JP2010122110A - Membrane protein function measuring substrate and membrane protein function measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a membrane protein function measuring substrate applicable both to measurement of transmission of ions or molecules other than ions in an ion channel type membrane protein, and to measurement of activity of a metabolic type membrane protein, and a membrane protein function measuring method using the substrate. <P>SOLUTION: The membrane protein function measuring substrate 1, which is a substrate used for measuring a function of a membrane protein utilizing fluorescence, has a hole part 11, wherein an opening 11a of the hole part 11 is covered with a lipid bimolecular membrane 12 containing a membrane protein 13, and a fluorescent material (I) emitting fluorescence or a material (II) enhancing fluorescence intensity by being bonded to the fluorescent material (I) is arranged inside 14 the hole part 11. The membrane protein function measuring method using the membrane protein function measuring substrate 1 is also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a membrane protein function measuring substrate and a membrane protein function measuring method.

  In measuring the function of membrane proteins expressed on the cell membrane, the membrane protein is stimulated with an agonist (agonist), and the inflow of ions from the outside of the cell to the inside of the cell, the outflow of ions from the inside of the cell to the outside of the cell, and the cytoplasm. Detecting changes in the concentration of intracellular second messengers present.

Membrane proteins that cause inflow of ions from the outside of the cell into the cell and outflow of ions from the inside of the cell to the outside of the cell are called ion channel membrane proteins. Among these, a membrane protein that controls the opening and closing of a channel by selectively binding a molecule (hereinafter referred to as “ligand”) to the ion channel membrane protein is called an ion channel receptor. Among ion channel receptors, there are calcium ions, sodium ions, chloride ions, potassium ions, and the like, and those that pass molecules larger than the aforementioned ions called organic ions under specific conditions.
For example, calcium ions, sodium ions, chloride ions, potassium ions and the like are used as indicators for measuring the function of such membrane proteins. By using a fluorescent dye that emits fluorescence by binding to these ions, ion transmission through the membrane protein can be measured by a change in fluorescence intensity. In addition to the measurement using the fluorescent dye, there is an electrophysiological method for determining ion permeability by measuring the current value inside and outside the lipid bilayer membrane.

  As a factor called the intracellular second messenger, calcium ion derived from the endoplasmic reticulum is well known. Intracellular second messengers are produced by the activation of various enzymes present in cells and the metabolism of intracellular molecules. Therefore, a membrane protein involved in such a metabolic reaction is called a metabotropic membrane protein. Among them, those having a ligand in particular are called metabotropic receptors. A typical example of a metabotropic receptor is a G protein-coupled receptor (GPCR). Metabotropic receptors are often expressed in electrically inactive cells. In such a case, since the electrophysiological method is not suitable for measuring the function of a metabotropic receptor, the intracellular second messenger, that is, a fluorescent dye that emits fluorescence by binding to calcium ions, is used, and the fluorescence intensity is measured. As an indicator, the activity of metabotropic receptors is measured.

  Thus, the function of the membrane protein can be measured using the change in intracellular ion concentration as an index. Membrane proteins (especially receptors) are said to account for 45% of drug targets in FY2000. In the 2000 sales of the top 20 drugs in the United States, membrane proteins (including GPCR and ion channel type) account for about 50% of the total (Non-patent Document 1). Therefore, it is very important to accurately measure the function of membrane proteins related to the occurrence of various diseases and their treatment.

  However, there are usually multiple membrane proteins other than the target membrane protein on the cell membrane, and even when stimulated with a drug, transactivity from the target membrane protein to another membrane protein adjacent to the membrane protein In some cases, information transmission is performed by a mechanism called conversion. Therefore, it may be unclear whether the reaction obtained by stimulating with a drug is only through the target membrane protein. Therefore, there is a need for an in vitro measurement system that can individually measure membrane proteins using purified membrane proteins.

In the functional measurement of the purified ion channel membrane protein, it is necessary to pay attention to the following. That is, the ion channel membrane protein plays a role different from the normal function under various conditions including during a disease state. For example, an ion channel plays a role of receiving an extracellular signal by transmitting a ligand in addition to an ion or transmitting a signal to a neighboring cell (Non-Patent Documents 2 to 4). On the other hand, when the ion channel is activated and passes a larger molecule than usual, it acts as a mechanism for inducing cell death, causes inflammation of surrounding tissues, and pathological conditions such as hyperalgesia It may act as a mechanism that causes (Non-Patent Documents 5 to 7). Although these cases are special, ion channel function is often closely related to the pathological mechanism, and measuring ion channel function under such conditions is a drug discovery study. It is very important from the point of view.
In the measurement of the purified ion channel membrane protein, permeation of ions can be detected as changes in current and voltage by a measurement system using an electrophysiological technique. However, although it is important to measure the activity of an ion channel using ion permeation as an index, it is difficult to measure the permeation of molecules other than ions as described above by electrophysiological techniques. It is also difficult to observe simultaneously the inflow of ions through membrane proteins observed under physiological conditions and the permeation of ligands or the like observed under special conditions. It is very important to measure these multiple reactions simultaneously in order to examine the function of the ion channel in more detail.

In the case of purified metabotropic membrane proteins, it is difficult to measure the activity by electrophysiological techniques. This is because the second messenger is used for measuring the activity of the metabotropic membrane protein as described above, but the second messenger cannot be produced by the metabotropic membrane protein alone.
For example, the following steps (1) to (5) are involved in the release of calcium ions from the endoplasmic reticulum. (1) GPCR is activated, (2) guanosine triphosphate (GTP) is bound to G protein bound to GPCR, (3) phospholipase C is activated, (4) inositol phospholipid present in cell membrane Inositol 1,4,5-triphosphate (IP ₃ ) and diacylglycerol (DG) are produced from phosphatidylinositol 4,5-diphosphate (PI (4,5) P ₂ ), which is a kind of (5) IP ₃ binds to the IP ₃ receptor present on the endoplasmic reticulum membrane, and calcium ions are released from the endoplasmic reticulum to the cytoplasm (Non-patent Document 8). Thus, in the case of a metabotropic membrane protein, in order to measure the activity using a second messenger as an index, not only the target protein but also a related intracellular signal transduction factor is required.
For the above reasons, there is a demand for a method that can measure not only the permeation of ions but also the permeation of molecules other than ions in ion channel membrane proteins, and can also be applied to the functional measurement of metabotropic membrane proteins.
Drews J. Science, 287; 1960-1964 (2000) Suadicani SO et al. J. Neurosci., 26; 1378-1385 (2005) Zhao HB et al. Proc. Natl. Acad. Sci. 102; 18724-18729 (2005) Ye ZC et al. J. Neurosci., 23: 3588-3596 (2003) Pelegrin P. and Surprenant A. EMBO J., 25: 5071-5082 (2006) Pelegrin P. and Surprenant AJ Biol. Chem., 282: 2386-2394 (2007) Chung MK. Et al. Nat. Neurosci. 11: 555-564 (2008) Mikoshiba KJ Neurochem., 105: 1426-1446 (2007)

  Therefore, in the present invention, a membrane protein function measurement substrate that can be applied to both the measurement of permeation of ions and molecules other than ions in ion channel membrane proteins and the measurement of the activity of metabotropic membrane proteins, and membrane protein functions using the same An object is to provide a measurement method.

The present invention employs the following configuration in order to solve the above problems.
[1] A substrate used for measuring the function of a membrane protein using fluorescence, having a hole, the opening of the hole being covered with a lipid bilayer membrane containing a membrane protein, A membrane protein function measuring substrate, characterized in that a fluorescent substance (I) that emits fluorescence or a substance (II) that binds to the fluorescent substance (I) and enhances fluorescence intensity is disposed therein.
[2] Inside the hole, as the fluorescent substance (I), an ion-sensitive fluorescent dye that changes its fluorescence intensity by binding to ions that pass through the membrane protein is disposed, and the membrane protein is an ion channel The membrane protein function measuring substrate according to [1], which is a type membrane protein.
[3] The membrane protein functional measurement substrate according to [1], wherein a nucleic acid is provided as the substance (II) inside the hole, and the membrane protein is an ion channel membrane protein.
[4] The inside of the hole, the fluorescent substance (I) is arranged in a state of being bound to an extracellular information transmission substance, and the membrane protein is an ion channel type membrane protein. Membrane protein functional measurement board.
[5] A cytoplasmic extract fraction and a calcium ion-sensitive fluorescent dye as a fluorescent substance (I) are arranged inside the hole, and the membrane protein is a G protein-coupled receptor. [1] The membrane protein function measuring substrate according to 1.
[6] Using the membrane protein function measuring substrate according to [2], a buffer solution containing ions that permeate the membrane protein is disposed outside the hole, and the ion permeability of the ion channel membrane protein. Is a method for measuring the function of a membrane protein that quantitatively measures the amount of fluorescence by a change in fluorescence intensity.
[7] A buffer solution comprising the membrane protein functional measurement substrate according to [3], and a nucleic acid-binding fluorescent dye that binds to the nucleic acid as the fluorescent substance (I) and enhances fluorescence outside the hole. A membrane protein that quantitatively measures the permeability of the nucleic acid binding fluorescent dye through the ion channel membrane protein and the change in the channel pore size of the ion channel membrane protein by the change in fluorescence intensity Function measurement method.
[8] Using the membrane protein function measurement substrate according to [4], the movement of the extracellular information transmitting substance from the inside of the hole to the outside through the ion channel type membrane protein is caused by a change in fluorescence intensity. Membrane protein function measurement method quantitatively measured by
[9] Using the membrane protein function measuring substrate according to [5], the release of calcium ions from the endoplasmic reticulum via an intracellular signal transduction system is detected as a change in fluorescence intensity by the calcium ion sensitive fluorescent dye. A membrane protein function measuring method for quantitatively measuring the activity of a G protein-coupled receptor.
[10] A membrane protein function measuring method for simultaneously measuring different reactions of membrane proteins using the membrane protein function measuring substrate according to [1] comprising two or more kinds of fluorescent substances (I) having different emission wavelengths.

The membrane protein functional measurement substrate of the present invention can be applied to the measurement of ion permeation of ion channel membrane proteins and the permeation of molecules other than ions, and can also be applied to the measurement of activity of metabotropic membrane proteins. .
Moreover, the membrane protein function measuring method of the present invention can measure the permeation of ions of ion channel membrane proteins and the permeation of molecules other than ions, and can also measure the activity of metabolic membrane proteins.

  The membrane protein function measuring substrate of the present invention is a substrate used for measuring the function of a membrane protein using fluorescence, and has a hole, and the opening of the hole is covered with a lipid bilayer membrane containing the membrane protein. A fluorescent substance (I) that emits fluorescence, or a substance (II) that enhances fluorescence by binding to the fluorescent substance (I) is arranged inside the hole. The fluorescence intensity in the present invention can be measured with a fluorescence microscope such as a confocal fluorescence microscope.

  In FIG. 1, the conceptual diagram of one Embodiment of the membrane protein function measurement board | substrate of this invention is shown. As shown in FIG. 1, the membrane protein function measuring substrate 1 (hereinafter referred to as “substrate 1”) has a hole 11 formed in the substrate body 10, and the opening 11 a of the hole 11 includes the membrane protein 13. It is covered with a lipid bilayer membrane 12. The substrate 1 can be used for measuring the function of an ion channel membrane protein.

The material of the substrate body 10 may be any material as long as a fluorescent image, a transmitted light image, or a reflected light image can be favorably acquired with a fluorescent microscope, and examples thereof include plastic, glass, and silicon.
The shape of the substrate body 10 is not particularly limited, and a substrate having a shape corresponding to the application can be used. For example, a flat substrate can be used. The thickness of the substrate body 10 is not particularly limited, and is preferably 0.3 mm to 2 mm.

The substrate body 10 has a hole 11. The number of holes 11 in the substrate body 10 is not particularly limited, and is preferably 1 to 10,000.
As a method for forming the hole 11 in the substrate body 10, for example, a fine processing technique such as a photolithography method or a dry etching method can be applied. Further, the plastic substrate body 10 may be formed by piercing a heated injection needle.

The hole 11 may be a concave hole or a through hole. In this example, the hole 11 is a concave hole.
The shape of the opening 11a of the hole 11 is not particularly limited, and is preferably a circular shape from the viewpoint that the lipid bilayer membrane 12 can be formed more stably.
The diameter of the opening 11a is preferably 100 nm to 1 mm. If the diameter of the opening 11a is 100 nm or more, the functional measurement of the ion channel protein (membrane protein 13) becomes easy. If the diameter of the opening 11a is 1 mm or less, the lipid bilayer membrane 12 can be formed more stably.
Moreover, what is necessary is just to set the depth of the hole part 11 according to the thickness of the board | substrate body 10, and it is preferable that it is 50 nm-2 mm.

The lipid bilayer membrane 12 is a membrane in which lipid molecules such as phospholipids that are amphipathic molecules form a bilayer structure, and is the most basic membrane of a biological membrane. Lipid molecules have a hydrophobic portion and a hydrophilic portion, and form a stable double membrane structure by associating the hydrophobic portions with each other.
The lipid molecule is not particularly limited as long as it has sufficient fluidity at room temperature, and the length and number of double bond alkyl chains in the hydrophobic portion of each lipid are not particularly limited. Examples of the lipid molecules include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidic acid (PA), phosphatidylglycerol ( PG) and sphingolipids.
These lipid molecules may be used alone or in combination of two or more.

  Examples of the method for forming the lipid bilayer membrane 12 include a method using lipid molecules dissolved in an organic solvent such as n-decane. First, an aqueous solution is placed in the hole 11, and lipid molecules dissolved in n-decane are placed on the top. Then, the hydrophilic part of the lipid molecule in n-decane gradually gathers at the interface between n-decane and the hole part 11 to form a uniform layer. Next, an aqueous solution such as a buffer solution is placed on the n-decane in the hole 11. Thereby, the hydrophilic part of a lipid molecule gradually concentrates also in the interface of n-decane and the aqueous solution above this n-decane (external 15 side). Since n-decane volatilizes at room temperature, a uniform lipid bimolecular film 12 that finally covers the opening 11a of the hole 11 is formed.

As the membrane protein 13, an ion channel membrane protein is used.
Examples of the ion channel type membrane protein include ion channel type receptors. TRP (Transient Receptor Potential) channels (subunits are TRPV1, TRPV2, TRPV3, which are involved in cell-to-cell information transmission, temperature sensitivity, inflammation and pain, for example. , TRPV4, TRPV5, TRPV6, TRPA1, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPML1, TRPML3, TRPP3PP , TRPP4, TRPP5), ATP receptor involved in cell-cell communication and pain (subunit _P2X 1, _P2X 2, _{2X 3, P2X 4, P2X 5} , P2X 6, P2X 7), serotonin receptor involved in cell-cell communication and emotion (subunit 5-HT1,5-HT2,5-HT4,5- HT6,5- HT7), NMDA receptors involved in intercellular communication and excitatory neurotransmission (subunits are NR1, NR2A, NR2B, NR2C, NR2D, NR3A, NR3B), AMPA involved in intercellular communication and excitatory neurotransmission Receptors (subunits are GluR1, GluR2, GluR3, GluR4), kainate receptors involved in intercellular signal transmission and excitatory neurotransmission (subunits are GluR5, GluR6, GluR7, KA-1, KA-2), GABA receptors involved in intercellular signal transmission and inhibitory neurotransmission (subtypes are GABA _A and GABA _C ) Alternatively, GABA receptors (subunits are α, β, γ) and the like can be mentioned.
These ion channel type receptors may be used alone or in combination of two or more.

  As a method for reconstituting the membrane protein 13 in the lipid bilayer membrane 12, a known method can be used, for example, a method using vesicle fusion. Specifically, by adding a spherical endoplasmic reticulum (vesicle) containing a membrane protein 13 called a proteoliposome onto the lipid bilayer membrane 12, the lipid bilayer membrane and the lipid bilayer membrane 12 of the proteoliposome can be obtained. A method of reconstituting the membrane protein 13 into the lipid bilayer membrane 12 by fusing is mentioned.

A fluorescent substance (I) that emits fluorescence, or a substance (II) that enhances fluorescence by binding to the fluorescent substance (I) is disposed in the inside 14 of the hole 11 of the substrate 1 as described above, and the membrane protein Used for function measurement.
Embodiments of the membrane protein function measuring substrate of the present invention and the membrane protein function measuring method of the present invention using the same will be described in detail below.

[First Embodiment]
As shown in FIG. 2, the membrane protein function measurement substrate 1 </ b> A (hereinafter referred to as “substrate 1 </ b> A”) of this embodiment transmits the membrane protein 13 as a fluorescent substance (I) into the inside 14 of the hole 11. An ion-sensitive fluorescent dye (IA in FIG. 2) that binds to the ions 16 and changes the fluorescence intensity is disposed. In the substrate 1A, the same parts as those of the substrate 1 are denoted by the same reference numerals and description thereof is omitted.
The substrate 1A can be used for measuring the ion permeability of the ion channel membrane protein.

  The ion sensitive fluorescent dye is contained in the buffer solution and arranged in the inside 14 of the hole 11. Any buffer may be used as long as it can detect sufficient fluorescence and does not inhibit the function of the membrane protein 13, and examples thereof include Tris-HCl.

  The ion 16 that permeates the membrane protein 13 may be any ion that permeates the cell membrane via the membrane protein in a living body, and examples thereof include calcium ions, sodium ions, chloride ions, and potassium ions.

As ion-sensitive fluorescent dyes, fura-2, fluo-3, fluo-4 capable of detecting calcium ions (Ca ²⁺ ), SBFI capable of detecting sodium ions (Na ⁺ ), CoroNa, and potassium ions (K ⁺ ) can be detected. PBFI, MQAE capable of detecting chloride ion (Cl ⁻ ) and the like can be mentioned.
The concentration of the ion sensitive fluorescent dye in the inside 14 of the hole 11 may be an amount that can quantitatively measure the ion permeability by the change of the fluorescence intensity, and is preferably 100 nM to 500 μM, and preferably 1 μM to 100 μM. Is more preferable.

(Measuring method)
The membrane protein function measuring method of the present embodiment is a method for quantitatively measuring the ion permeability of the ion channel membrane protein (membrane protein 13) by using the substrate 1A by changing the fluorescence intensity.
In this embodiment, a buffer solution containing ions 16 that permeate the membrane protein is disposed outside the hole 11 of the substrate 1A.
As the buffer solution containing the ions 16, the same buffer solution containing the ion sensitive fluorescent dye in the inside 14 of the hole 11 can be used.

  For example, calcium ion sensitive fluorescent dyes such as fura-2, fluo-3, and fluo-4 are disposed in the inside 14 of the hole 11, and a buffer solution containing calcium ions (ions 16) is disposed in the outside 15 of the hole 11. To do. As a result, by stimulating the ion channel membrane protein (membrane protein 13), when calcium ions permeate the lipid bilayer membrane 12 through the ion channel membrane protein and move from the outer 15 to the inner 14, the permeation is performed. Calcium ions and calcium ion-sensitive fluorescent dyes are combined in the interior 14 to enhance the fluorescence intensity. Thereby, the permeation | transmission of the calcium ion of an ion channel type | mold membrane protein can be measured quantitatively.

  The concentration of the ions 16 may be appropriately determined according to the purpose of measurement, and is preferably 1 nM to 10 mM, more preferably 10 μM to 2 mM.

  In addition, conventionally known methods can be used for stimulation of ion channel membrane proteins. When ion channel receptors are used, ligands such as adenosine triphosphate (ATP) and glutamic acid can be used. . In addition, chemical substances other than the ligand, potential, light, pressure, or the like may be used for stimulation.

  Similarly, when ions other than calcium ions are used as the ions 16, the ion transmission can be quantitatively measured by using an ion-sensitive fluorescent dye corresponding to the ions 16.

[Second Embodiment]
As shown in FIG. 3, the membrane protein measurement substrate 1 </ b> B (hereinafter referred to as “substrate 1 </ b> B”) of the present embodiment is combined with the fluorescent substance (I) in the inside 14 of the hole 11 to enhance the fluorescence intensity. A nucleic acid (II-B in FIG. 3) is arranged as the substance (II). In the substrate 1B, the same portions as those of the substrate 1 are denoted by the same reference numerals and description thereof is omitted.
The substrate 1B can be used for measurement of transmission of molecules (fluorescent molecules (I)) other than ions of the ion channel membrane protein.

  The nucleic acid is contained in the buffer solution and arranged in the inside 14 of the hole 11. Any buffer may be used as long as it can detect sufficient fluorescence and does not inhibit the function of the membrane protein 13, and examples thereof include Tris-HCl.

Examples of the nucleic acid include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid may be artificially synthesized or extracted from cells. Further, the number of base pairs of the nucleic acid is not particularly limited as long as the change in fluorescence intensity can be measured quantitatively.
The concentration of the nucleic acid in the inside 14 of the hole 11 varies depending on the type and length of the nucleic acid, but is preferably 1 ng / μl to 500 μg / μl, and more preferably 1 to 100 μg / μl.

(Measuring method)
The membrane protein function measuring method of the present embodiment uses the substrate 1B to change the permeability of the nucleic acid-binding fluorescent dye through the ion channel membrane protein (membrane protein 13) and the channel pore size of the ion channel membrane protein. Is quantitatively measured by a change in fluorescence intensity.
In the present embodiment, a buffer solution containing a nucleic acid-binding fluorescent dye (IB in FIG. 3) that binds to a nucleic acid as a fluorescent substance (I) and enhances fluorescence is disposed outside the hole 11 of the substrate 1B. To do.

As the buffer solution containing the nucleic acid-binding fluorescent dye, the same buffer solution containing the nucleic acid in the inside 14 of the hole 11 can be used.
Examples of the nucleic acid binding fluorescent dye include ethidium bromide and propidium iodide.

  By arranging a nucleic acid in the inside 14 of the hole 11 and a buffer containing a nucleic acid-binding fluorescent dye in the outside 15 of the hole 11, the ion channel membrane protein (membrane protein 13) is stimulated to bind the nucleic acid. When the fluorescent dye passes through the lipid bilayer membrane 12 through the membrane protein 13 and moves from the outer portion 15 to the inner portion 14, the permeated nucleic acid-binding fluorescent dye binds to the nucleic acid in the inner portion 14, and the fluorescence intensity in the inner portion 14 Is strengthened. Thereby, the permeation | transmission of the ion channel type | mold membrane protein of the nucleic acid binding fluorescent dye which is a molecule larger than an ion can be measured quantitatively. Further, by detecting the permeation of the nucleic acid-binding fluorescent dye, it is possible to detect that the channel pore size of the ion channel membrane protein is larger than the size of the nucleic acid-binding fluorescent dye.

The same method as described in the first embodiment can be used for the stimulation of the ion channel membrane protein.
The concentration of the nucleic acid-binding fluorescent dye may be appropriately determined according to the purpose of measurement, and is preferably 1 nM to 500 μM, and more preferably 1 to 10 μM.

[Third Embodiment]
As shown in FIG. 4, the membrane protein measurement substrate 1 </ b> C (hereinafter referred to as “substrate 1 </ b> C”) of the present embodiment has a fluorescent substance (I) bound to an extracellular information transmitting substance in the inside 14 of the hole 11. In this state (compound 17 in FIG. 4 (compound in which an extracellular information transmission substance and fluorescent substance (I) are combined)). In the substrate 1C, the same parts as those of the substrate 1 are denoted by the same reference numerals and description thereof is omitted.
The substrate 1C can be used for measurement of permeation of an extracellular information transmission substance of an ion channel type membrane protein.

  Compound 17 is contained in the buffer solution and arranged in the inside 14 of the hole 11. Any buffer may be used as long as it can detect sufficient fluorescence and does not inhibit the function of the membrane protein 13, and examples thereof include Tris-HCl.

Compound 17 is a compound in which the fluorescent substance (I) is bound to the extracellular information transmission substance.
Extracellular information transmission substances are substances used for information transmission between cells, and examples thereof include adenosine triphosphate (ATP) and glutamic acid.
As the fluorescent substance (I) that binds to the extracellular information transmission substance, NBD (nitrobenzofurazane), TexasRed, Alexa Fluor, fluorescein, rhodamine, and Cy dye are preferable.

That is, examples of the compound 17 include ATP-FITC and glutamate-FITC. A known method can be used as a method for binding FITC to an extracellular signal transmitting substance.
The concentration of compound 17 may be appropriately determined according to the purpose of measurement, and is preferably 1 nM to 10 mM, and more preferably 10 nM to 100 μM.

(Measuring method)
The membrane protein function measuring method of the present embodiment uses the substrate 1C to transfer the extracellular information transmission substance from the inside 14 to the outside 15 via the ion channel membrane protein (membrane protein 13). This is a method of quantitatively measuring the change in fluorescence intensity.
In the present embodiment, in the substrate 1C, the compound 17 is disposed in the inside 14 of the hole 11, and a ligand such as ATP or glutamic acid (not shown) in which the fluorescent substance (I) is not bound to the outside 15 of the hole 11 (not shown). Place buffer containing

  When the ion channel type membrane protein (membrane protein 13) is stimulated by the ligand of the outer 15 and the extracellular information transmitting substance permeates the lipid bilayer membrane 12 through the membrane protein 13 and moves from the inner 14 to the outer 15, The amount of the fluorescent substance (I) in the inside 14 of the part 11 is reduced, and the fluorescence intensity in the inside 14 is reduced. By this change in fluorescence intensity, it is possible to quantitatively measure the permeation of the extracellular information transmitting substance through the ion channel membrane protein.

In addition, the membrane protein function measurement substrate and the membrane protein function measurement method of the present invention can be used not only for the function measurement of the ion channel type membrane protein described above but also for the measurement of the activity of the metabotropic membrane protein.
FIG. 5 shows a conceptual diagram of an embodiment of a membrane protein function measurement substrate used for measuring the activity of metabotropic membrane proteins. As shown in FIG. 5, the membrane protein function measurement substrate 2 (hereinafter referred to as “substrate 2”) has a hole 21 formed in the substrate body 20, and the opening 21 a of the hole 21 includes the membrane protein 23. It is covered with a lipid bilayer membrane 22. The substrate body 20, the hole portion 21, the opening 21a, and the lipid bilayer membrane 22 are the same as the substrate body 10, the hole portion 11, the opening 11a, and the lipid bilayer membrane 12 in the substrate 1 described above.

In the substrate 2 of the present embodiment, a metabotropic membrane protein is used as the membrane protein 23. An example of a metabotropic membrane protein is a G protein-coupled receptor (GPCR).
Examples of G protein-coupled receptors include adenosine receptors (A ₁ , A _2A , A _2B , A ₃ ) involved in cell-to-cell information transmission, and ATP receptors (P2Y ₁ , P2Y involved in cell-to-cell information transmission). ₂ , P2Y ₄ , P2Y ₆ , P2Y ₁₁ , P2Y ₁₂ , P2Y ₁₃ , P2Y ₁₄ ) Serotonin receptor involved in intercellular signal transmission (5-HT3) Adrenergic receptor (α1, α2, β1, β2), metabotropic glutamate receptors involved in intercellular communication (mGluR1, mGluR2, mGluR3, mGluR5, mGluR6, mGluR7, mGluR8), GABA receptors involved in intercellular communication, intercellular information transmission And opioid receptors (ν receptor, δ receptor, κ receptor) involved in analgesia.
These G protein-coupled receptors may be used alone or in combination of two or more.

  As a method for reconstituting the membrane protein 23 in the lipid bilayer membrane 22, a known method can be used as in the case of the ion channel membrane protein of the first embodiment. For example, a method using vesicle fusion is used. Can be mentioned.

In the present invention, the activity of the metabotropic membrane protein can be measured by using the substrate 2 and utilizing fluorescence. Hereinafter, an example of the embodiment will be shown and described.
[Fourth Embodiment]
As shown in FIG. 6, the membrane protein measurement substrate 2D (hereinafter referred to as “substrate 2D”) of the present embodiment has a cytoplasmic extract fraction 26 disposed in the inside 24 of the hole 21, and the fluorescent substance. As (I), a calcium ion sensitive fluorescent dye (FIG. 6, ID) is arranged. In the substrate 2D, the same portions as those of the substrate 2 are denoted by the same reference numerals and description thereof is omitted.
With the substrate 2D, the activity of a G protein-coupled receptor that is a metabotropic membrane protein can be measured using calcium ions released from the endoplasmic reticulum as an index.

The cytoplasmic extract 26 can be prepared by a known method from cells or tissues that express a G protein-coupled receptor.
For example, in the case of cells, the cells seeded in a culture dish (diameter 10 cm) are cultured until there is no gap between the cells. Next, the cells are washed three times with a phosphate buffer (PBS), scraped off with a cell scraper, placed in a 1.5 ml safe lock tube, and centrifuged at 5000 rpm (4 ° C.) to recover the cells. Next, 50 μl of pure water is added to the obtained cell pellet, and the mixture is allowed to stand on ice for 10 minutes, and the cell membrane is destroyed by low osmotic pressure stimulation. Thereafter, the supernatant is taken so as not to suck the pellet, and a protease inhibitor is added to obtain a cytoplasmic extract fraction 26.

Examples of calcium ion-sensitive fluorescent dyes include fura-2, fluo-3, and fluo-4.
The concentration of the calcium ion sensitive fluorescent dye in the inside 24 of the hole 21 may be an amount that can quantitatively measure the activity of the G protein-coupled receptor by a change in fluorescence intensity, and is preferably 100 nM to 500 μM. More preferably, it is 1 μM to 100 μM.

(Measuring method)
The membrane protein measurement method of the present embodiment uses a substrate 2D to detect the release of calcium ions from the endoplasmic reticulum via an intracellular signal transduction system as a change in fluorescence intensity using a calcium ion-sensitive fluorescent dye. This is a method for quantitatively measuring the activity of a conjugated receptor. It is preferable to arrange a buffer solution in the outside 25 of the hole portion 21 of the substrate 2D from the viewpoint that the membrane protein 23 is stably present and the activity is easily expressed in the same state as in the living body.

  In the substrate 2D, components necessary for the intracellular signal transmission system are supplied by the cytoplasm extraction fraction 26, and the components present in the cytoplasm are arranged in the hole 24.

The activity is measured as follows. When a ligand is added to the buffer solution of the outer 25, the GPCR (membrane protein 23) reconstituted in the lipid bilayer membrane 22 is activated, and guanosine triphosphate (GTP) binds to the G protein bound to the GPCR in the hole 24. To do. Next, phospholipase C is activated, and phosphatidylinositol 4,5-diphosphate (PI (4,5) P ₂ ), which is a kind of inositol phospholipid in the lipid bilayer membrane 22, is converted into inositol 1,4,5. -Triphosphate (IP ₃ ) and diacylglycerol (DG) are produced, IP ₃ binds to the IP ₃ receptor present on the endoplasmic reticulum membrane, and calcium ions are released into the interior 24 from the endoplasmic reticulum. And in the inside 24, the calcium ion discharge | released from the endoplasmic reticulum and the calcium ion sensitive fluorescent dye couple | bond together, and fluorescence intensity increases. Thereby, the activity of the G protein-coupled receptor can be quantitatively measured by detecting the release of calcium ions from the endoplasmic reticulum as a change in fluorescence intensity.

In the membrane protein function measuring method of the present invention, different reactions can be simultaneously measured by using two or more kinds of fluorescent substances (I) having different emission wavelengths.
For example, an ion-sensitive fluorescent dye (fluorescent substance IA) and a nucleic acid (substance II-B) are arranged in the inside 14 of the hole 11, and ions 16 corresponding to the ion-sensitive fluorescent dye and the outside 15 of the hole 11 and By arranging the nucleic acid-binding fluorescent dye (fluorescent substance IB), it is possible to simultaneously measure the permeation of ions in the ion channel membrane protein and the permeation of the nucleic acid-binding fluorescent dye.
Further, by arranging the ion sensitive fluorescent dye (fluorescent substance IA) and the compound 17 in the inside 14 of the hole 11 and arranging the ions 16 corresponding to the ion sensitive fluorescent dye in the outside 15 of the hole 11, The permeation of ions in the ion channel membrane protein and the permeation of extracellular signal transmitter can be measured simultaneously.
Similarly, the permeation of the nucleic acid-binding fluorescent dye and the permeation of the extracellular information transmission substance may be measured simultaneously, and the permeation of the ions, the nucleic acid-binding fluorescent dye, and the extracellular information transmission substance may be measured. You may make it measure simultaneously.
In addition, the ion bilayer membrane protein and the metabolite membrane protein are both reconstituted in the lipid bilayer membrane, and the function measurement of the ion channel membrane protein as in the first to third embodiments and the fourth embodiment are performed. The measurement of the activity of the metabotropic membrane protein such as the form may be performed simultaneously.

The membrane protein function measuring substrate and the membrane protein function measuring method using the same according to the present invention described above measure the function of the membrane protein using the change in fluorescence intensity as an index, and through a single kind of membrane protein. The reaction can be detected. In the case of an ion channel membrane protein, it can be used for analysis of permeation of ions and permeation of molecules larger than ions such as ligands and nucleic acid binding dyes. In addition, by using two or more kinds of fluorescent substances having different excitation wavelengths and emission wavelengths, different reactions of membrane proteins can be simultaneously measured.
The target for drug discovery is not a healthy membrane protein, but a diseased membrane protein. Therefore, it is a very important technique in drug discovery screening that normal ion permeation occurring under physiological conditions and dye permeation occurring under conditions corresponding to the pathological condition can be observed simultaneously or separately.
In the present invention, the activity of the metabotropic membrane protein can also be measured by disposing the cytoplasmic extract fraction in the hole of the substrate, so that it is a very useful measurement system.

  In addition, the membrane protein function measuring substrate and the membrane protein function measuring method using the same of the present invention can use an inexpensive material such as plastic for the substrate. Since it is not necessary, it is very low cost and simple. In drug discovery screening, it is necessary to measure the activity of an enormous number of compounds contained in a compound library, so it is necessary to lower the unit cost for each measurement. In this respect as well, the measurement system of the present invention is Very useful.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited by the following description.
In this example, observation with transmitted light using a fluorescence microscope and measurement of fluorescence intensity were performed using a confocal laser scanning microscope (trade name: LSM510, manufactured by Zeiss).

Example 1
Hereinafter, measurement of ion permeability of the ion channel membrane protein using the substrate 1A illustrated in FIG. 2 will be described.
A 25 gauge (diameter 500 μm) injection needle was blown with a gas burner, and a hole (hole 11) having a diameter of about 500 μm was made in a plastic plate (substrate body 10). Plastic burrs around the hole were removed with a razor. The transmitted light image of the obtained substrate is shown in FIG.
Next, an aqueous solution (about 0.2 μl) containing fluo-3 (50 μM) is added to the inside 14 of the hole 11, and a lipid mixture (phosphatidylcholine: phosphatidylsein = 1: 1, 2 mM) is added to n-decane. The lipid solution (2 μl) dissolved in the solution is added, and buffer X (10 mM Tris-HCl, pH 7.4, 2 μl) is further added thereto, and the mixture is allowed to stand at room temperature for 5 minutes to volatilize decane. A lipid bilayer 12 covering the 11 openings 11a was formed.
Next, a lipid vesicle (hereinafter referred to as “proteoliposome”) reconstituted with P2X ₄ receptor (ion channel type membrane protein, purified from 1321N1 human astrocytoma cells, 10 ng), which is membrane protein 13, on the outside 15 of the hole 11. Buffer Y (30 mM HEPES, 5 mM EDTA (disodium ethylenediaminetetraacetate), 1 mM EGTA (ethylene glycol bis (β-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid)), 0.02% NaN ₃ ) 0.5 μl was added and allowed to stand for an additional 5 minutes. Thereafter, buffer Y containing external 15 proteoliposomes was replaced with buffer Z containing ₂ mM CaCl ₂ (10 mM Tris-HCl, pH 7.4, 2 μl).
When the fluorescence intensity was measured in this state, no fluorescence was observed in the hole 14 (FIG. 8A).

The buffer Z external 15 of the hole 11, adenosine triphosphate (ATP, 100 [mu] M) is a ligand of P2X ₄ receptor was stimulated by the addition of fluorescence intensity (green interior 14 of the hole 11, the excitation wavelength 503 nm and emission wavelength of 530 nm) were remarkably enhanced (FIG. 8B, after 300 seconds from the stimulation).
Further, this fluorescence enhancement was significantly suppressed by adding TNP-ATP (100 μM), which is a P2X receptor antagonist, to the buffer solution Z. Fluorescence increase in the hole 14 by ATP stimulation from this is believed to be due to transmission of calcium ions through the P2X ₄ receptor.
Further, FIG. 8C shows the fluorescence intensity in the hole 14 after 300 seconds from the stimulation by ATP. As shown in FIG. 8 (c), the fluorescence intensity increased after ATP was added at 0 second, and the increased fluorescence intensity was sustained thereafter. From this result, it was shown that the lipid bilayer 12 was stably maintained without being broken within a measurement time of at least about 5 minutes.

(Example 2)
P2X ₄ receptor, in addition to the calcium ion transmission, when there is no outer calcium ions have been reported to transmit large molecules such as dyes (Khakh BS et al, Nat Neurosci 2:... 322 -330 (1999)). Therefore, the permeability of the nucleic acid-binding fluorescent dye of the ion channel membrane protein was measured using the substrate 1B exemplified in FIG.

DNA (purified from rat cerebral cortex-derived primary astrocytes, 5 μg / μl) is placed in the hole 11 of the substrate used in Example 1, and the ion channel membrane protein membrane (protein 13) in the same manner as in Example 1. After forming the lipid bilayer membrane 12 containing EDTA or EGTA by adding 5 mM of EDTA or EGTA to the buffer solution Z disposed outside the hole 11, the CaCl ₂ is removed with the nucleic acid binding fluorescent dye. Some ethidium bromide (20 μM) was added.
When the fluorescence intensity was measured in this state, almost no fluorescence was observed in the hole 14 (FIG. 9A).

Then, when stimulated with buffer Z to ATP (100 [mu] M) were added P2X ₄ receptor, in the hole 11, the fluorescence of the ethidium bromide-derived bound to DNA (red, excitation wavelength 488 nm, emission wavelength 590 nm) is significantly It was enhanced (FIG. 9 (b), 300 seconds after stimulation). Since no fluorescence derived from ethidium bromide was observed before stimulation with ATP, it is considered that the lipid bilayer 12 was formed sufficiently stably.
Further, FIG. 9C shows the fluorescence intensity in the hole 14 from 300 seconds after the stimulation by ATP. As shown in FIG. 9C, the red fluorescence intensity gradually increased after ATP stimulation.
Further, when the lipid bilayer 12 was broken with a surfactant or the like, the fluorescence intensity was instantaneously increased. Furthermore, when the P2X receptor antagonist TNP-ATP (100 μM) was added to the buffer Z, the enhancement of the fluorescence intensity was suppressed. These results, an increase in loose fluorescence intensity in FIG. 9 (c), believed to be via a P2X ₄ receptor.

  The membrane protein function measuring substrate and the membrane protein function measuring method using the same according to the present invention can individually measure the function of a specific membrane protein using a change in fluorescence intensity as an index, and can be easily and inexpensively The function of channel-type membrane proteins and metabotropic membrane proteins can be measured. In addition, ion channel membrane proteins are very useful for drug discovery screening because both normal ion permeation that occurs under physiological conditions and dye permeation that occurs under conditions corresponding to the pathological condition can be observed simultaneously or separately. is there.

It is the conceptual diagram which showed a mode that the ion channel type | mold membrane protein was reconfigure | reconstructed in the lipid bilayer membrane in the membrane protein function measurement board | substrate of this invention. It is the conceptual diagram which showed an example of embodiment which measures the function of the ion channel type | mold membrane protein of the membrane protein function measurement board | substrate of this invention. It is the conceptual diagram which showed the other example of embodiment which measures the function of the ion channel type | mold membrane protein of the membrane protein function measurement board | substrate of this invention. It is the conceptual diagram which showed the other example of embodiment which measures the function of the ion channel type | mold membrane protein of the membrane protein function measurement board | substrate of this invention. It is the conceptual diagram which showed a mode that the metabotropic membrane protein was reconfigure | reconstructed to the lipid bilayer membrane in the membrane protein function measurement board | substrate of this invention. It is the conceptual diagram which showed an example of embodiment which measures the function of the metabolite type membrane protein of the membrane protein function measurement board | substrate of this invention. It is the transmitted light image by the confocal laser scanning microscope of the board | substrate in which the hole part was formed in Example 1. FIG. FIG. 3 shows the results of functional measurement of ion channel membrane proteins in Example 1. (A) Confocal laser scanning microscope image before stimulation, (b) Confocal laser scanning microscope image after 300 seconds from stimulation, (c) Fluorescence intensity from 300 seconds after stimulation. It is the figure which showed the result of the function measurement of the ion channel type membrane protein in Example 2. (A) Confocal laser scanning microscope image before stimulation, (b) Confocal laser scanning microscope image after 300 seconds from stimulation, (c) Fluorescence intensity from 300 seconds after stimulation.

Explanation of symbols

  1, 1A, 1B, 1C Membrane protein functional measurement substrate 10 Substrate body 11 Hole 12 Lipid bilayer membrane 13 Membrane protein 14 Inside of hole 15 Outside of hole 17 Cytoplasmic extract fraction 2, 2D Membrane protein functional measurement substrate 20 Substrate body 21 Hole 22 Lipid bilayer membrane 23 Membrane protein 24 Inside of hole 25 Outside of hole IA, IB, IC Fluorescent substance (I) II-A substance (II)

Claims (10)

A substrate used for measuring the function of a membrane protein using fluorescence,
A fluorescent substance (I) that emits fluorescence, or the fluorescent substance (I) that has a hole, the opening of the hole being covered with a lipid bilayer membrane containing a membrane protein, and the fluorescent substance (I) A membrane protein function measuring substrate, comprising a substance (II) that binds and enhances fluorescence intensity.   Inside the hole, an ion-sensitive fluorescent dye whose fluorescence intensity is changed by binding to ions that pass through the membrane protein is disposed as the fluorescent substance (I), and the membrane protein is an ion channel membrane protein. The membrane protein function measuring substrate according to claim 1, wherein   The membrane protein functional measurement substrate according to claim 1, wherein a nucleic acid is provided as the substance (II) inside the hole, and the membrane protein is an ion channel membrane protein.   2. The membrane protein according to claim 1, wherein the fluorescent substance (I) is disposed inside the hole portion in a state of being bound to an extracellular information transmission substance, and the membrane protein is an ion channel membrane protein. Functional measurement board.   The cytoplasmic extract fraction and a calcium ion sensitive fluorescent dye as the fluorescent substance (I) are arranged inside the hole, and the membrane protein is a G protein-coupled receptor. Membrane protein functional measurement board.   The membrane protein function measurement substrate according to claim 2, wherein a buffer solution containing ions that permeate the membrane protein is disposed outside the hole, and the ion permeability of the ion channel membrane protein is measured by fluorescence intensity. Membrane protein function measurement method that measures quantitatively based on changes in temperature.   Using the membrane protein functional measurement substrate according to claim 3, a buffer solution containing a nucleic acid-binding fluorescent dye that enhances fluorescence by binding to the nucleic acid as the fluorescent substance (I) is disposed outside the hole. The membrane protein function measuring method for quantitatively measuring the permeability of the nucleic acid binding fluorescent dye through the ion channel membrane protein and the change in the channel pore size of the ion channel membrane protein by the change in the fluorescence intensity .   Using the membrane protein function measuring substrate according to claim 4, the movement of the extracellular information transmitting substance from the inside to the outside of the hole through the ion channel membrane protein is quantitatively determined by a change in fluorescence intensity. Method for measuring membrane protein function to be measured.   A release of calcium ions from the endoplasmic reticulum via an intracellular signal transduction system is detected as a change in fluorescence intensity with the calcium ion sensitive fluorescent dye using the membrane protein function measuring substrate according to claim 5, A membrane protein function measuring method for quantitatively measuring the activity of a conjugated receptor.   A membrane protein function measuring method for simultaneously measuring different reactions of a membrane protein using the membrane protein function measuring substrate according to claim 1 comprising two or more kinds of fluorescent substances (I) having different emission wavelengths.

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