JP2011022076A - System and method for measuring film protein function - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a system for measuring a film protein function capable of activating a film protein by the extension of a lipid bimolecular membrane and measuring the relationship between variations in density of a receptor in the lipid bimolecular membrane by the extension and activity, by the reconfiguration of the film protein in a lipid bimolecular membrane that is developed on a micropore, and a method for the same. <P>SOLUTION: The system 1 for measuring a film protein includes a film protein function measuring substrate 10A in which the opening 11a of a hole 11 on a substrate body 12 with the hole 11 is covered with the lipid bimolecular membrane 14 containing the film protein 13 and a buffer solution 15 is arranged on the lipid bimolecular membrane 14 and an extending means 20 for extending the lipid bimolecular membrane 14. Also, the method for measuring a film protein uses the system 1 for measuring a film protein. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

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

  To understand the functions of biomolecules including proteins, it is essential to know their structures. When a biomolecule functions, it functions by changing its structure in response to a stimulus from the outside (extracellular).

As a technique for observing the structure of biomolecules such as proteins, lipids, and nucleic acids under conditions close to those in the living body, a method using a scanning probe microscope that can be observed in an aqueous solution, particularly an atomic force microscope (AFM) is widely used. ing. In recent years, the technology of AFM has greatly advanced, and further contributions to the structural analysis of biomolecules and the observation of dynamic structural changes of biomolecules due to the binding of low molecular compounds and peptides added to the solution are expected. Has been.
As a measuring apparatus and measuring method using AFM, for example, a measuring apparatus and measuring method (non-patent document 1) in which resolution at a molecular level or atomic level is achieved in a solution, or observation at a video rate is possible. A measuring device (nonpatent literature 2) is mentioned.

Examples of stimuli that biomolecules receive from the outside world include cell membrane extension in some membrane proteins. It is known that the membrane protein is activated by cell membrane extension and exhibits physiological activities such as phosphorylation and calcium ion permeation (Non-Patent Documents 3 to 5). In addition to these membrane proteins, there may be proteins that are activated by cell membrane extension.
In addition, it is known that some receptors have a correlation between receptor density and activity (Non-patent Document 6). For this reason, it is also important to analyze the relationship between the distance between neighboring receptors and the function in detail and clarify the relationship.
However, there is no measurement system for detailed evaluation of cell membrane extension and protein activation due to changes in the distance between receptors. Searching for new proteins that are activated by cell membrane extension can also be performed by measuring the distance between receptors. It is also difficult to analyze the relationship between the function and its function in detail.

T. Fukuma, M. J. Higgins, S. P. Jarvis, Direct imaging of lipid-ion network formation under physiological conditions by frequency modulation atomic force microscopy. Phys. Rev. Lett. 98 (2007) 10601-10603. T. Ando, N. Kodera, E. Takai, D. Maruyama, K. Saito, A. Toda, A high-speed atomic force microscope for stdying biological macromolecules. Proc. Natl. Acad. Sci. USA. 98 (2001) 12468-12472. A. Kloda, L. Kua, R. Hall, DJ Adams, B. Martinac, Liposome reconstitution and modulation of recombinant N-methyl-D-aspartate receptor channels by membrane stretch, Proc. Natl. Acad. Sci. USA. 104 ( 2007) 1540-1545. Y. Sawada, M. Tamada, B. J. Dubin-Thaler, O. Cherniavskaya, R. Sakai, S. Tanaka, M. P. Sheetz, Force sensing by mechanical extension of the Src family kinase substrate p130Cas, Cell 127 (2006) 1015-1026. K. Yamamoto, T. Sokabe, T. Matsumoto, K. Yoshimura, M. Shibata, N. Ohura, T. Fukuda, T. Sato, K. Sekine, S. Kato, M. Isshiki, T. Fujita, M. Kobayashi, K. Kawamura, H. Masuda, A. Kamiya, J. Ando, Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice, Nat Med. 12 (2006) 133-137. Y. Fujiwara, Y. Kubo, Density dependent changes of the pore properties of P2X2 receptor channel, J. Physiol. 558 (2004) 31-43.

  The present invention reconstitutes a membrane protein in a lipid bilayer membrane developed on a micropore, activation of the membrane protein by extension of the lipid bilayer membrane, and density of receptors in the lipid bilayer membrane by extension An object of the present invention is to provide a membrane protein function measuring system and a membrane protein function measuring method capable of measuring the relationship between change and activity.

The present invention employs the following configuration in order to solve the above problems.
[1] A membrane protein function measuring substrate in which an opening of a substrate body having a hole is covered with a lipid bilayer membrane containing a membrane protein, and a buffer solution is disposed on the lipid bilayer membrane And a membrane protein function measuring system comprising: stretching means for stretching the lipid bilayer membrane.
[2] The membrane protein function measuring system according to [1], wherein the extension means is a deep needle of a scanning probe microscope.
[3] The membrane protein function measuring system according to [1] or [2], further comprising observation means for observing the surface structure of the lipid bilayer membrane.
[4] The method according to any one of [1] to [3], further including a first electrode provided outside the hole in the substrate and a second electrode provided inside the hole. The membrane protein function measuring system according to any one of the above.
[5] A method for measuring the function of a membrane protein using the membrane protein function measuring system according to any one of [1] to [3], wherein the membrane protein is an ion channel membrane protein, and the hole An ion-sensitive fluorescent dye that emits fluorescence by binding to ions that pass through the membrane protein is placed inside the unit, and a buffer solution that contains ions that pass through the membrane protein is placed on the lipid bilayer membrane. A membrane protein function measuring method in which the lipid bilayer is stretched by the stretching means, the ion permeation of the ion channel membrane protein is measured from the change in fluorescence intensity, and the function of the membrane protein is measured.
[6] A method for measuring the function of a membrane protein using the membrane protein function measuring system according to any one of [1] to [3], wherein the membrane protein is an ion channel membrane protein, and the hole A buffer solution containing a nucleic acid-binding fluorescent dye that binds to a nucleic acid and enhances fluorescence is disposed on the lipid bilayer membrane, and a nucleic acid-binding fluorescent dye is disposed on the lipid bilayer membrane. A membrane protein function measuring method for measuring the function of a membrane protein by extending a molecular membrane, measuring the penetration of the nucleic acid-binding fluorescent dye of the ion channel membrane protein from a change in fluorescence intensity.
[7] A method for measuring a function of a membrane protein using the membrane protein function measuring system according to any one of [1] to [3], wherein the membrane protein is an ion channel membrane protein, and the hole A fluorescently labeled extracellular information transmission substance is disposed inside the part, the lipid bilayer is extended by the extension means, and the extracellular information transmission substance is transferred from the inside to the outside of the hole part. A membrane protein function measuring method for measuring the function of a membrane protein by measuring movement through an ion channel type membrane protein from a change in fluorescence intensity.
[8] A method for measuring a function of a membrane protein using the membrane protein function measuring system according to any one of [1] to [3], wherein the membrane protein is a metabotropic membrane protein, and the hole portion In the inside, a cytoplasmic extract fraction and a calcium ion sensitive fluorescent dye that emits fluorescence by binding to calcium ions that permeate membrane proteins are arranged, and the lipid bilayer is stretched by the stretching means, A membrane protein function measuring method for measuring the function of a membrane protein by measuring the release of calcium ions from the endoplasmic reticulum via an internal signal transduction system from the change in fluorescence intensity.
[9] A method for measuring a function of a membrane protein using the membrane protein function measuring system according to [4], wherein the membrane protein is an ion channel membrane protein, and the membrane is formed on the lipid bilayer membrane. A buffer solution containing ions that pass through the protein is disposed, the lipid bilayer is stretched by the stretching means, and a current flowing between the first electrode and the second electrode is measured, A membrane protein function measuring method for measuring protein function.

  According to the membrane protein function measuring system and the membrane protein function measuring method of the present invention, the activation of membrane protein by extension of the lipid bilayer membrane, and the relationship between the density change of the receptor in the lipid bilayer membrane and the activity by extension. It can be measured.

It is the schematic sectional drawing which showed an example of embodiment of the membrane protein function measuring system of this invention. It is the schematic sectional drawing which showed other embodiment examples of the membrane protein function measuring system of this invention. It is the schematic sectional drawing which showed 1 process of the membrane protein function measuring method of this invention. It is the schematic sectional drawing which showed 1 process of the membrane protein function measuring method of this invention. It is the schematic sectional drawing which showed 1 process of the membrane protein function measuring method of this invention. It is the schematic sectional drawing which showed 1 process of the membrane protein function measuring method of this invention. It is the schematic sectional drawing which showed 1 process of the membrane protein function measuring method of this invention. It is the schematic sectional drawing which showed 1 process of the membrane protein function measuring method of this invention. It is a fluorescence image using the confocal fluorescence microscope of the board | substrate in Experimental example 1. FIG. 6 is an AFM image of a substrate in Experimental Example 2. AFM image (a) of substrate in Example 1, graph (b) showing height information of lipid bilayer membrane in broken line part in AFM image (a), and enlarged view (c) of AFM image (a) It is. AFM image (a) of the substrate having a hole in Example 1, graph (b) showing the height information of the lipid bilayer membrane at the broken line in AFM image (a), and enlargement of AFM image (a) It is a figure (c). It is the graph (a) which showed the graph (a) which showed the change of the fluorescence intensity in Example 2, the fluorescence image (b) using the confocal fluorescence microscope of a board | substrate, and the change of the electric current value in Example 3. FIG.

<Membrane protein function measurement system>
As shown in FIG. 1, a membrane protein function measuring system 1 (hereinafter referred to as “system 1”) of the present invention includes a membrane protein function measuring substrate 10A (hereinafter referred to as “substrate 10A”) and an extension means 20. And.
The substrate 10A includes a substrate body 12 having a hole portion 11, a lipid bilayer membrane 14 containing a membrane protein 13 that covers the opening 11a of the hole portion 11, and a buffer solution 15 disposed on the lipid bilayer membrane 14. Have. The extension means 20 is means for extending the lipid bilayer membrane 14.

The substrate 10A is used for measuring the function of the membrane protein 13 by measuring the structural change of the membrane protein 13 activated by stretching the lipid bilayer membrane 14, observing the localization in the lipid bilayer membrane 14, and measuring the change in fluorescence intensity. It is a substrate.
In the substrate 10 </ b> A, the buffer solution 15 is disposed on the lipid bilayer membrane 14 covering the hole portion 11, and the outside of the hole portion 11 and the inside of the hole portion 11 are separated by the lipid bilayer membrane 14.

The surface of the substrate body 12 is preferably flat.
The material of the substrate body 12 is preferably a material whose surface can be processed flat, and examples thereof include mica, glass, and silicon.
The shape of the substrate main body 12 is not particularly limited, and a shape according to the application can be used. For example, a flat substrate can be mentioned.
The thickness of the substrate body 12 is not particularly limited, and is preferably 0.1 mm to 2 mm.

The substrate body 12 has a hole 11.
The number of the hole portions 11 in the substrate body 12 is not particularly limited, and is preferably 1 to 10,000.
The shape of the hole 11 is such that the lipid bilayer membrane 14 can be stably disposed in the opening 11a, and if necessary, a fluorescent image of the hole 11 can be obtained with a fluorescence microscope, and the surface structure can be observed with an AFM. That's fine. Examples of the shape of the hole 11 include a cylindrical shape.
The hole 11 may be a concave hole or a through hole. In this example, the hole 11 is a concave hole.

The diameter of the opening 11a is preferably 10 nm to 10 μm. If the diameter of the opening 11a is 10 nm or more, observation of the surface structure and the like can be facilitated by AFM, and functional measurement of the membrane protein 13 can be facilitated. If the diameter of the opening 11a is 10 μm or less, the lipid bilayer membrane 14 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 12, and 50 nm-2 mm are preferable.

  As a method of forming the hole 11 in the substrate body 12, for example, a fine processing technique such as a photolithography method or a dry etching method can be applied. Further, the plastic substrate body 12 may be formed by piercing a heated injection needle.

Examples of the membrane protein 13 include an ion channel membrane protein and a metabotropic membrane protein.
Examples of the ion channel membrane protein include an ion channel receptor, and TRP (Transient Receptor Potential) (subunits are TRPV1 to TRPV6, TRPA1, TRPC1 to TRPC7, TRPM1 to TRPM8, TRPML1 to TRPML3, TRPP1 to TRPP5). ), ATP receptor (subunits are P2X ₁ to P2X ₇ ), setronin receptor (subunit is 5-HT ₃ ), NMDA receptor (subunits are NR1, NR2A to NR2D, NR3A, NR3B), AMPA receptor (Subunits are GluR1 to GluR4), kainate receptors (subunits are GluR5 to GluR7, KA-1, KA-2), GABA receptors (subunits are α, β, γ) or ABA receptor (subtype GABA _A, GABA _C) and the like.
Examples of metabotropic membrane proteins include G protein-coupled receptors, such as adenosine receptors (A ₁ , A _2A , A _2B , A ₃ ), ATP receptors (P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y _6). , P2Y ₁₁ , P2Y ₁₂ , P2Y ₁₃ , P2Y ₁₄ ), serotonin receptor (subunits are 5-HT1, 5-HT2, 5-HT4, 5-HT6, 5-HT7), metabotropic glutamate receptors (mGluR1- mGluR3, mGluR5 to mGluR8), adrenergic receptors (α, β), protease activated receptors (PAR-1 to PAR-4), bradykinin receptors (B1, B2), GABA _B receptors, opioid receptors (ν) Receptor, δ receptor, κ receptor) and the like.
These receptors may be used alone or in combination of two or more.

  As a method of reconstituting the membrane protein 13 in the lipid bilayer membrane 14, a known method can be used, and examples thereof include a vesicle fusion method.

The lipid bilayer membrane 14 is a membrane in which lipid molecules such as phospholipids that are amphipathic molecules form a bilayer structure, and is the most basic structure of a biological membrane. Lipid molecules have a hydrophobic part and a hydrophilic part, and form a stable bilayer structure when the hydrophobic parts are associated with each other.
The lipid molecule is not particularly limited as long as it has sufficient fluidity at room temperature, and the alkyl chain length and the number of double bonds in the hydrophobic part of each lipid are not particularly limited.
Examples of lipid molecules include egg yolk-derived phosphatidylcholine (egg-yolk PC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP). ), Phosphatidic acid (PA), phosphatidylglycerol (PG), sphingolipid and the like.
These lipid molecules may use only 1 type and may use 2 or more types.

  Examples of the method for forming the lipid bilayer membrane 14 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. Thereby, the hydrophilic part of the lipid molecule in n-decane gathers gradually in the interface of n-decane and the hole part 11, and forms 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 side). Since n-decane volatilizes at room temperature, a uniform lipid bilayer membrane 14 that finally covers the opening 11a of the hole 11 is formed.

The buffer solution 15 may be any buffer that can detect sufficient fluorescence and does not inhibit the function of the membrane protein 13, and includes, for example, Tris-HCl.
The amount of the buffer solution 15 may be an amount that sufficiently covers the lipid bilayer membrane 14.

  The extension means 20 may be any means as long as it can press the lipid bilayer membrane 14 and partially apply mechanical stimulation to extend the lipid bilayer membrane 14, and a deep needle of a scanning probe microscope such as AFM is preferable.

In addition, the system 1 preferably includes observation means for observing the surface structure of the lipid bilayer membrane 14.
Examples of the observation means include AFM. Further, if the membrane protein 13 or the lipid bilayer 14 is fluorescently labeled, a confocal laser scanning microscope can be used.

In addition, when the opening 11a of the hole 11 is too large and the lipid bilayer 14 is bent even if it does not extend and it is difficult to observe the structure on the lipid bilayer, the opening 11a of the hole 11 is covered. Thus, a network structure may be formed, and the lipid bilayer 14 may be formed on the network structure.
The network structure is a structure composed of nano linear bodies, and specifically, the nano linear body has a large number of branches to form a network.
Examples of the nanolinear body include nanotubes, nanowires, and nanofibers, and the material thereof is not particularly limited. However, carbon is preferable in that the nanolinear body can be easily formed.
Here, the nano linear body is a linear body having a diameter of 1 to 20 nm in a cross section perpendicular to the length direction. If the diameter of the cross section perpendicular to the length direction of the nanowire is not less than the lower limit, the lipid bilayer membrane can be sufficiently supported. Moreover, if it is below the said upper limit, even if a mesh structure is arrange | positioned on the hole part 11, it will not interfere with AFM observation of the biological sample for observation.
In the case of using carbon nanotubes as the nanowires, in order to make the diameter within the above range, the type, particle diameter and density of the metal catalyst used, and the temperature at the time of formation are determined in the method for forming the network structure described later. What is necessary is just to select suitably. For example, the larger the catalyst particle diameter, the larger the diameter.

Examples of the method for forming the network structure include a method of forming by a chemical vapor deposition method (CVD method). Examples of the carbon source when the chemical vapor deposition method is applied include methane, ethane, acetylene, methanol, ethanol, and the like.
Further, when the network structure is formed by chemical vapor deposition, it is preferable to use a metal catalyst such as iron because a nano linear body can be easily formed. In order to form a nano-linear body on the surface of the substrate body 12, it is preferable that a metal catalyst is attached to the surface of the substrate body 12 in advance.

Moreover, other embodiment of the membrane protein function measuring system of this invention is shown in FIG.
As shown in FIG. 2, the membrane protein function measuring system 2 (hereinafter referred to as “system 2”) includes a membrane protein function measuring substrate 10B (hereinafter referred to as “substrate 10B”) and an extension means 20. It has. In the system 2, the same parts as those in the system 1 are denoted by the same reference numerals and description thereof is omitted.
The substrate 10B includes a substrate body 12 having a hole portion 11, a lipid bilayer membrane 14 containing a membrane protein 13 that covers the opening 11a of the hole portion 11, a buffer solution 15 disposed on the lipid bilayer membrane 14, and It has the 1st electrode 16 which is provided in the exterior of the hole part 11, and contacts the buffer solution 15, and the 2nd electrode 17 provided in the inside of the hole part 11. FIG. The extension means 20 is means for extending the lipid bilayer membrane 14.

The first electrode 16 and the second electrode 17 are provided so as to be in contact with the buffer solution outside and inside the hole 11, respectively. The first electrode 16 and the second electrode 17 are each connected to an ammeter (not shown) so that a current flowing between the electrodes can be measured.
The second electrode 17 is preferably provided at the bottom of the hole 11.
Examples of the material for the electrode include metals such as copper, aluminum, gold, silver, silver chloride, platinum, chromium, and nickel, and conductive substances such as conductive resins.
The thickness of the electrode is preferably 50 to 500 nm.

  As in the system 1, the system 2 preferably includes observation means for observing the surface structure of the lipid bilayer 14 such as an AFM or a confocal laser scanning microscope. Alternatively, a network structure may be formed so as to cover the opening 11a of the hole 11, and the lipid bilayer membrane 14 may be formed on the network structure.

<Method for measuring membrane protein function>
The membrane protein function measuring method of the present invention is a method for measuring the function of a membrane protein when the lipid bilayer is stretched, and the localization of the membrane protein (receptor) in the lipid bilayer. The method using the membrane protein function measuring system of the present invention.
The membrane protein function measuring method of the present invention includes the following method (I), method (II) or method (III) depending on the function measuring means.
(I) When measuring the surface structure of a lipid bilayer by observation means such as AFM.
(II) When the function of a membrane protein is optically measured by a change in fluorescence intensity.
(III) When the function of the membrane protein is electrically measured by a change in current value.

The method (II) includes the following methods (II-1) to (II-4) depending on the type of membrane protein and fluorescent substance used for measurement.
(II-1) When the membrane protein is an ion channel type membrane protein and an ion sensitive fluorescent dye is used as a fluorescent substance.
(II-2) The membrane protein is an ion channel membrane protein and a nucleic acid-binding fluorescent dye is used as the fluorescent substance.
(II-3) When the membrane protein is an ion channel type membrane protein and an extracellular signal transmitting substance labeled with fluorescence is used as the fluorescent substance.
(II-4) When the membrane protein is a metabotropic membrane protein and an ion-sensitive fluorescent dye is used as the fluorescent substance.

Method (I):
Using system 1A using ion channel membrane protein 13A and metabotropic membrane protein 13B as membrane protein 13 in system 1, as shown in FIG. 3, lipid bilayer membrane 14 is extended by extension means 20 such as an AFM probe. At the same time, the surface structure of the lipid bilayer membrane 14 having the ion channel membrane protein 13A and the metabotropic membrane protein 13B is observed by an observation means (not shown). The lipid bilayer 14 is extended by extension means 20 such as an AFM probe.
Thereby, the structural change of the ion channel type membrane protein 13A and the metabotropic membrane protein 13B due to the extension of the lipid bilayer membrane 14 can be examined. Similarly, changes in the localization of the ion channel membrane protein 13A and the metabotropic membrane protein 13B in the lipid bilayer membrane 14 due to the extension of the lipid bilayer membrane 14 can be observed.

Method (II-1):
In the system 1, the membrane protein 13 is an ion channel type membrane protein 13A, an ion-sensitive fluorescent dye 32 that emits fluorescence by binding to ions 31 that permeate the membrane protein 13 is disposed in the hole portion 11, and lipids Using the system 1B in which the buffer solution 15 containing the ions 31 that permeate the membrane protein 13 is arranged on the bilayer membrane 14, as shown in FIG. The transmission of the ions 31 of the channel-type membrane protein 13A is measured from the change in fluorescence intensity.

When the lipid bilayer 14 is stretched by the stretching means 20, the structure of the ion channel membrane protein 13 </ b> A changes with the stimulation, the ion channel at the transmembrane portion opens, and the ions 31 flow into the hole 11. Then, the ion 31 that has flowed into the hole 11 and the ion-sensitive fluorescent dye 32 are combined to emit fluorescence, whereby a change in the activity of the ion channel membrane protein 13A due to the extension of the lipid bilayer membrane 14 can be measured.
The ion sensitive fluorescent dye 32 is contained in the buffer solution and disposed inside the hole 11. Any buffer solution may be used as long as it can detect sufficient fluorescence and does not inhibit the function of the ion channel membrane protein 13A. Examples thereof include Tris-HCl.

Examples of the ions 31 include calcium ions, sodium ions, chloride ions, and potassium ions.
What is necessary is just to determine the density | concentration of the ion 31 in the buffer solution 15 suitably according to a measurement, 1 nM-500 mM are preferable, and 10 micromol-150 mM are more preferable.

The ion-sensitive fluorescent dye 32 is a substance that emits fluorescence by binding to the ions 31, and can be combined with calcium ions (Ca ²⁺ ) to emit fluorescence, such as fura-2, fluo-3, fluo-4, sodium ions ( Examples thereof include SBFI that emits fluorescence by binding to Na ⁺ ), CoroNa, PBFI that emits fluorescence by binding to potassium ion (K ⁺ ), MQAE that emits fluorescence by binding to chloride ion (Cl ⁻ ), and the like.
The concentration of the ion-sensitive fluorescent dye 32 inside the hole 11 may be an amount sufficient to measure the activity of the ion channel membrane protein 13A by a change in fluorescence intensity, and is preferably 100 nM to 500 μM, preferably 1 μM to 100 μM. Is more preferred

Method (II-2):
In the system 1, the membrane protein 13 is an ion channel type membrane protein 13A, the nucleic acid 33 is disposed inside the hole 11, and the nucleic acid binding that binds to the nucleic acid 33 and enhances fluorescence on the lipid bilayer membrane 14 As shown in FIG. 5, using the system 1C in which the buffer solution 15 containing the fluorescent fluorescent dye 34 is disposed, the lipid bilayer membrane 14 is extended by the extension means 20, and the nucleic acid-binding fluorescence of the ion channel membrane protein 13A is obtained. The transmission of the dye 34 is measured from the change in fluorescence intensity.

When the lipid bilayer membrane 14 is stretched by the stretching means 20, the structure of the ion channel membrane protein 13A changes with the stimulation, the channel pore size changes, and the nucleic acid binding fluorescent dye 34 becomes the ion channel membrane protein 13A. It flows into the inside of the hole part 11 via. Then, the nucleic acid-binding fluorescent dye 34 and the nucleic acid 33 that have flowed into the hole 11 are combined to emit fluorescence, whereby the change in the activity of the ion channel membrane protein 13A due to the extension of the lipid bilayer membrane 14 can be measured.
The nucleic acid 33 is contained in the buffer solution and disposed inside the hole 11. Any buffer solution may be used as long as it can detect sufficient fluorescence and does not inhibit the function of the ion channel membrane protein 13A. Examples thereof include Tris-HCl.

Examples of the nucleic acid 33 include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid may be artificially synthesized or extracted from cells. The number of base pairs of the nucleic acid 33 is not particularly limited as long as the change in fluorescence intensity can be measured quantitatively.
The concentration of the nucleic acid 33 inside 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.

Examples of the nucleic acid binding fluorescent dye 34 include ethidium bromide and propidium iodide.
The concentration of the nucleic acid-binding fluorescent dye 34 inside the hole 11 may be an amount sufficient to measure the activity of the ion channel membrane protein 13A by a change in fluorescence intensity, and is preferably 100 nM to 500 μM, preferably 1 μM to 100 μM is more preferable

Method (II-3):
In the system 1, the membrane protein 13 is an ion channel type membrane protein 13A, and a fluorescently-labeled extracellular information transmission substance 35 (hereinafter referred to as “ligand molecule 35”) is disposed inside the hole 11. As shown in FIG. 6, the lipid bilayer membrane 14 is stretched by the stretching means 20, and the movement of the ligand molecule 35 from the inside of the hole 11 to the outside through the ion channel membrane protein 13A is fluorescence intensity. Measure from changes in

When the lipid bilayer membrane 14 is stretched by the stretching means 20, the ion channel membrane protein 13A is activated and the structure is changed in accordance with the stimulation, and the ligand molecule 35 passes through the ion channel membrane protein 13A. It flows out from the inside to the outside, and the fluorescence intensity is reduced inside the hole 11. Therefore, the change in the activity of the ion channel membrane protein 13A due to the extension of the lipid bilayer membrane 14 can be measured by measuring the change in the fluorescence intensity due to the ligand molecule 35 inside the hole 11.
The ligand molecule 35 is contained in the buffer solution and arranged inside the hole 11. Any buffer solution may be used as long as it can detect sufficient fluorescence and does not inhibit the function of the ion channel membrane protein 13A. Examples thereof include Tris-HCl.

The ligand molecule 35 is a compound in which a fluorescent substance is bound to an 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 that binds to the extracellular information transmission substance, NBD (nitrobenzofurazane), Texas Red, Alexa Fluor, fluorescein, rhodamine, and Cy dye are preferable.
Specific examples of the ligand molecule 35 include ATP-FITC and glutamate-FITC. A known method can be used as a method for binding FITC to an extracellular signal transmitting substance.
What is necessary is just to determine the density | concentration of the ligand molecule 35 suitably according to the objective of a measurement, 1 nM-10 mM are preferable, and 10 nM-100 micromol are more preferable.

Method (II-4):
In the system 1, the membrane protein 13 is a metabotropic membrane protein 13B, and a cytoplasmic extract fraction 36 and a calcium ion sensitive fluorescent dye 37 that emits fluorescence by binding to calcium ions are arranged inside the hole 11. Using 1E, as shown in FIG. 7, the lipid bilayer 14 is stretched by the stretching means 20, and the release of calcium ions from the endoplasmic reticulum via the intracellular signal transduction system is measured from the change in fluorescence intensity.

When the lipid bilayer 14 is extended by the extension means 20, the metabotropic membrane protein 13B is activated. Then, the guanosine diphosphate (GDP) -binding G protein existing in the hole 11 and associated with the metabotropic membrane protein 13B is replaced with a guanosine triphosphate (GTP) -binding G protein. Activates phospholipase C (PLC) downstream of the signal transduction pathway. PLC is to metabolize PI (4,5) _{P 2} which is one of phosphatidylinositol lipid bilayer produce inositol triphosphate (IP _3). IP ₃ changes its structure by binding to the IP ₃ receptor of the endoplasmic reticulum, and releases calcium ions present in the endoplasmic reticulum into the cytoplasm (hole 11 in this method (II-4)). The released calcium ions bind to the calcium ion-sensitive fluorescent dye 37 and increase the fluorescence intensity. Thus, the activity of the metabotropic membrane protein 13B activated by the extension of the lipid bilayer membrane 14 can be measured through these series of reactions.

The cytoplasmic extract 36 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 following methods can be mentioned. 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 36.

Examples of the calcium ion sensitive fluorescent dye 37 include fura-2, fluo-3, and fluo-4.
The concentration of the calcium ion sensitive fluorescent dye 37 inside the hole 11 may be an amount that can measure the activity of the G protein-coupled receptor by a change in fluorescence intensity, preferably 100 nM to 500 μM, and more preferably 1 μM to 100 μM. .

Method (III):
In the system 2, the membrane protein 13 is an ion channel type membrane protein 13A, and a buffer solution 15 containing ions 38 that permeate the membrane protein is disposed on the lipid bilayer membrane 14, and as shown in FIG. The lipid bilayer 14 is stretched by the stretching means 20 and the current between the first electrode 16 and the second electrode 17 is measured.
When the lipid bilayer 14 is stretched by the stretching means 20, the structure of the ion channel membrane protein 13 </ b> A changes with the stimulation, and the ion channel at the transmembrane portion opens and ions 38 flow into the hole 11. Since the current is detected between the first electrode 16 and the second electrode 17 due to the inflow of the ions 38, the change in the activity of the ion channel membrane protein 13A due to the extension of the lipid bilayer membrane 14 can be measured.

As the ions 38, the same ions as the ions 31 mentioned in the method (II-1) can be used.
The concentration of the ions 38 in the buffer solution 15 may be an amount that can sufficiently detect the current, preferably 1 nM to 500 mM, more preferably 10 μM to 150 mM.
A buffer solution is placed inside the hole 11. The buffer solution arranged inside the hole 11 is preferably the same buffer solution as the buffer solution 15 arranged on the lipid bilayer membrane 14.

  According to the membrane protein function measuring system and the membrane protein function measuring method of the present invention described above, the change in the activity of the membrane protein when the lipid bilayer provided on the hole of the membrane protein function measuring substrate is extended is measured. can do. Further, by combining the method (I) with the method (II) or the method (III), that is, using an observation means such as AFM and measurement of optical or electrical activity, a lipid bilayer membrane is obtained. The relationship between the density change of the receptor in the lipid bilayer when stretched and the activity can be measured.

  In addition, the membrane protein function measuring method of this invention is not limited to the above-mentioned method. For example, in the method (I), only the ion channel membrane protein may be used, or only the metabolite membrane protein may be used. Further, in the methods (II) and (III), measurement may be performed using both ion channel membrane proteins and metabotropic membrane proteins as membrane proteins.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description.
The fluorescence image in this example was observed using a confocal laser scanning microscope (trade name: LSM510, manufactured by Zeiss).

[Experimental Example 1]
Phosphatidylcholine derived from egg yolk was dissolved in chloroform, dried under an argon gas atmosphere, and dissolved in a buffer solution (10 mM Tris-HCl, pH 7.4) at a final concentration of 160 μg / mL. The buffer solution was treated with an ultrasonic crusher to prepare lipid vesicles. Next, lipid (fluorescein-bound dihexadecanoylglycerophosphoethanolamine) fluorescently labeled with 20% by mass of fluorescein is added to the lipid vesicles to a final concentration of 40 μg / mL to further prepare lipid vesicles. By doing so, a lipid bilayer membrane that can be observed by fluorescence was prepared. A fluorescent dye (Alexa 647, maximum excitation wavelength 650 nm, maximum fluorescence wavelength 668 nm, He-Ne laser used, final concentration 500 μM) is mixed with the buffer containing the lipid vesicles, and a substrate having a hole (nanowell) in that state A vesicle fusion was conducted. Then, when the fluorescence image of this board | substrate was observed, the fluorescence derived from Alexa 647 was observed in the hole of the board | substrate (FIG. 9 (a), measurement area | region 500 * 500 micrometer). The buffer solution arranged on the substrate was replaced 10 times with a lipid-free buffer solution (10 mM Tris-HCl, pH 7.4) to remove unnecessary substances. Thereafter, when the fluorescence image of the substrate was observed, it was observed that the lipid bilayer membrane was adhered to the substrate, and the alexa was only in the hole portion covered with the lipid bilayer membrane (FIG. 9B). From the fact that fluorescence derived from 647 was observed, it was found that the hole was sufficiently covered and sealed with a lipid bilayer membrane.

[Experiment 2]
Dipalmitoyl phosphatidylcholine, 1,2-dilauroyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol were mixed at a ratio of 75: 23: 2. Lipid vesicles were prepared by dissolving in a buffer solution (10 mM Tris-HCl, pH 7.4) at a concentration of 200 μg / mL. The buffer containing the lipid vesicles is dropped onto a substrate having a hole (nanowell) to produce a lipid bilayer membrane covering the hole, and the lipid bilayer is extended with an AFM deep needle, Observed (FIG. 10). The number of seconds in FIG. 10 means the observation elapsed time when the start of extension is 0 second.
The lipid bilayer membrane covering the hole was found to be formed flat from the height information (FIG. 10 (a), 15 seconds). When the mechanical stimulation by the AFM deep needle was gradually increased, the lipid bilayer membrane covering the hole was stretched to the inside of the hole (FIG. 10 (a), 15 seconds to 215 seconds). . Thereafter, when the mechanical stimulation was weakened, the stretched lipid bilayer became flat as it was (FIG. 10 (a), 215 to 255 seconds). Thus, it was found that the lipid bilayer did not move and adhered to the substrate even under the condition of applying the extension stimulus under this condition.
On the other hand, when mechanical stimulation is further applied to the lipid bilayer membrane that has returned to the flat state, the lipid bilayer membrane covering the hole is finally broken (FIG. 10 (b), 110 seconds). The lipid bilayer was peeled toward the left of the part (FIG. 10 (b), 140 seconds). At this time, it was found from the height information that the lipid bilayer was broken in the hole.

[Example 1]
A complex membrane (purple membrane) of 75% by mass of bacteriorhodopsin (BR) and 25% by mass of lipid bilayer membrane was dissolved in a buffer solution (10 mM Tris-HCl, pH 8.0, 1MKCl) at a concentration of 5 μg / mL, and a mica substrate It was dripped on and left still at room temperature for 30 minutes. Thereafter, the membrane was replaced 10 times with a buffer solution containing no purple membrane, and the purple membrane not adhered to the mica substrate was washed away. The purple film adhered to the mica substrate has a film-like structure with uniform height (FIGS. 11A and 11B), and when enlarged, it was observed that the molecules are regularly arranged (FIG. 11 ( c)). When this observation result was compared with the research result of DJ Muller et al. (Journal of Molecular Biology (1999) 285, 1903-1909), the molecule in region X in FIG. 11 (c) is a trimeric BR protein. It was considered.
On the other hand, a purple membrane was adhered to a substrate having a hole as described above, and the BR protein on the hole was observed (FIG. 12, (a)). The purple membrane covering the hole was not flat but bent (FIG. 12, (b)). Even in this state, trimers of individual BR proteins could be observed (FIG. 12 (c), region Y). In this observation, a significant structural change of the BR protein was not observed by the stimulation of extending the lipid bilayer with the AFM deep needle, and the trimeric structure was maintained. In contrast, the distance between the trimer structures was greatly changed, and it was revealed that the localization of the BR protein in the lipid bilayer membrane was changed.
Thus, with the measurement method of the present invention, the structure of the membrane protein and the positional relationship between the membrane proteins could be observed with the lipid bilayer stretched.

[Example 2]
Egg yolk-derived phosphatidylcholine is dissolved in a buffer solution (10 mM Tris-HCl, pH 7.4) at a concentration of 160 μg / mL, and dropped onto a substrate having a hole to produce a lipid bilayer film on the hole. Then, the receptor was inserted by fusing a proteoliposome containing NMDA receptor to the lipid bilayer. Fluoro-4 (10 μM), which is a calcium-sensitive fluorescent dye, was added to the inside of the hole, and ₂ mM CaCl ₂ was added to the buffer solution (10 mM Tris-HCl, pH 7.4) outside the hole. When the lipid bilayer was stretched (starting at 0 second), the fluorescence intensity in the observation region increased (FIG. 13 (a)). When the state of the substrate at this time was observed with fluorescence, fluorescence associated with the extension of the lipid bilayer was observed in the hole (FIG. 13B). As a result, it was clarified that the NMDA receptor was activated by the extension of the lipid bilayer and permeated calcium ions existing outside the hole into the hole.

[Example 3]
A lipid bilayer membrane containing an NMDA receptor in the hole was prepared in the same manner as in Example 2 except that electrodes were provided inside and outside the hole, respectively. When the lipid bilayer was extended with an AFM deep needle, the current value between the electrodes changed (FIG. 13C).
Since the purified NMDA receptor was inserted into the lipid bilayer membrane, this change in current value was thought to be due to the activation of the NMDA receptor by the extension of the lipid bilayer membrane.

  INDUSTRIAL APPLICABILITY The present invention is useful as a measurement system and a measurement method for analyzing the function of a membrane protein related to the extension of a lipid bilayer, and relates to the extension of a lipid bilayer in a body such as myocardial infarction and hypertension. It can be suitably used as a membrane protein screening system.

  1, 1A-1E, 2 Membrane Protein Function Measurement System 10 Membrane Protein Function Measurement Substrate 11 Hole 12 Substrate Body 13 Membrane Protein 13A Ion Channel Type Membrane Protein 13B Metabolic Membrane Protein 14 Lipid Bilayer 15 Buffer 16 Electrode 17 second electrode 20 extension means 31 ion 32 ion sensitive fluorescent dye 33 nucleic acid 34 nucleic acid binding fluorescent dye 35 ligand molecule 36 cytoplasmic extract fraction 37 calcium ion sensitive fluorescent dye 38 ion

Claims (9)

A substrate for measuring a membrane protein function, wherein the opening of the substrate body having a hole is covered with a lipid bilayer membrane containing a membrane protein, and a buffer solution is disposed on the lipid bilayer membrane,
Stretching means for stretching the lipid bilayer;
A membrane protein function measuring system comprising:   The membrane protein function measuring system according to claim 1, wherein the extension means is a deep needle of a scanning probe microscope.   The membrane protein function measuring system according to claim 1 or 2, further comprising observation means for observing the surface structure of the lipid bilayer membrane.   Furthermore, the film | membrane in any one of Claims 1-3 which comprises the 1st electrode provided in the exterior of the said hole part in the said board | substrate, and the 2nd electrode provided in the inside of the said hole part. Protein function measurement system. A method for measuring the function of a membrane protein using the membrane protein function measuring system according to any one of claims 1 to 3,
The membrane protein is an ion channel membrane protein;
An ion-sensitive fluorescent dye that emits fluorescence by binding to ions that permeate the membrane protein is disposed inside the hole, and a buffer containing ions that permeate the membrane protein is disposed on the lipid bilayer membrane. And
A membrane protein function measuring method comprising: measuring the function of a membrane protein by extending the lipid bilayer membrane by the extension means, measuring the ion permeation of the ion channel membrane protein from a change in fluorescence intensity. A method for measuring the function of a membrane protein using the membrane protein function measuring system according to any one of claims 1 to 3,
The membrane protein is an ion channel membrane protein;
Inside the hole, a nucleic acid is arranged, and on the lipid bilayer membrane, a buffer solution containing a nucleic acid-binding fluorescent dye that binds to the nucleic acid and enhances fluorescence is arranged,
A membrane protein function measuring method for measuring the function of a membrane protein by extending the lipid bilayer membrane by the extension means, measuring the transmission of the nucleic acid binding fluorescent dye of the ion channel membrane protein from a change in fluorescence intensity . A method for measuring the function of a membrane protein using the membrane protein function measuring system according to any one of claims 1 to 3,
The membrane protein is an ion channel membrane protein;
Inside the hole, a fluorescently labeled extracellular information transmitting substance is disposed,
The lipid bilayer is stretched by the stretching means, and the movement of the extracellular information transmitting substance from the inside of the hole to the outside through the ion channel membrane protein is measured from a change in fluorescence intensity, A membrane protein function measuring method for measuring protein function. A method for measuring the function of a membrane protein using the membrane protein function measuring system according to any one of claims 1 to 3,
The membrane protein is a metabotropic membrane protein;
Inside the hole, a cytoplasmic extract fraction, and a calcium ion sensitive fluorescent dye that emits fluorescence by binding to calcium ions are arranged,
Membrane protein function measurement in which the lipid bilayer is stretched by the stretching means, and the release of calcium ions from the endoplasmic reticulum via an intracellular signal transduction system is measured from the change in fluorescence intensity to measure the function of the membrane protein. Method. A method for measuring the function of a membrane protein using the membrane protein function measuring system according to claim 4,
The membrane protein is an ion channel membrane protein;
A buffer solution containing ions that permeate membrane proteins is disposed on the lipid bilayer membrane,
A membrane protein function measuring method for measuring a function of a membrane protein by extending the lipid bilayer by the extension means and measuring a current flowing between the first electrode and the second electrode.