JP4808952B2 - IP3 indicator - Google Patents

Description

The present invention relates to a highly sensitive method for measuring IP ₃ concentration.

IP ₃ receptor, tetramer of intracellular IP ₃ - inducible Ca ²⁺ release channels ^{_{(IP 3 -gated Ca 2+ release channel}} ), in mammals, there are three different IP ₃ receptors To do. Type 1 IP ₃ receptor (IP ₃ R1) is highly expressed in the central nervous system, particularly in the cerebellum. Mouse IP ₃ R1 consists of 2749 amino acids (see Non-Patent Document 1) and is divided into three functionally different regions. That is, there is an IP ₃ binding domain near the N-terminus, a channel-forming domain having a 6-transmembrane region near the C-terminus, and a control region between these two regions. From deletion mutant analysis of IP ₃ binding domain, that IP ₃ amino 224-578 residues of the receptor is a region required for ligand binding specificity and high affinity shown, IP ₃ binding core and (See Non-Patent Document 2). By IP ₃ binds to the IP ₃ binding core, IP ₃ receptor undergoes a conformational change, IP ₃ receptor and thereby are thought the opening of the channel is induced, induced by IP ₃ The detailed mechanism of body channel opening is still unknown.

Increased cytoplasmic Ca ²⁺ concentration due to activation of the IP ₃ receptor controls the activity of a wide variety of downstream target molecules. These downstream target molecules play an important role in a variety of cellular responses such as fertilization, development, proliferation, secretion, and synaptic plasticity, and the IP ₃ molecule is an important second messenger that controls various cellular functions. The intracellular concentration is thought to change in a time-space-controlled manner. Measuring such changes in intracellular IP ₃ concentration is extremely important in elucidating the mechanisms of cell functions and various cellular responses, and the development of a measurement method has been an urgent task.

However, although a method using [ ³ H] IP ₃ has been developed as an effective indicator for quantifying IP ₃ in vitro, it is not convenient in terms of using a radioisotope. Furthermore, as a method for detecting changes in IP ₃ concentration in living cells over time, there is a method of detecting by monitoring the transition of the GFP-pleckstrin homology domain from the cell membrane to the cytoplasm. It has already been reported that the method is not always effective because it is indirect and has problems in terms of quantitativeness and spatial resolution (J. van der Wal, R. Habets, P. Varnai, T. Balla, K. Jalink, J Biol Chem 276, 15337-44 (2001)., C. Xu, J. Watras, LM Loew, J Cell Biol 161, 779-91 (2003)., R. Irvine, Curr Biol 14 , R308-10 (2004).). In addition, as in the present inventors, there is a report of an IP ₃ indicator using an IP ₃ binding core (Tanimura, A et.al., J Biol Chem, 279, 38095-8). Since the IP ₃ indicator does not function unless it is localized in the plasma membrane, the existing method can only detect local changes in IP ₃ concentration.

The present inventors have found an IP ₃ receptor binding protein IRBIT (IP ₃ R binding protein released with inositol 1,4,5-trisphospate) that binds to IP ₃ R1. IRBIT binds to IP ₃ R1 in vitro and in vivo and dissociates from IP ₃ R1 by physiological concentrations of IP ₃ . Using this IRBIT characteristic, a method for measuring IP ₃ concentration was developed (see Patent Document 1).

In the method of measuring intracellular IP ₃ concentration using IRBIT, a protein complex of IRBIT or a protein containing its IP ₃ receptor binding domain and a protein containing at least the IP ₃ binding domain of IP ₃ receptor The body needs to be prepared.

JP 2004-129612 A Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., Mikoshiba, K. (1989) Nature 342, 32-38 Yoshikawa, F., Morita, M., Monkawa, T., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 18277-18284 J. van der Wal, R. Habets, P. Varnai, T. Balla, K. Jalink, J Biol Chem 276, 15337-44 (2001). C. Xu, J. Watras, L. M. Loew, J Cell Biol 161, 779-91 (2003)., R. Irvine, Curr Biol 14, R308-10 (2004). Tanimura, A et.al., J Biol Chem, 279, 38095-8

The present invention provides a simpler method for measuring the concentration of IP ₃ in living cells.

When IP ₃ binds to IP ₃ R1, based on the hypothesis that the three-dimensional structure of IP ₃ R1 is changed, we tried to develop a method for measuring the concentration of IP ₃ utilizing this change in conformation. An energy acceptor protein or an energy donor protein that can be used in the FRET method at both ends of a protein consisting of the IP ₃ core region of IP ₃ R1 (in this specification, IRIS: IP ₃ R-based IP ₃ sensor) A protein fused with is produced. The obtained protein was found to be soluble and localized in the cytoplasm. It was found that when an IP ₃ molecule was bound to the protein, the fluorescence of the energy accepting protein in the protein was reduced. A method for measuring the concentration of IP ₃ using the decrease in fluorescence of the protein as an index has been established, and the present invention has been completed.

Accordingly, the present invention provides the following aspects:
1. Fluorescence that is an energy donor that can be used in the fluorescence energy transfer (FRET) method at both ends of the protein containing the IP ₃ binding core of the inositol 1,4,5-triphosphate receptor (IP ₃ R) A fusion protein in which a protein and a fluorescent protein serving as an energy acceptor are fused;
2. 2. The fusion protein according to 1 above, wherein IP ₃ R is mouse type 1 inositol 1,4,5-triphosphate receptor (IP ₃ R1);
3. _3. The fusion protein according to 2 above, wherein the IP ₃ binding core of mouse IP ₃ R1 consists of the amino acid sequence of amino acids 224 to 575 of the IP ₃ R1;
4). 1 to ₃ above, wherein the protein containing the IP ₃ binding core is fused with a fluorescent protein serving as the energy acceptor at the N-terminus and a fluorescent protein serving as the energy donor at the C-terminus. A fusion protein according to any of the above;
5. The above-mentioned 1-4, wherein the fluorescent protein as the energy acceptor is selected from the group consisting of Venus, EYFP and ReAsh, and the fluorescent protein as the energy donor is ECFP or FlAsH A fusion protein according to any of the above;
6). 6. The fusion protein according to 5 above, wherein the fluorescent protein serving as an energy acceptor is Venus, and the fluorescent protein serving as an energy donor is ECFP;
7). The fusion protein according to 1 above, which has the amino acid sequence of SEQ ID NO: 1;
8). A nucleic acid molecule encoding the fusion protein according to any one of 1 to 7 above;
9. A vector comprising the nucleic acid molecule according to 8 above;
10. A cell comprising the nucleic acid molecule according to 8 above in a form capable of expressing the encoded protein;
11. A method of detecting the IP ₃ concentration using a protein containing the IP ₃ binding core of inositol 1,4,5-triphosphate receptor (IP ₃ R), that IP ₃ binds to the IP ₃ binding core and detecting the FRET method conformational changes of a protein containing the IP ₃ binding core caused by the above method;
12 A method for detecting IP ₃ concentration comprising:
a) preparing the fusion protein according to any one of 1 to 7 above, and bringing it into contact with a sample suspected of containing IP ₃ ; and
b) detecting a change in fluorescence from the fusion protein before and after the contact;
Comprising the above method;
13. A method for detecting IP ₃ concentration comprising:
Venus the N-terminus of a protein containing an IP ₃ binding core of a) mouse IP ₃ R1 amino acid No. 224 to 575 of the amino acid sequence, providing a fusion protein ECFP the C-terminus is fused respectively, which Contacting with a sample suspected of containing IP ₃ , and
b) Before and after the contact, the protein is irradiated with light having an excitation wavelength of ECFP, and the fluorescence intensity by ECFP and the fluorescence intensity by Venus are measured to determine the ratio between the two before and after the contact. Detecting a change in fluorescence from the fusion protein before and after the contact,
Wherein the change in the ratio reflects the concentration of IP _{3 in} the sample;
14 A method for detecting intracellular IP ₃ concentration comprising:
a) introducing the fusion protein according to any one of 1 to 7 above into a cell; and
b) detecting a change in fluorescence from the fusion protein;
Comprising the above method;
15. A method for detecting intracellular IP ₃ concentration comprising:
Venus the N-terminus of a protein containing an IP ₃ binding core of a) a mouse IP ₃ R1 amino acid No. 224 to 575 of the amino acid sequence, the fusion proteins ECFP the C-terminus is fused respectively, into the cell Introducing steps, and
b) irradiating the cells with light having an excitation wavelength of ECFP, measuring a fluorescence intensity by ECFP and a fluorescence intensity by Venus to obtain a ratio between the two, and detecting a change in fluorescence from the fusion protein;
Wherein the change in the ratio reflects the concentration of IP ₃ in the cell;
16. 16. The method according to 14 or 15, wherein the step of introducing the fusion protein of a) into a cell is achieved by injecting the protein into the cell;
17. 16. The method according to 14 or 15, wherein the step of introducing the fusion protein of the above a) into the cell is achieved by introducing the nucleic acid molecule encoding the protein into the cell in an expressible form. A method as described above, characterized in that
18. A nucleic acid molecule encoding a protein comprising the amino acid sequence of amino acids 224 to 575 of mouse type 1 inositol 1,4,5-triphosphate receptor (IP ₃ R1) for producing an IP ₃ indicator;
19. Use of a nucleic acid molecule encoding a protein containing the amino acid sequence from amino acids 224 to 578 of type 1 inositol 1,4,5-triphosphate receptor (IP ₃ R1) for producing an IP ₃ indicator;
20. An IP ₃ detection kit comprising the fusion protein according to any one of 1 to 7 above;
21. 21. An IP ₃ detection kit comprising the nucleic acid molecule according to 8 above; An IP ₃ detection kit comprising the vector according to 9 above;
About.

According to the present invention, highly sensitive measurement of IP ₃ concentration has become possible. The method of the present invention only needs to prepare one kind of fusion protein, and can be carried out simply and inexpensively as compared with the conventional method. In addition, by forcibly expressing the fusion protein in living cells, fluctuations in IP ₃ concentration in living cells can be measured with high sensitivity, and temporal and spatial analysis becomes possible.

  The FRET method used in the present invention is a technique developed for analyzing protein-molecule interaction using fluorescence resonance energy transfer (FRET). FRET means that when two fluorophores are close to each other in an appropriate positional relationship, one fluorophore (energy donor (donor)) and another longer wavelength fluorophore (energy acceptor ( acceptor)). By labeling two molecules with two specific fluorescent molecules, changes in the interaction between the two molecules can be measured as changes in FRET efficiency.

In the present invention, both ends of protein IRIS having an amino acid sequence that is the IP ₃ core region of mouse IP ₃ R1 are labeled with an energy donor and an energy acceptor, respectively, and IP ₃ binds to IRIS, A technique of detecting a change in the fluorescence intensity of the energy acceptor is used. That is, in the FRET method used in the present invention, an energy acceptor and an energy donor are linked to the N-terminus and C-terminus of one protein, respectively, rather than detecting the intermolecular interaction between two protein molecules, The change in the fluorescence intensity due to the variation in the distance between them is used as an index.

The IP ₃ binding core region of mouse IP ₃ R1 that has already been reported is amino acids 226 to 578, and the IRIS of the present invention includes amino acid sequences of positions 224 to 275 including almost the entire region. Was used. The IRIS is, because it binds to IP ₃ has been confirmed by experiments of the present inventors, IP ₃ binding core region required for IP ₃ binding was also found to be sufficient in the area of amino acids No. 224-575.

Therefore, a fusion protein in which a fluorescent protein serving as an energy donor for FRET method and a fluorescent protein serving as an energy acceptor are fused to both ends of a protein having the amino acid sequence of amino acid positions 224 to 575 of mouse IP ₃ R1, It can be used as an IP ₃ indicator.

The IP ₃ R1 used in the present invention is a mouse, but the IP ₃ binding core region of IP ₃ R1 derived from other animal species (eg, rat or human) may be used. Furthermore, the IP ₃ binding core region of IP ₃ R of type 2 (IP ₃ R2) or type 3 (IP ₃ R3) can also be used. The amino acid sequences of IP ₃ R1, IP ₃ R2 and IP ₃ R3 of each animal species can be obtained from known databases. Each IP ₃ binding core region can also be easily estimated by one skilled in the art. For example, a technique of searching for a region corresponding to amino acid positions 224 to 575 of mouse IP ₃ R1 by homology analysis using computer software (Blast or the like) is also effective. In mouse type II and type III IP ₃ R, it is already known that the amino acid sequence portion at positions 224 to 604 has an IP ₃ binding ability. A sequence containing an amino acid sequence having IP ₃ binding ability can also be used.

  The fluorescent molecule used as an energy donor for the FRET method used in the present invention is preferably cyan fluorescent protein (CFP) or an improved product thereof, ECFP or FlAsH, particularly ECFP. The fluorescent molecule serving as the energy acceptor is preferably yellow fluorescent protein (YFP), Venus, or ReAsh, and Venus is particularly preferable. Furthermore, it is expected that various fluorescent molecules for use in the FRET method will be developed in the future, and they can also be used in the practice of the present invention. In addition, Proc. Natl. Acad. Sci. 101, 10554-10559 Use as an energy acceptor is also considered effective.

  Either the energy donor or acceptor may be linked to either the N or C terminus of IRIS. It is preferable that an energy acceptor is linked to the N-terminal side and an energy donor is linked to the C-terminal side.

  The most preferred example of such a fusion protein is shown in SEQ ID NO: 1 as the amino acid sequence of a fusion protein in which Venus is fused to the N-terminus of IRIS and ECFP is fused to the C-terminus.

Such a fusion protein is obtained by ligating each of DNA encoding an energy donor, DNA encoding the IP ₃ binding domain of IP ₃ R, and DNA encoding an energy acceptor in frame, and expressing them in an expression vector. It can be obtained by inserting it into a host and expressing it in a host. The amino acid sequence of the protein having the amino acid sequence of amino acids 224 to 578 of IRIS, that is, IP ₃ R1, is the sequence of amino acids 242 to 593 of the amino acid sequence shown in SEQ ID NO: 1, and the DNA encoding this is a person skilled in the art If so, the DNA encoding the fusion protein can be obtained by linking the DNA encoding the energy donor and the acceptor to both ends in-frame. Techniques for linking these in-frame are also known to those skilled in the art. IRIS containing amino acid positions 224 to 575 of IP ₃ R1 has EcoRI sites at both ends, and can be fused with DNA encoding an energy donor and an acceptor using these sites. A linker consisting of 1 to 5 amino acid residues, preferably 1 or 2 amino acid residues, may be inserted between IRIS and each energy donor and acceptor.

  It is easy for those skilled in the art to obtain the protein from the DNA encoding the obtained fusion protein.

  Furthermore, based on the amino acid sequence of the fusion protein, those skilled in the art can easily determine the RNA sequence that encodes it, and it is also possible to synthesize RNA having the sequence and prepare the protein therefrom. .

Accordingly, nucleic acids (including DNA and RNA) encoding the fusion protein are also included in the scope of the present invention. Also, proteins comprising IP ₃ binding core region of IP ₃ R, for example, be a nucleic acid encoding a protein having the amino acid sequence of amino acid No. 224 to 575 of the mouse IP ₃ R1, it may be included in the scope of the present invention. Using these nucleic acids and proteins, reagents for detecting the concentration of detection or IP ₃ and IP _3, i.e. capable of producing IP ₃ indicator will be understood from the above.

Furthermore, an IP ₃ detection kit containing the fusion protein of the present invention or a nucleic acid encoding the fusion protein of the present invention is also encompassed in the scope of the present invention. Such kit comprises IP ₃ in a predetermined concentration, IP ₃ standard solution or assay buffer (e.g., EDTA, 2-mercaptoethanol, and / or NP-40 the appropriate concentration, for titration curve produced for IP ₃ And 10 mM HEPES (pH 7.2) containing 100 mM NaCl. Furthermore, it may further contain an inactive form of the fusion protein of the present invention or a nucleic acid encoding it for control experiments. In the case of the above kit containing a nucleic acid encoding the fusion protein of the present invention, the nucleic acid may be contained in the form of an expression cassette or an expression vector. The kit may further contain a transfection reagent.

Detection of IP ₃ concentration using the fusion protein of the present invention can be performed not only in a cell-free system but also in a living cell. The “detection of IP ₃ concentration” in the present invention includes both qualitative detection and quantitative detection of IP ₃ . It also includes detecting changes in IP ₃ concentration relative and / or over time. The method of the present invention can detect IP ₃ in a pH environment within the physiological range and / or in the presence of Ca ²⁺ . The effects of the presence of the IP ₃ metabolites 1,4-IP ₂ and 1,3,4,5-IP ₄ are sufficiently low to be negligible and can specifically detect IP ₃ . Therefore, the method of the present invention is very useful for quantifying IP ₃ in any biological sample (including cell lysate and cell extract), detecting changes in IP ₃ concentration in living cells, and the like.

When detecting the IP ₃ concentration in a cell-free system, prepare the above fusion protein, contact it with a sample suspected of containing IP ₃ of interest, and use the fusion protein from the energy donor and acceptor at that time. Measure the change in fluorescence spectrum. More specifically, before the contact with the sample, the fluorescence intensity of the fusion protein by the energy donor and the fluorescence intensity by the energy acceptor are measured, and the ratio is obtained. It is good to measure and obtain ratio similarly and to compare it. “Fluorescence intensity by energy donor” is a fluorescence intensity of a specific wavelength emitted from the fluorophore when irradiated with light having a fluorescence excitation wavelength of the fluorophore that is an energy donor. Alternatively, when ECFP is used, it refers to a fluorescence spectrum peak when irradiated with light having an excitation wavelength of about 433 nm, or a wavelength in the vicinity thereof, that is, a fluorescence intensity of 460 to 510 nm, preferably 470 to 478 nm, for example, 475 nm. “Fluorescence intensity by energy acceptor” means light emitted from the fluorophore, which is excited by receiving the fluorescence of the energy donor, when irradiated with light of the fluorescence excitation wavelength of the fluorophore, which is the energy donor. The fluorescence intensity of a specific wavelength. That is, for example, when the energy acceptor is Venus or YFP, it refers to a fluorescence intensity at or near the peak of the fluorescence spectrum, ie, 515 to 560 nm, preferably 525 to 533 nm, for example, 530 nm. These wavelengths vary depending on the type of fluorophore and may vary slightly depending on the surrounding environment (buffer, temperature, etc.) of the fusion protein, but the fluorescence spectrum using the fusion protein under the conditions used for sample testing The wavelength at which a peak can be obtained can be determined by acquiring.

For example, ECFP the energy donor, when the energy donor fusion protein using Venus, by its IP ₃ IP ₃ binding core region binds, that the peak intensity of the fluorescence spectrum of Venus decreases, the Found by the inventors. Therefore, when the ratio of the fluorescence intensity is the Venus fluorescence intensity with respect to the ECFP fluorescence intensity, the ratio increases as IP ₃ binds, so the ratio increases as the IP ₃ concentration in the sample increases. .

Before testing the sample, obtain the ratio of fluorescence intensity to IP ₃ of several predetermined concentrations as described above, obtain a titration curve of the fluorescence intensity ratio to concentration, and determine the IP ₃ concentration in the sample from the titration curve. By determining, quantitative IP ₃ concentration can be detected.

According to the method of the present invention, it is possible to quantitatively detect IP ₃ at a concentration of 10 ⁻⁷ to 10 ⁻⁵ M. The concentration of IRIS-575 at the time of measurement can be appropriately set by those skilled in the art.

  The fusion protein to be used is preferably substantially isolated, and preferably has a high purity. However, the fusion protein does not have to be completely isolated, and a solution containing the fusion protein at an appropriate concentration should be used. Can do.

Such tests can be performed in any environment as long as the binding of IRIS and IP ₃ and the excitation of the energy donor fluorophore and the fluorescence of both the donor and acceptor fluorophores are sufficiently obtained. At a temperature of about 10 to 40 ° C., preferably 15 to 30 ° C., for example about 20 ° C., in a physiological buffer. Various physiological buffers are known and any can be used, and examples include 10 mM HEPES (pH 7.4: 100 mM NaCl). Furthermore, chelating agents, protein denaturation inhibitors, protease inhibitors, etc. (eg EDTA, 2-mercaptoethanol, nonionic surfactants (eg NP-40)) are added, and contaminants other than IRIS and IP ₃ It can be easily understood by those skilled in the art that the influence can be suppressed, and it is easy for those skilled in the art to select an appropriate one according to the environment to be carried out and add an appropriate amount to the reaction solution.

Furthermore, by using an appropriate set of excitation wavelength filter and fluorescence wavelength filter according to the fluorophore used as the energy donor and acceptor, the fluorescence intensity can be monitored as an image, and the image is analyzed. Thus, quantitative detection of IP ₃ can be performed.

When detecting the change in intracellular IP ₃ concentration using the fusion protein of the present invention, the fusion protein may be injected into the target cell or the fusion protein may be expressed in the target cell.

  Protein injection into cells can be performed using any method known in the art. In addition, in order to express the fusion protein in the target cell, the nucleic acid encoding the protein may be introduced into the cell in a form in which the protein can be expressed, and may be performed by various known methods. A person skilled in the art can select an appropriate method. For example, an expression vector can be used to transfect cells.

  The concentration of the protein at this time can be appropriately set by those skilled in the art. For example, the protein concentration may be finally 10 to 200 μg / ml, preferably 40 to 100 μg / ml. The fusion protein of the present invention is a soluble protein and can be evenly distributed in the cytoplasm.

A cell expressing the protein can be irradiated with an excitation wavelength in the same manner as described above to detect fluorescence derived from the protein. As the intracellular IP ₃ concentration increases, the fluorescence intensity of the energy receptor changes. Further, by monitoring the said change in the specific site of the cell, it can also be detected locally IP ₃ concentration change in the cell, the further image analysis under a microscope cells, IP ₃ concentration change in the cell It is also possible to monitor changes in the localization of the.

Furthermore, since the fusion protein of the present invention is a soluble protein, it is uniformly distributed in the cytoplasm. By using a protein in which a signal peptide such as a nuclear translocation signal peptide or a cell membrane localization signal peptide is added to the fusion protein. It is possible to localize in a specific site in a cell such as a nucleus or a cell membrane. Thereby, it is also possible to detect a change in IP ₃ concentration at a specific site in the cell.

The cDNA of IP ₃ R1 IP ₃ binding core region of (224 to 575 position amino acid sequence portion of the IP ₃ R1) was obtained by a known method. A cDNA was prepared by ligating DNA encoding Venus on the 5 ′ side and DNA encoding ECFP on the 3 ′ side so as to be in-frame with this. Such a cDNA encodes the fusion protein shown in the upper part of FIG. 1a (herein, IRIS-575). The sequence is shown in SEQ ID NO: 1.

Furthermore, a dominant negative mutant in which the threonine at position 267 in the IP ₃ binding core region (IRIS) of IP ₃ R1 is mutated to alanine and the lysine at position 508 is mutated to glutamic acid (ie, mutations that cannot bind to IP _3). In the same manner, a cDNA encoding a protein in which Venus and ECFP were fused was prepared (Sawano, A and Miyawaki, A., Nucleic Acids Res 28 E78 (2000)). A cDNA encoding a histidine tag was further ligated upstream of the cDNA.

  The cDNA is inserted into pFastBac1 (GIBCO / BRL), expressed in the SF9 baculovirus expression system (GIBCO / BRL), and purified using a Ni-agarose column and HiTrap using a parin column (Amersham Biosciences). -575 and IRIS-Dmut were obtained.

In vitro, changes in fluorescence due to IP ₃ of IRIS-575 were measured. Add 10 μM IP ₃ in 10 mM HEPES (pH 7.2: 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.5% NP-40) at 20 ° C., and excite it at 420 nm. Measured (Figure 1b). It was found that the fluorescence peak at 525 nm decreased when compared to the control without IP ₃ added. IRIS-Dmut was also measured in the same manner (FIG. 1c). With IRIS-Dmut, no decrease in fluorescence peak at 525 nm was observed. Therefore, reduction of the fluorescence peak is due to binding of IRIS and IP _3. The binding of IP ₃ to change conformation of IRIS, change the positional relationship between the Venus and ECFP is considered due to occur.

In order to confirm the IP ₃ concentration dependency of this fluorescence spectrum change, titration with IP ₃ was performed. The relationship between the ratio of the peak intensity at 475 nm (cyan) to the peak intensity at 525 nm (yellow) (C / Y) and the IP ₃ concentration is shown in FIG. When an excessive amount of IP ₃ was added, the increase in C / Y was 25.1 ± 0.8% (n = 6). Furthermore, when the sensitivity of IP ₃ was determined from the titration curve in FIG. 1d, it was Kd = 437 nM ± 30 nM, n = 6), and the binding constant of the IP ₃ binding domain of IP ₃ R1 (Kd = 336 nM; Natsume, T et al., Biochem Biophys Res Commun 260, 527-33, (1999)). This also suggests that IRIS-575 can detect physiological concentrations of IP _{3 at} which IP ₃ R1 is activated.

In order to examine the change in the fluorescence spectrum in more detail, the change in the fluorescence spectrum of IRIS-575 due to the IP ₃ metabolites 1,4-IP ₂ and 1,3,4,5-IP ₄ was examined (Fig. 1e). The change in C / Y ratio with IP ₃ was 50 and 400 times that with IP ₄ and IP ₂ , respectively. This indicates that IRIS-575 can measure IP ₃ concentration with high sensitivity and specificity. Furthermore, the influence by Ca ²⁺ was examined (FIG. 1f). The addition of 1 μM Ca ²⁺ into the reaction solution had no effect on the C / Y ratio. Therefore, it is possible to measure the IP ₃ concentration with IRIS-575 without being affected by the calcium concentration in the living cells. Furthermore, although the influence by pH was also examined, C / Y ratio was not influenced in the range of pH 7.0-7.8 which is the range of physiological pH (FIG. 1g).

Therefore, even when the intracellular Ca ²⁺ concentration or pH varies within the physiological pH range, the intracellular IP ₃ concentration can be measured by IRIS-575.

Based on this novel IP ₃ indicator, it is also possible to introduce indicator mutations having various binding strengths to IP ₃ by introducing a point mutation of the gene into the IP ₃ binding site. As shown in FIG. 1h, arginine (R) and lysine (K), which are basic amino acids present at the IP ₃ binding site, were converted to glutamine (Q), which is a neutral amino acid. Four indicators of R241Q, K249Q, R504Q, R506Q (the numbers of 241 249 504, 506 are the numbers of each amino acid counted from the first amino acid of mouse IP ₃ R1) were prepared. It showed a different binding strength to IP ₃ respectively, (IRIS-575 containing no mutation) wild-type, K249Q, R504Q, R506Q, binding strength to the IP ₃ in the order of R241Q was high.

Detection of IP ₃ concentration in sea squirt cells Eggs of sea squirt Ciona savignyi were purified using purified IRIS-575 and Indo-1 according to known methods (Yoshida, M. et al., Dev. Biol. 203, 122-33). After injection, increases in IP ₃ concentration and Ca ²⁺ concentration after insemination were detected. Ascidian fertilized eggs are known to undergo Ca ²⁺ release from an IP ₃ -induced intracellular store immediately after fertilization (Miyazaki, S. et al., Science 257, 251-5 (1992), and Runft, LL Dev. Biol. 245, 237-54 (2002)). The profiles of Ca ²⁺ concentration and IP ₃ concentration are shown in FIGS. 2a and b. An increase in IP ₃ concentration immediately after fertilization and a subsequent gradual decrease were observed, followed by a re-elevation around 30 seconds after fertilization and a subsequent gradual decrease. This was almost consistent with the change in intracellular Ca ²⁺ concentration detected by Indo-1 over time, confirming that the fluorescence change of IRIS-575 reflects the intracellular IP ₃ concentration. . In addition, the variation in IP ₃ concentration in the egg as measured by IRIS-575 was very gradual. This is in contrast to Ca ²⁺ , where the concentration oscillates. The IP ₃ variation measured by the GFP-pleckstrin homology domain oscillated similarly to Ca ²⁺ . The discrepancy between the results is shown in (J. van der Wal, R. Habets, P. Varnai, T. Balla, K. Jalink, J Biol Chem 276, 15337-44 (2001)., C. Xu, J. Watras, LM Loew, J Cell Biol 161, 779-91 (2003)., R. Irvine, Curr Biol 14, R308-10 (2004).) as indicated by al GFP- pleckstrin homology domain of IP ₃ It is presumed that the change in the precursor PIP ₂ is more easily reflected than the change.

Further, detection of intracellular IP ₃ concentration was performed. Microinjection between IRIS-575 and Indo-1 was carried out in the same manner as described above so that 40-100 μg of protein was introduced and fertilized. Obtain an image with an objective magnification of 10 x (na0.4) or 20 x (na0.75) with an IX-71 inverted microscope (Olympus) equipped with a cooled CCD camera ORCA-ER (Hamamatsu Photonics) at 20 ° C. It was. For fluorescence detection of IRIS-575, fluorescence from IRIS-575 was detected using an excitation filter of 425 to 445 nm and a filter for emission of 460 to 510 and 515 to 560 nm. The change in the concentration of IP ₃ detected by IRIS-575 was indicated by the value obtained by dividing the fluorescence intensity at 460 to 510 nm (Cyan: C) by the fluorescence intensity at 515 to 560 nm (Yellow: Y) (C / Y). A 450 nm beam splitter mirror was inserted into the optical path. Indo-1 fluorescence was detected using a 333 to 348 nm excitation filter and 420 to 440 and 460 to 510 nm emission filters. A 400 nm beam splitter mirror was inserted into the optical path. The change in Ca ²⁺ concentration detected by Indo-1 was shown by the value obtained by dividing the fluorescence intensity at 425 to 445 nm by the fluorescence intensity at 460 to 510 nm (Indo-1 ratio). Analysis was performed with TI Workbench software.

An image obtained for a fertilized egg immediately after fertilization is shown in FIG. Changes over time are monitored from the upper left to the second line. The upper side of each stage is the C / Y ratio image of IRIS-575, and the lower side is the Indo-1 ratio image. After fertilization, an increase in intracellular IP ₃ concentration and an increase in Ca ²⁺ concentration are observed from the images. Furthermore, the intracellular localization of IP ₃ could be reflected in the image.

IP ₃ concentration detection in mammalian cells
IRIS-575 protein was forcibly expressed in COS-7 cells using TransIT transfection reagent (Mirus) according to a conventional method. The localization of IRIS-575 in the cell is shown in FIG. 4a. It can be seen that IRIS-575 is uniformly localized in the cytoplasm. Cells 12-36 hours after transfection were used in the following experiments. As a general method of incorporating Ca ²⁺ dye, cells were immersed in 3 μM Indo-1 AM solution immediately before the experiment. Cells were maintained at 37 ° C. in a physiological concentration of HEPES buffer flow supplemented with 1 g / l glucose (flow rate 2 ml / min) and stimulated with 0.3 μM or 1.0 μM ATP. In response to the ATP stimulation, the fluorescence of IRIS-575 changed (FIG. 4b). The uppermost part of FIG. 4b shows cyan fluorescence (C) at 460 to 510 nm, and the upper middle part shows (Y) fluctuation of yellow fluorescence at 515 to 560 nm. These ratios (C / Y) are shown in the lower middle part. C / Y increases in response to ATP stimulation, and an increase in intracellular IP ₃ concentration due to ATP stimulation has been detected. In addition, Ca2 + fluctuations could be measured in parallel by simultaneous imaging with Indo-1 as in the experiments with squirt eggs (bottom).

Furthermore, in order to prove the sensitivity of detection of intracellular IP ₃ concentration by IRIS-575, it was examined whether or not the oscillation of intracellular IP ₃ concentration was artificially induced and detected. The cells were permeabilized by treatment for 3 minutes at beta-escin of 60 [mu] M, and a water tank containing the IP _3-free buffer with the cells, even aquarium (both containing the buffer containing 500 [mu] M IP ₃ flow rate 4ml ) And every 30 seconds, IRIS-575 fluorescence was detected. The results are shown in FIG. The solid line in the graph upper end, indicating the time at which the cells had been in contact with the buffer containing 500 nM IP _3. As can be seen from FIG. 4c, the C / Y ratio of IRIS-575 can reflect changes in intracellular IP ₃ concentration very sensitively and with high sensitivity. Furthermore, this experiment can show that the result in eggs that IP ₃ does not vibrate in sync with Ca ²⁺ is not wrong. In fact, if the intracellular IP ₃ concentration is oscillating, IRIS-575 can correctly reflect the fluctuation.

The above results indicate that the C / Y ratio of IRIS-575 can detect cell-free and intracellular IP ₃ concentration changes in real time with high sensitivity. The method of the present invention, as compared to conventional IP ₃ concentration detection methods, very accurately it is possible to detect the change in the IP ₃ concentration of cytoplasm.

Local measurement of IP3 concentration in cells
A myristylation / palmitylation target signal (MP-IRIS-575) and a nuclear translocation signal (NLS-IRIS-575) were added to the 5 ′ end of the gene of IRIS-575. These genes were forcibly expressed in HeLa cells and localized in the nucleus immediately below the cell membrane, respectively (FIGS. 5a and b). Compared to the fluorescence image of Indo-1 in which the whole cell is stained (Fig. 5a center, b right), IRIS-575 signal is observed directly under the cell membrane (Fig. 5a right) and in the nucleus (Fig. 5b right). Has been. The right figure of FIG. 5a is the image which observed the cell which expressed MP-IRIS-575 with the confocal laser microscope. It can be seen that many MP-IRIS-575 are present directly under the cell membrane. By stimulating these cells with 10 μM histamine, it was possible to simultaneously measure changes in IP ₃ concentration in the cell (upper figures in FIGS. 5c and d) simultaneously with Ca ²⁺ (lower figures in FIGS. 5c and d). .

FIG. 1a schematically shows the structure of the fusion protein of the present invention (IRIS-575: top) used in the Examples and the dominant negative type (IRIS-Dmut: top) of the fusion protein of the present invention. . Figure 1b and c, respectively, showing a variation of the fluorescence spectrum of IRIS-575 and IRIS-Dmut by IP _3. The presence of IP ₃ reduces the fluorescence intensity of IRIS-575 at about 525 nm of Venus, the energy acceptor. FIG. 1 d shows the IP ₃ concentration dependent IRIS-575 fluorescence change. The ratio (C / Y) of the fluorescence intensity (C) of 475 nm of the energy donor ECFP to the fluorescence intensity (Y) of Venus at about 525 nm. The C / Y value increased depending on the concentration of IP ₃ at a concentration of 10 ⁻⁵ or less. Figure 1e is a IP _3, shows the change in fluorescence IRIS-575 by l, 4-IP ₂ and 1, 3, 4, 5-IP ₄ respectively a metabolite of IP ₃ in the C / Y ratio. The change in C / Y ratio with IP ₃ was 50 and 400 times that with IP ₄ and IP ₂ , respectively. FIG. 1 f shows the change in fluorescence of IRIS-575 in the presence of Ca ²⁺ as a C / Y ratio. The fluorescence change of IRIS-575 was not affected by 1 μM Ca ²⁺ . FIG. 1g shows the results of examining the effect of pH on the fluorescence change of IRIS-575. In the range of pH 7.0 to 7.8, the C / Y ratio was not affected. Figure 1h is a diagram showing an IP ₃ dependent IRIS-575 and fluorescent change of IP ₃ indicator introduced point mutations IP ₃ binding site. Red is IRIS-575 with wild type IP ₃ binding site, yellow is 249th lysine converted to glutamine (K249Q), blue is 504th arginine converted to glutamine (R504Q), light blue is 506 The data of the 1st arginine converted to glutamine (R506Q), and the green color the 241st arginine converted to glutamine (R241Q) are shown. FIG. 2 shows changes in IP ₃ and Ca ²⁺ of fertilized eggs. In both FIGS. 2a and 2b, the arrows indicate the time when sperm was added. The graph is a graph of the data acquired every 4 seconds. FIG. 2a is the change in Ca ²⁺ concentration measured by Indo-1 and the upper bar shows the initial transient Ca ²⁺ increase. FIG. 2b is the concentration change of IP ₃ measured by IRIS-575. FIG. 3 is an image in which changes in IP ₃ (C / Y) and Ca ²⁺ (Indo-1 ratio) during the initial transient Ca ²⁺ increase in a fertilized egg are detected. Monitored over time from the top left in two steps. The upper side is detection of IP ₃ by IRIS-575, and the lower side is detection of Ca ²⁺ by Indo-1. FIG. 4 shows the results of detecting changes in IP ₃ concentration in COS-7 cells in which IRIS-575 was forcibly expressed. FIG. 4a is a fluorescence image of IRIS-575 forcibly expressed in the COS-7 cells. FIG. 4b shows changes in fluorescence upon stimulation with ATP at concentrations of 0.3 μM and 1.0 μM, respectively. Single COS-1 cells are excited with 420-440 nm light, and the fluorescence intensity (C) of 460-510 nm and the fluorescence intensity (Y) of 515-450 are shown in the top and upper middle, respectively. These ratios C / Y are shown in the lower middle part. The bottom row shows the ratio change of Indo-1. Figure 4c is artificially varying the IP ₃ concentration of the COS-7 cells shows the fluorescence changes IRIS-575 with C / Y. The upper bar indicates the time during which 500 nM IP ₃ was present in the extracellular fluid. Figure 5 shows the intracellular localization of IRIS-575 and the change in the fluorescence ratio of IRIS-575 and Indo-1 induced by histamine stimulation in HeLa cells in which IRIS-575 was forced to express cell membrane and nuclear translocation. It is the plot shown. FIG. 5a is a fluorescence image of IRIS-575 (MP-IRIS-575, left end) with a myristylation / palmitylation target signal added to the N-terminus and Indo-1 (center). The rightmost image shows a tomographic image of cells expressing MP-IRIS-575 by exciting Venus using a confocal laser microscope with the existing method. FIG. 5b is a fluorescence image of IRIS-575 (NLS-IRIS-575, left) and Indo-1 (right) with the SV-40 • T antigen nuclear translocation signal added to the N-terminus. FIG. 5c shows the time course of (C / Y ratio) (left) and Indo-1 ratio (right) of MP-IRIS-575 when cells were stimulated with 10 uM histamine. FIG. 5d shows the change over time of the C / Y ratio (left) and Indo-1 ratio (right) of NLS-IRIS-575 when the same stimulus as c was applied. The bars shown on the graphs of FIGS. 5c and 5d indicate the time during which histamine was added to the measurement solution.

Claims (20)

Mammal corresponding to amino acids 224 to 575 according to the IP ₃ binding core region consisting of amino acids 224 to 575 of mouse type 1 inositol 1,4,5-triphosphate receptor ( mouse IP ₃ R 1 ) or homology analysis A fluorescent protein that serves as an energy donor and an energy acceptor that can be used in the fluorescence energy transfer (FRET) method at both ends of the protein comprising the IP ₃ binding core region of IP ₃ R 1 Are fusion proteins each fused via a linker sequence consisting of 1 or 2 amino acids. Wherein the protein consisting of the IP ₃ binding core region, fluorescent protein of the energy donor fluorescent protein and C-terminal to be the energy acceptor to the N-terminus is fused respectively, in claim 1 The fusion protein described. And wherein the fluorescent protein of the energy acceptor is Venus, are those selected from the group consisting of EYFP and ReAsH, and fluorescent protein of the energy donor is ECFP or FlAsH, claim 1 or 2. The fusion protein according to 2 . The fusion protein according to claim 3, wherein the fluorescent protein serving as an energy acceptor is Venus, and the fluorescent protein serving as an energy donor is ECFP. The fusion protein according to any one of claims 1 to 4 , wherein the linker sequence consisting of 2 amino acids is an NS sequence. Wherein the nuclear localization to a protein consisting of the IP ₃ binding core domain signal peptide or cell membrane localization signal peptide is added, the fusion protein according to any one of claims 1-5. The fusion protein of claim 1 having the amino acid sequence of SEQ ID NO: 1. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 7 . A vector comprising the nucleic acid molecule of claim 8 . A cell comprising the nucleic acid molecule according to claim 8 in a form capable of expressing the encoded protein. A method for detecting IP ₃ concentration comprising:
a) preparing a fusion protein according to any one of claims 1 to 7 and contacting it with a sample suspected of containing IP ₃ ; and
b) detecting a change in fluorescence from the fusion protein before and after the contact;
Including the above method. A method for detecting IP ₃ concentration comprising:
a) Mouse IP ₃ R1 consists of amino acid No. 224 to 575 of the amino acid sequence IP ₃ binding core region or homology IP ₃ binding core region of IP ₃ R 1 mammalian corresponding to the amino acid No. 224 to 575 Analysis A fusion protein in which Venus is fused at the N-terminus of the protein consisting of ECFP at the C-terminus via a linker sequence consisting of 1 or 2 amino acids, and a sample suspected of containing IP ₃ Contacting, and
b) Before and after the contact, the protein is irradiated with light having an excitation wavelength of ECFP, and the fluorescence intensity by ECFP and the fluorescence intensity by Venus are measured to determine the ratio between the two before and after the contact. Detecting a change in fluorescence from the fusion protein before and after the contact,
Wherein the change in the ratio reflects the concentration of IP _{3 in} the sample. A method for detecting intracellular IP ₃ concentration comprising:
a) introducing the fusion protein according to any one of claims 1 to 7 into a cell; and
b) detecting a change in fluorescence from the fusion protein;
Including the above method. A method for detecting intracellular IP ₃ concentration comprising:
a) Mouse IP ₃ R1 consists of amino acid No. 224 to 575 of the amino acid sequence IP ₃ binding core region or homology IP ₃ binding core region of IP ₃ R 1 mammalian corresponding to the amino acid No. 224 to 575 Analysis Introducing into a cell a fusion protein in which Venus at the N-terminus of the protein consisting of ECFP and CFP at the C-terminus are fused via a linker sequence consisting of 1 or 2 amino acids, and
b) irradiating the cells with light having an excitation wavelength of ECFP, measuring the fluorescence intensity by ECFP and the fluorescence intensity by Venus to obtain a ratio between the two, and detecting a change in fluorescence from the fusion protein;
It includes, wherein a change in the ratio is reflective of the concentration of IP ₃ in a cell, the method described above. 15. The method according to claim 13 or 14 , wherein the step of introducing the fusion protein of a) into the cell is achieved by injecting the protein into the cell. 15. The method according to claim 13 or 14 , wherein the step of introducing the fusion protein of a) into the cell is achieved by introducing the nucleic acid molecule encoding the protein into the cell in an expressible form. The method described above, wherein Amino acids of the mouse IP ₃ R1 consists of amino acid No. 224 to 575 of the amino acid sequence IP ₃ binding core region or homology IP ₃ binding core region of IP ₃ R 1 mammalian corresponding to the amino acid No. 224 to 575 Analysis Use of a nucleic acid molecule encoding a protein consisting of a sequence to produce an IP ₃ indicator. A kit for detecting IP ₃ comprising the fusion protein according to any one of claims 1 to 7 . An IP ₃ detection kit comprising the nucleic acid molecule according to claim 8 . A kit for detecting IP ₃ comprising the vector according to claim 9 .

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