cggggatgggacaataacaaccaaggagctggggacggtgatgcggtctctggggcagaaccccacagaagcagagctgcaggacatgatcaatgaagtagatgcc gacggtaatggcacaatcgacttccctgagttcctgacaatgatggcaagaaaaatgaaagacacagacagtgaagaagaaattagagaagcgttccgtgtgtttgataa ggatggcaatggctacatcagtgcagcagagcttcgccacgtgatgacaaaccttggagagaagttaacagatgaagaggttgatgaaatgatcagggaagcagacatc gatggggatggtcaggtaaactacgaagagtttgtacaaatgatgacagcgaagtga SEQ ID NO: 7 NUCLEOTIDE SEQUENCE FOR L134C/T201C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgcagtcagagctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgaacttcaagatccgccacaacatcgaggacggcggcgtgcagctcgcctaccactaccagcagaacacccccatcggcgacggccccgtgctgctgccc gacaaccactacctgagcacccagtgcaaactttcgaaagaccccaacgagaagcgcgatcacatggtcctgtgcgagttcgtgacagctgccgggatcactctcggca tggacgagctgtacaagggcggtaccggagggagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaa cggccacaagttcagcgtgtccggcgagggtgagggcgatgccaccaacggcaagctttgcctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccac cctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacatccaggag cgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaag gaggacggcaacatcctggggcacaagctggagtacaacacgcgtgaccaactgactgaagagcagatcgcagaatttaaagaggctttctccctatttgacaaggacg gggatgggacaataacaaccaaggagctggggacggtgatgcggtctctggggcagaaccccacagaagcagagctgcaggacatgatcaatgaagtagatgccga cggtaatggcacaatcgacttccctgagttcctgacaatgatggcaagaaaaatgaaagacacagacagtgaagaagaaattagagaagcgttccgtgtgtttgataagg atggcaatggptacatcagtgcagcagagcttcgccacgtgatgacaaaccttggagagaagttaacagatgaagaggttgatgaaatgatcagggaagcagacatcga tggggatggtcaggtaaactacgaagagtttgtacaaatgatgacagcgaagtga SEQ ID NO: 8
NUCLEOTIDE SEQUENCE FOR T331C/T365C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgcagtcagagctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgaacttcaagatccgccacaacatcgaggacggcggcgtgcagctcgcctaccactaccagcagaacacccccatcggcgacggccccgtgctgctgccc gacaaccactacctgagcacccagtgcaaactttcgaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggc atggacgagctgtacaagggcggtaccggagggagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaa acggccacaagttcagcgtgtccggcgagggtgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacatccagg agcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttca aggaggacggcaacatcctggggcacaagctggagtacaacacgcgtgaccaactgactgaagagcagatcgcagaatttaaagaggctttctccctatttgacaagga cggggatgggacaatatgcaccaaggagctggggacggtgatgcggtctctggggcagaaccccacagaagcagagctgcaggacatgatcaatgaagtagatgcc gacggtaatggctgcatcgacttccctgagttcctgacaatgatggcaagaaaaatgaaagacacagacagtgaagaagaaattagagaagcgttccgtgtgtttgataag gatggcaatggctacatcagtgcagcagagcttcgccacgtgatgacaaaccttggagagaagttaacagatgaagaggttgatgaaatgatcagggaagcagacatcg atggggatggtcaggtaaactacgaagagtttgtacaaatgatgacagcgaagtga SEQ ID NO: 9
NUCLEOTIDE SEQUENCE FOR T332C/E348C REDOX-SENSITIVE PROTEIN atgatgggttctcatcatcatcatcatcatggtatggctagcatgactggtggacagcaaatgggtcgggatctgtacgacgatgacgataaggatctcgccaccatggtcg actcatcacgtcgtaagtggaataagacaggtcacgcagtcagagctataggtcggctgagctcactcgagaacgtctatatcatggccgacaagcagaagaacggcat caaggcgaacttcaagatccgccacaacatcgaggacggcggcgtgcagctcgcctaccactaccagcagaacacccccatcggcgacggccccgtgctgctgccc gacaaccactacctgagcacccagtgcaaactttcgaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggc atggacgagctgtacaagggcggtaccggagggagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaa acggccacaagttcagcgtgtccggcgagggtgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacatccagg agcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttca aggaggacggcaacatcctggggcacaagctggagtacaacacgcgtgaccaactgactgaagagcagatcgcagaatttaaagaggctttctccctatttgacaagga cggggatgggacaataacatgcaaggagctggggacggtgatgcggtctctggggcagaaccccacatgcgcagagctgcaggacatgatcaatgaagtagatgccg acggtaatggcacaatcgacttccctgagttcctgacaatgatggcaagaaaaatgaaagacacagacagtgaagaagaaattagagaagcgttccgtgtgtttgataag gatggcaatggctacatcagtgcagcagagcttcgccacgtgatgacaaaccttggagagaagttaacagatgaagaggttgatgaaatgatcagggaagcagacatcg atggggatggtcaggtaaactacgaagagtttgtacaaatgatgacagcga SEQ ID NO: 10
ENHANCED GFP VARIANT
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLE Y YNSHNVYIMADKQKNGIKANFKIRH IEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSTQCKLSKDPNE
KRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 11
NATIVE GFP
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFS RYPDHM QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV RIELKGIDFKEDGNILGHKLEY NY SH VYIMADKQ NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEK RDHMVLLEFVTAAGITHGMDELY SEQ ID NO: 12
ENHANCED GFP VARIANT
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV RIELKGIDFKEDGNILGHKL EY YNSH VYIMADKQKNGIKVNF IRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPN EKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 13

Claims

CLAIMS What is claimed is:
1. An isolated nucleic acid comprising a nucleotide sequence encoding a calcium sensor protein comprising, from N terminus to C terminus, an Ml 3 domain, a circularly permuted green fluorescent protein domain, and a calmodulin (CaM) domain, wherein at least one pair of amino acid residues located at N77 and Y95, LI 34 and T201 , T331 and T365, and/or T332 and E348 in the calcium sensor protein is/are replaced with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO: 1 , and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
2. The nucleic acid of claim 1 , wherein the calcium sensor protein comprises an amino acid sequence with at least 90% or at least 95% identity to SEQ ID NO: 1.
3. The nucleic acid of claim 1 , wherein the calcium sensor protein comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
4. A vector comprising the nucleic acid of any one of claims 1 -3.
5. The vector of claim 4, wherein the vector is an expression vector.
6. The vectors of claim 3 or claim 4, wherein the vector is derived from a bacterium, a virus, a cosmid, a yeast, or a plant.
7. The vectors of claim 3 or claim 4, wherein the expression vector is a viral vector.
8. The vector of claim 7, wherein the viral vector is selected from the group consisting of: an AAV vector, a retroviral vector, an adenoviral vector, a HSV vector, and a lentiviral vector.
9. The vector of any one of claims 4-8, wherein the vector is a low copy vector or a high copy vector.
10. An isolated cell comprising the nucleic acids of any one of claims 1 -3.
1 1. An isolated cell comprising the vectors of any one of claims 4-8.
12. The cell of claim 10 or claim 1 1 , wherein the cell is selected from the group consisting of an animal cell, a bacterial cell, an insect cell, a nematode cell, and a yeast cell.
13. The cell of claim 12, wherein the animal cell is a human cell or a non-human cell.
14. The cell of claim 13, wherein the human cell is in a cell culture.
15. The cell of claim 12, wherein the non-human cell is selected from the group consisting of a rodent cell, a mouse cell, a rat cell, and a non-human primate cell.
16. The cell of claim 15, wherein the non-human cell is in a cell culture or in a non-human animal.
17. The cell of any of claims 13-16, wherein the animal cell is a muscle cell, a gamete, a neural cell, a cancer cell, or an endocrine cell.
18. The cell of claim 17, wherein the muscle cell is selected from the group consisting of smooth muscle, skeletal muscle, and cardiac muscle.
19. The cell of any of claims 10-18, wherein the nucleic acid encoding the calcium sensor protein is integrated into the cell's genome.
20. The cell of claims 10-19, further comprising one or more plasma membrane proteins selected from the group consisting of a G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RT ), an ion channel protein, and an ion pump protein.
21. The cell of claim 20, wherein said one or more plasma membrane proteins are
heterologously expressed or endogenously expressed.
22. The cell of claim 20, wherein the cell further comprises a nucleic acid encoding the GPCR located on a vector or in the genome of the cell.
23. The cell of claim 22, wherein the GPCR is selected from the group consisting of alpha- 1 adrenegic receptors (cd-AR), urotensin (UT) receptors, 5-HT2 and 5-HT6 serotonin receptors, hypocretic (orexin) receptors, histamine HI receptors, bradykinin Bl and B2 receptors, bombesin BB2 receptors, P2Y purinergic receptors, acetycholine receptors, mGluR5 glutamate receptors, vasopressin V2 and VI receptors, angiotensin AGTR1 receptors, cholecystokinin CCKAR and CCKBR receptors, endothelin ENDRA receptors, ghrelin GHSRla receptors, melatonin MTNR1 A receptors, neurotensin NTSR1 receptors, platelet-activating factor PTAFR receptors, prolactin releasing peptide receptor PRLHR receptors, G- coupled 5-HT2, 5-HT2A, 5-HT6, and 5-Ηχ7 serotonin receptors, Gj-coupled GABA-B, histamine H3, and mGluR2/4 glutamate receptors.
24. The cell of any one of claims 10-14 or 19-23 wherein the cell is a Hela cell, a KEK293 cell, or a human embryonic kidney (HEK) cell.
25. The cell of claim 24, wherein the cell is a KEK293 cell.
26. A population of cells comprising the cell of any one of claims 10-25.
27. A non-human animal comprising the cell of any one of claims 10-25.
28. A tissue slice comprising the cell of any one of claims 10-25.
29. An isolated calcium sensor protein comprising an amino acid sequence comprising, from N terminus to C terminus, an Ml 3 domain, a circularly permuted green fluorescent protein domain, and a calmodulin (CaM) domain, wherein at least one pair of amino acid residues located at N77 and Y95, LI 34 and T201, T331 and T365, and/or T332 and E348 is/are replaced with cysteine residues, wherein the amino acid residue position corresponds to the position in SEQ ID NO:l, and wherein the calcium sensor protein exhibits reduced fluorescence under oxidative conditions in comparison to the level of fluorescence exhibited under reducing conditions.
30. The calcium sensor protein of claim 29, comprising an amino acid sequence with at least 90% or at least 95% identity to SEQ ID NO: 1.
31. The calcium sensor protein of claim 29, comprising the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
32. A method of screening for an agent that is capable of increasing or decreasing
intracellular calcium concentrations in a cell comprising:
(i) contacting the agent with a cell expressing a calcium sensor protein encoded by the nucleic acids of any one of claims 1-3; and (ii) determining a level of fluorescence, wherein an increase in fluorescence indicates that the agent is capable of increasing intracellular calcium concentrations and a decrease in fluorescence indicates that the agent is capable of decreasing intracellular calcium concentrations.
33. The method of claim 32, wherein the cell is selected from the group consisting of an animal cell, a bacterial cell, an insect cell, a nematode cell, and a yeast cell.
34. The method of claim 33, wherein the animal cell is a human cell or a non-human cell.
35. The method of claim 34, wherein the human cell is in a cell culture.
36. The method of claim 35, wherein the non-human cell is selected from the group
consisting of a rodent cell, a mouse cell, a rat cell, and a non-human primate cell.
37. The method of claim 36, wherein the non-human cell is in a cell culture or in a non- human animal.
38. The method of any of claims 32-37 wherein the animal cell is a muscle cell, a gamete, a neural cell, a cancer cell, or an endocrine cell.
39. The method of claim 38, wherein the muscle cell is selected from the group consisting of smooth muscle, skeletal muscle, and cardiac muscle.
40. The method of any of claims 32-39, wherein the cell further comprises one or more
plasma membrane proteins selected from the group consisting of a G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RT ), an ion channel protein, and an ion pump protein.
41. The method of claim 40, wherein said one or more plasma membrane proteins are
heterologously expressed or endogenously expressed.
42. The method of claim 40, wherein the nucleic acid is integrated into the cell's genome.
43. The method of any of claims 32-42, wherein the cell is in a cell culture.
44. The method of any of claims 32-42, wherein the cell is in a non-human animal.
45. The method of any of claims 32-44, wherein the agent is a small molecule chemical compound, an antibody, a protein, an inhibitory nucleic acid, or any combination thereof.
46. The method of claim 45, wherein the inhibitory nucleic acid is selected from the group consisting of a triplex forming oligonucleotide, an aptamer, a ribozyme, a short interfering RNA (siRNA), an antisense oligonucleotide, and a micro-RNA (miRNA).
47. The method of claim 45, wherein the small molecule chemical compound is a compound from a chemical library.
48. The method of any of claims 32-47, wherein the method is a high-throughput method.
49. A kit comprising one or more of the following:
0) one or more of the vectors of claims 4-9;
(ϋ) more of the calcium sensor proteins of claims 29-31 ;
(iii) one or more of the cells of claims 10-25; the non-human animal of claim 27; and/or
(v) the tissue slice of claim 28.
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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104198689A (en) * 2014-08-25 2014-12-10 李洁 Method for detecting response of G protein-coupled receptor to vaso-excitor material
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999064859A1 (en) * 1998-06-06 1999-12-16 University Court Of The University Of Glasgow Detection of neural activity
WO2010018545A1 (en) * 2008-08-15 2010-02-18 Universite De Lausanne Detection and use of pheromones

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1287171A (en) * 1999-09-07 2001-03-14 复旦大学 Human neuron calcium sensing protein and its code sequence, preparation and use
EP1700865A1 (en) * 2005-03-11 2006-09-13 AXXAM S.r.l. Photoproteins with enhanced bioluminescence and their use as intracellular calcium indicators
EP2496697B1 (en) * 2009-11-06 2017-04-12 Howard Hughes Medical Institute Genetically encoded calcium indicators and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999064859A1 (en) * 1998-06-06 1999-12-16 University Court Of The University Of Glasgow Detection of neural activity
WO2010018545A1 (en) * 2008-08-15 2010-02-18 Universite De Lausanne Detection and use of pheromones

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NAKAI, J. ET AL.: "A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein", NATURE BIOTECH., vol. 19, 28 February 2001 (2001-02-28), pages 137 - 141, XP002187231, DOI: doi:10.1038/84397 *
QI WANG ET AL.: "Structural basis for calcium sensing by GCaMP2", STRUCTURE, vol. 16, no. 12, 12 December 2008 (2008-12-12), pages 1817 - 1827, XP026079390, DOI: doi:10.1016/j.str.2008.10.008 *
RODRIGUEZ GUILBE MM ET AL.: "Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2", ACTA CRYSTALLOGR SECT F STRUCT BIOL CRYST COMMUN., vol. 64, no. PT 7, 1 July 2008 (2008-07-01), pages 29 - 631 *

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