Ion channel protein evaluation model, ion channel current measuring device, and method for manufacturing the ion channel protein evaluation model

Abstract

To provide an ion channel protein evaluation model and a method for producing the same, which allows for the incorporation of multiple channel proteins into a single lipid bilayer. [Solution] An ion channel protein evaluation model in which an ion channel protein is provided penetrating a lipid bilayer, and the ion channel protein is linked to gel beads.

Description

Technical Field

[0001] The present invention relates to an ion channel protein evaluation model, an ion channel current measurement device, and a method for manufacturing an ion channel protein evaluation model.

Background Art

[0002] Ion channel proteins are transmembrane proteins that penetrate the cell's biomembrane (lipid bilayer), and are proteins that are involved in various information transmissions inside and outside the cell by controlling the permeation of ions inside and outside the cell, and play extremely important roles in vivo.

[0003] It is known that when an abnormality occurs in an ion channel protein (hereinafter also referred to as "channel protein" in this specification), serious diseases are often induced. For example, muscular dystrophy that develops when an abnormality occurs in a calcium channel, alternating hemiplegia of childhood that develops when an abnormality occurs in Na + / K + -countertransport ATPase, cystic fibrosis that develops when an abnormality occurs in a chloride ion channel, periodic paralysis that develops when an abnormality occurs in a voltage-gated potassium channel, etc. are known.

[0004] These diseases are so-called "channelopathies", and the development of effective therapeutic agents is an urgent task. It is said that about 15% of the 6,650 human genes that can be targets for pharmaceuticals are channel proteins, and a highly efficient and highly accurate screening method is required.

[0005] As a screening method from the In vitro perspective of such channelopathy therapeutic agents, a method of creating an artificial biomembrane (lipid bilayer), incorporating a channel protein, and measuring current can be cited. While this method has many advantages such as not being limited by cell types, being applicable to intracellular membrane system channels, and having a high degree of freedom in the measurement environment, it has the disadvantage of low efficiency.

[0006] Conventional methods for measuring the current of such channel proteins involve artificially forming a lipid bilayer in aqueous solution, inserting ion channel proteins into the lipid bilayer via membrane fusion, and then measuring the ionic current. However, this method requires a considerable amount of time for the artificial formation of the lipid bilayer in aqueous solution. Furthermore, the process of inserting ion channel proteins into the formed lipid bilayer also requires a lot of time, and it is difficult to insert multiple ion channel proteins simultaneously, making it extremely inefficient.

[0007] Therefore, a method has been proposed in which channel proteins are bound to gold electrodes coated with a hydrophilic membrane and inserted into an artificially fabricated lipid bilayer (Patent Document 1). While this method can shorten the time required for lipid bilayer formation, there are still obstacles to inserting multiple ion channel proteins, making multi-channelization practically impossible. Therefore, there is a need for a more efficient ion channel protein evaluation model that can incorporate multiple ion channel proteins into a lipid bilayer. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 6632826 [Overview of the project] [Problems that the invention aims to solve]

[0009] In view of the above circumstances, the object of the present invention is to provide an ion channel protein evaluation model and a method for producing the same, which can incorporate multiple channel proteins into a single lipid bilayer. [Means for solving the problem]

[0010] The inventors diligently conducted research to solve the above problems and discovered that multi-channelization is possible by linking ion channel proteins to gel beads and inserting them into an artificially formed lipid bilayer. Based on this finding, the inventors conducted further research and completed the present invention.

[0011] In other words, the present invention provides the following ion channel protein evaluation model, ion channel current measuring device, and method for manufacturing the ion channel protein evaluation model. Section 1. An ion channel protein evaluation model in which an ion channel protein is provided penetrating a lipid bilayer, and the ion channel protein is linked to gel beads. Section 2. An ion channel current measuring device, including the ion channel protein model described in item 1. Section 3. The ion channel current measuring device according to claim 2, having one or more suction ports for aspirating and fixing one or more gel beads, a space for holding the one or more gel beads, and a space for holding a water-soluble solvent on the side opposite to the one or more suction ports. Section 4. A step of bringing ion channel proteins immobilized on gel beads into contact with the surface of a lipid dissolution layer laminated on a water-soluble solvent layer, and A method for producing the ion channel protein evaluation model according to item 1, comprising the step of inserting the ion channel protein to the interface between the water-soluble solvent layer and the lipid dissolution layer. Section 5. The method for producing the product according to item 4, wherein the ion channel protein is contained in a membrane vesicle linked to the gel beads. Section 6. The manufacturing method according to item 4 or 5, wherein the membrane vesicles are linked to the gel beads by His-tag or Biotin-tag. [Effects of the Invention]

[0012] The ion channel protein evaluation model according to the present invention thus formed can incorporate a plurality of ion channel proteins into an artificially formed lipid bilayer membrane.

Brief Description of Drawings

[0013] [Figure 1] Schematic diagram of lipid and lipid bilayer membrane. [Figure 2] Explanatory drawing of the method for manufacturing the ion channel protein evaluation model of the present invention. [Figure 3] Explanatory drawing of the method for manufacturing the ion channel protein evaluation model of the present invention. [Figure 4] Schematic diagram of an ion channel protein linked to gel beads. [Figure 5] Schematic diagram of a membrane vesicle. [Figure 6] Schematic diagram of an example of the ion channel current measuring device of the present invention. [Figure 7] Schematic diagram for manufacturing the ion channel current measuring device of the present invention. [Figure 8] Measurement results of Example 1. [Figure 9] Measurement results of Example 2. [Figure 10] Measurement results of Example 3. [Figure 11] Measurement results of Example 4. [Figure 12] Measurement results of Examples 5 to 8. [Figure 13] An example of an embodiment of the ion channel current measuring device and test results. [Figure 14] An example of another embodiment of the ion channel current measuring device and test results.

Modes for Carrying Out the Invention

[0014] In this specification, "contains" is a concept that encompasses all of the following: "contains," "consist essentially of," and "consist of." Furthermore, in this specification, when a numerical range is indicated as "A~B," it means A or greater and B or less.

[0015] (1. Ion channel protein evaluation model) The ion channel protein evaluation model of the present invention is an evaluation model in which ion channel proteins are provided penetrating a lipid bilayer, and one or more of these ion channel proteins are linked to gel beads.

[0016] The ion channel protein evaluation model of the present invention is an evaluation system for simulating the function of transmembrane proteins present in lipid bilayers that constitute biological membranes, and for example, the activity of ion channel proteins can be measured by measuring membrane current.

[0017] Ion channels are membrane proteins located in the plasma membrane or intima-membrane system that play a role in ion permeability. They are responsible for all physiological phenomena involving ions, such as the generation of action potentials in nerve cells, muscle contraction, neurotransmitter release, hormone secretion, and sensation. Since the lipid bilayer that makes up biological membranes is almost impermeable to ions, ion channels are essential proteins for biological functions to allow ions to pass in and out of the membrane, and are expressed in all cells, from bacteria to higher animals. Ion channels have an ion permeability pathway, which functions to allow ions to flow in and out according to the gradient of concentration and potential. In addition, the ion permeability pathway usually has a gate that opens and closes in response to stimuli such as membrane potential and ligands. Furthermore, the type and size of ions that can pass through are determined by the ion-selective filter present in the ion permeability pathway.

[0018] Due to these unique properties, ion channels can be classified according to the type of ions they pass through, the type of gate, and their topology (how many times they penetrate the membrane). They can also be classified by their gate, including voltage-gated channels that open and close depending on the membrane potential, ligand-gated ion channels (ion channel receptors) that open when a ligand binds, and mechanostimulatory channels.

[0019] Examples of ion channels used herein include, but are not limited to, voltage-gated potassium channels, voltage-gated sodium channels, voltage-gated calcium channels, calcium-activated potassium channels, members of the voltage-gated channel family including HCN (Hyperpolarization-activated cyclic nucleotide-gated) channels, CNG (Cyclic nucleotide-gated) channels, TRP (Transient receptor potential) channels, and voltage-gated proton channels, as well as inwardly rectified potassium channels, two-pore channels, acid-sensitive ion channels, epithelial sodium channels, chlorine channels, ligand-gated channels including Cys-loop receptors, glutamate receptors, and P2X receptors, intracellular membrane ion channels including ryanodine receptors, IP3 receptors, and TRIC channels, and connexins.

[0020] Lipids that are the main components of biological membranes can be suitably used as lipids for lipid bilayers. Specifically, phospholipids, glycolipids, cholesterol, etc., can be used. Among these, phospholipids are lipids that have a phosphate ester moiety in their structure, and a wide range of known phospholipids can be used without particular limitations. More specifically, phospholipids in which fatty acids and phosphate are bonded to a central skeleton such as glycerol or sphingosine, and an alcohol is further ester-bonded to the phosphate moiety, are preferred. In other words, glycerophospholipids or sphingophospholipids can be suitably used. These may be used individually or in combination of multiple types.

[0021] As shown in Figure 1A, lipids such as phospholipids are composed of a hydrophilic head and a hydrophobic tail. As shown in Figure 1B, multiple phospholipids 2 are arranged in parallel with their hydrophilic heads facing the solvent and their hydrophobic tails facing away from the solvent, thereby forming a lipid bilayer 3 that forms the cell membrane in living organisms.

[0022] As shown in Figure 1B, ion channel protein 1 is located across the lipid bilayer.

[0023] The gel constituting the gel beads is preferably a hydrophilic gel, and more preferably a hydrophilic polysaccharide gel composed of polysaccharides. Among these, polyacrylamide gel, dextran gel, or agarose gel is more preferable, and agarose gel is particularly preferable.

[0024] The gel beads are preferably approximately spherical in shape, and in order to facilitate the formation of a lipid bilayer, the diameter of the gel beads is preferably 1.0 μm or more, more preferably 10 μm or more, and even more preferably 50 μm or more. Furthermore, if the gel beads are too large, the lipid bilayer described later will become unstable, so the diameter of the gel beads is preferably 1000 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less.

[0025] The amount of crosslinking agent contained in the gel constituting the gel beads is, for example, when a polysaccharide gel is used as the gel, preferably the amount of polysaccharide (for example, the amount of agarose in the case of agarose gel beads) is 0.5% by mass or more, more preferably 1.0% by mass or more, and even more preferably 2.0% by mass or more, relative to the total mass of the gel beads. On the other hand, preferably the amount of polysaccharide is 10% by mass or less, more preferably 8% by mass or less, and even more preferably 6% by mass or less, relative to the total mass of the gel beads.

[0026] Furthermore, it is preferable to chemically modify the gel beads to improve their retention of the tag protein, as described later. A wide range of known methods can be used for this chemical modification, and there are no particular limitations. Specifically, known chemical modifications such as epoxy activation and glyoxylation are preferable.

[0027] It is preferable that the surface of the gel beads is flexible, and in order to impart flexibility to the surface of the gel beads, it is preferable to use non-crosslinked gel beads, and in particular, it is preferable to use non-crosslinked agarose gel beads.

[0028] (2. Method for manufacturing an ion channel protein evaluation model) This invention includes an invention relating to a method for producing an ion channel protein evaluation model.

[0029] The present invention provides a method for producing an ion channel protein, comprising the steps of: contacting an ion channel protein immobilized on gel beads with the surface of a lipid dissolution layer laminated on a water-soluble solvent layer; and inserting the ion channel protein up to the interface between the water-soluble solvent layer and the lipid dissolution layer.

[0030] The water-soluble solvent constituting the water-soluble solvent layer can be any known hydrophilic solvent, and there are no particular limitations. Specifically, aqueous solutions of sodium chloride, calcium chloride, and potassium chloride can be used. These may be used individually or in combination of two or more. The concentration can be, for example, 0.3 to 10% by mass, and there are no particular limitations.

[0031] The lipid dissolution is a solution in which the above-mentioned lipids are dissolved in an oily solvent. As shown in Figure 2, the lipid dissolution layer 4 is layered on top of the water-soluble solvent layer 5. At this stage, at the interface between the lipid dissolution layer 4 and the water-soluble solvent layer 5, a single layer of first lipid layer 6 is formed, with the hydrophilic heads facing the water-soluble solvent layer and the hydrophobic tails facing away from the water-soluble solvent layer, arranged in a line.

[0032] Regarding the oily solvent used in the lipid dissolution, a wide range of known lipophilic solvents can be used, and there are no particular limitations. Specifically, examples include decane, hexadecane, and squalene. These may be used individually or in combination in combination.

[0033] The lipid content in the lipid dissolution is preferably 0.1% by mass or more, more preferably 1.0% by mass or more, and even more preferably 1.5% by mass or more. Furthermore, the lipid content in the lipid dissolution is preferably 10.0% by mass or less, more preferably 7.5% by mass or less, and even more preferably 5.0% by mass or less.

[0034] As shown in Figure 3, the ion channel protein immobilized on the gel bead is brought into contact with the surface of the lipid dissolution layer laminated on the water-soluble solvent layer, that is, the side of the lipid dissolution layer that is not in contact with the water-soluble solvent layer. In this case, by using hydrophilic gel beads, the hydrophilic heads of the lipids in the lipid dissolution layer are attracted toward the ion channel side, and conversely, the hydrophobic tails face the opposite side, thereby forming a second lipid layer 9 and completing the lipid bilayer 10.

[0035] In this case, as shown in Figures 4A and 4D, the ion channel protein may be directly immobilized on the gel beads via a tag such as a His-tag or Biotin-tag. However, as shown in Figures 4B and 4C, the method of incorporating and encapsulating the ion channel protein in a membrane vesicle is preferable because it shortens the time required to complete the model.

[0036] Membrane vesicles are spherical vesicles mainly composed of phospholipids, as shown in Figure 5 (omitted). They are generally called vesicles or liposomes, and are composed of a lipid bilayer, and may also contain other proteins, etc., as long as they do not hinder the objectives of the present invention.

[0037] Here, as shown in Figure 4B, the gel beads and the ion channel protein may be directly immobilized, or as shown in Figure 4C, the gel beads may be immobilized with the membrane lipids constituting the membrane vesicles. However, the method of immobilizing the gel beads with the ion channel protein in the membrane vesicles is particularly preferred because it allows for highly efficient and rapid integration of the ion channel protein into the lipid bilayer shown in Figure 5. Even when using membrane vesicles, tags such as His-tag or Biotin-tag can be suitably used to immobilize the membrane vesicles or ion channel protein with the gel beads.

[0038] For convenience, Figures 4B and 4C illustrate a configuration in which one ion channel protein is linked to one gel bead; however, in reality, multiple ion channel proteins are likely to be linked to a single gel bead, preferably via tags.

[0039] By inserting the ion channel protein to the interface between the water-soluble solvent layer and the lipid dissolution layer, the lipids constituting the membrane vesicles are incorporated into the lipid bilayer shown in Figure 5. As a result, an ion channel protein evaluation model like the one shown in Figure 3 is completed.

[0040] (3. Ion channel current measuring device) This invention includes an invention relating to an ion channel current measuring device.

[0041] The above-described ion channel protein evaluation model can be fabricated in multiples simultaneously, and it is preferable to utilize it as an ion channel current measuring device by providing one or more of these models. Conventional ion channel protein evaluation models are difficult to make multi-channel and require several tens of minutes to several hours to fabricate, whereas the ion channel protein evaluation model of the present invention can be fabricated in tens of seconds and is easy to make multi-channel, making it extremely useful in in vitro screening for the development of new drugs for channel diseases.

[0042] The specific configuration of such an ion channel current measuring device is not particularly limited, as long as it is capable of performing ion channel current measurement using the ion channel protein evaluation model described above.

[0043] As an example of a specific embodiment of such an ion channel current measuring device, one can be given a configuration having one or more suction ports for suctioning and fixing gel beads, as shown in Figure 6.

[0044] In this embodiment, as shown in Figures 6(A) to (D), it is preferable to provide a suction port for fixing the gel beads, a space for holding the gel beads, and a space for filling with an aqueous solvent on the side opposite to the suction port (the opposite side via the gel beads).

[0045] The specific procedure for preparing the ion channel current measuring device is as follows: as shown in Figure 6(A), fill the suction port 13 for fixing the gel beads, the space 12 for holding the gel beads, and part or all of the space 11 provided on the side opposite the suction port with a water-soluble solvent, and then, as shown in Figure 6(B), add the gel beads and perform suction from the suction port to fix the gel beads to the suction port.

[0046] In this case, it is preferable to pre-attach one or more ion channel proteins to each gel bead. In particular, having multiple ion channel proteins attached to each gel bead is preferable because when the gel beads are aspirated and fixed, there is a high probability that ion channel proteins will be present on the side opposite the aspiration port.

[0047] Next, as shown in Figure 6(C), for example, it is preferable to inject the lipid dissolving solution into the space for holding the gel beads while the suction port for fixing the gel beads and the space on the opposite side of the suction port remain filled with a water-soluble solvent. As a result, the hydrophilic heads of the lipids contained in the lipid dissolving solution layer face the respective water-soluble solvent layers.

[0048] Although embodiments of the present invention have been described above, the present invention is not limited in any way to these examples, and can be implemented in various forms without departing from the spirit of the invention. [Examples]

[0049] The embodiments of the present invention will be described in more detail below based on examples, but the present invention is not limited to these.

[0050] Preparation of gel beads The gel beads used in the ion channel current measuring device of the present invention can be commercially available. Sepharose 4B (4% agarose, average diameter 90 μm) and Sepharose 6B (6% agarose, average diameter 90 μm) were obtained from GE Healthcare. These gel beads were used either as is or after being chemically modified according to a standard method as appropriate.

[0051] Chemical modification of gel bead surface <Synthesis of short spacer beads (1)> Based on reaction equation 1 below, 2 ml / wet of Sepharose 6B was suspended in 10 ml of 1 M NaOH, NaBH4 (2 mg / ml) was added, glycidol was added dropwise at a temperature below 25°C, and the mixture was gently shaken overnight at room temperature. After thoroughly washing the bead suspension with Milli-Q water and 1 M NaCl, 0.1 M NaIO4 was added to the bead suspension at a ratio of 10%, and the mixture was gently shaken at room temperature for 1 hour to obtain glyoxyl agarose beads. 2 ml / wet of glyoxyl agarose beads was suspended in 10 ml of MeOH-AcOH (volume ratio 10:1), and AB-NTA or streptavidin was added. Then, pic-BH3 (10 mg / ml) was added to the suspension, and the mixture was gently shaken at room temperature for 24 hours. The product was thoroughly washed with Milli-Q water to obtain NTA beads or avidin beads. NTA beads were converted into NTA-N beads by immersing them in a NiCl2 solution to remove the Ni deposit.

[0052] [ka]

[0053] <Synthesis of long spacer (6B-NTA) beads> 500 μl / wet of epoxy-activated Sepharose beads was added to 2 ml of 0.1 M borate buffer (pH 10.5) with either AB-NTA or streptavidin, and the mixture was gently shaken overnight at room temperature. The reaction mixture was blocked for free epoxy groups with 0.5 M ethanolamine, and the product was thoroughly washed with Milli-Q water to obtain NTA beads or avidin beads. NTA beads were converted to NTA-N beads by immersion in NiCl2 solution to remove the Ni.

[0054] Immobilization of ion channel proteins onto modified beads <Immobilization of biotin-binding ion channel proteins onto avidin beads> Biotin tags were conjugated to the KcsA E71A mutant using conventional methods. Similarly, biotin-binding ion channel proteins were prepared using human BK channels, RP-CNG channels, KcsA / RP-CNG channels, and SthK channels.

[0055] Binding of His-tagged ion channel proteins to Ni-NTA beads The KcsA(E71A) mutant was conjugated with a His tag using a standard method. Similarly, His tag-binding ion channel proteins were prepared using human BK channels, RP-CNG channels, KcsA / RP-CNG channels, and SthK channels.

[0056] (Channel current measurement test) A glass capillary (GC150T-10, Harvard Apparatus, Holliston, MA, USA) was pulled using a pipette puller (P-97, Sutter Instrument, Novato, CA, USA) to create a pipette (tip diameter less than 1 μm), and then the tip was cut to create a larger opening (diameter greater than 10 μm). The tip was treated with a microforge (MF-900, Narishige, Tokyo, Japan) to smooth the surface. The glass pipette was set in a pipette holder, and beads were drawn through a tube from the holder using a syringe and fixed to the tip of the pipette.

[0057] Integration of ion channel proteins into artificial membranes using membrane fusion method Figure 7 shows a schematic diagram of the experimental apparatus. A cut glass tube (8 mm inner diameter) attached to a glass slide was used as the bath. An aqueous recording solution (200 mM KCl, 10 mM succinic acid (pH 4)) was poured into the bath chamber, and a lipid solution (30 mg lipid / ml n-decane or hexadecane) was placed on top of the recording solution layer. A bilayer was created by moving a bead, fixed by suction to the tip of a pipette, through this lipid solution layer into the recording solution (Explanation (3) in Figure 7). The bead was moved downward using a manipulator until it came into contact with the lipid / aqueous solution interface. The depth to which the bead was immersed in the aqueous solution was usually 10-100 μm. Simply moving the bead with a coarse manipulator resulted in the spontaneous formation of a lipid bilayer immediately after contact.

[0058] (Example 1: Short spacer (6B-NTA) bead-KcsA channel) KcsA (E71A E146Q D149N), solubilized with a surfactant (decyl maltoside), was brought into contact with 4B-NTA beads, fixed with His-tags, and the channel current was measured. From this, channel-mediated current fluctuations were confirmed within several tens of minutes. A step-like trace with a high probability of opening was observed, which is consistent with the characteristics of the KcsA channel. Furthermore, the results of measuring the channel current using gel beads are shown in Figure 8. After forming the artificial membrane, the current was recorded while changing the membrane potential in increments of several tens of mV, and an IV graph was created. From the IV graph, the single-channel conductance was estimated to be 116.8 pS. The single-channel conductance of KcsA (E71A E146Q D149N) at 200 mM KCl is reported to be 100-200 pS, which is consistent with the value obtained here. From the above, it is concluded that the channel current of KcsA (E71A E146Q D149N) was measured in this experiment.

[0059] (Example 2: Long spacer (6B-NTA) bead-KcsA channel) KcsA (E71A E146Q D149N), solubilized with a surfactant (decyl maltoside), was brought into contact with 6B-NTA beads, fixed with His-tags, and the channel current was measured. The measurement results are shown in Figure 9. After the formation of the artificial membrane, channel-mediated current fluctuations could be confirmed within a few minutes to several tens of minutes. A step-like trace with a high open-channel probability, characteristic of KcsA, was obtained. Furthermore, from the IV graph created by changing the membrane potential every 10 mV, the single-channel conductance was estimated to be 152.4 pS. From the above, it can be said that the KcsA channel current was measured in this experiment.

[0060] (Example 3) Furthermore, the His-tag of KcsA (E71A) reconstituted into vesicles was immobilized on beads and measured (Figure 10). The step-like trace characteristic of KcsA was confirmed. In addition, the single-channel conductance was estimated to be 129.6 pS from the IV graph. From the above, it was found that channels reconstituted into vesicles can be immobilized on beads and measured. Since KcsA is reconstituted into vesicles, it is thought that KcsA is incorporated into the artificial membrane via vesicle fusion. When using vesicle fusion, the time until activity begins to appear was generally several tens of seconds, which was faster than when only the channel was immobilized, so it can be said that immobilizing together with vesicles is a more efficient measurement method.

[0061] (Example 4: Avidin bead-KcsA channel mutant) Biotin vesicles reconstituted with the KcsA (E71A) mutant (see Figure 4C) were immobilized on streptavidin beads and measured (Figure 11). From the IV graph, the single-channel conductance was estimated to be 152.4 pS. Therefore, it was found that biotin vesicles reconstituted with KcsA can be immobilized and measured using avidin beads. Since the vesicles are immobilized on beads, channel integration is thought to occur via vesicle fusion. Because the measurement could be completed in approximately several tens of seconds, it can be said that measurement using vesicle fusion is effective.

[0062] (Examples 5-8) For human BK channels, RP-CNG channels, KcsA / RP-CNG channels, and SthK channels, the same method as in Examples 1, 2, or 3 above was used to immobilize agarose beads with His-tags or biotin-tags, and the agarose gels were brought into contact with the measurement solution to obtain the results. The measurement results are shown in Figure 12.

[0063] (Evaluation test using an ion channel current measuring device: Embodiment 1) As shown in Figure 13, the beads and recording solution were flowed together in one direction (lateral direction) through the channel, and then the beads were aspirated and fixed at the intersection with a narrow vertical channel (hole). After filling the channel with lipid solution, the recording solution was flowed into the channel and brought into contact with the beads. When the beads came into contact with the recording solution, a KcsA channel current was observed.

[0064] Channel current was measured using long spacer (6B-NTA) beads immobilized with His-tags of KcsA (E71A) mutants reconstituted into vesicles (Figure 13). Channel-mediated current fluctuations were observed within approximately 10 minutes after the formation of the artificial membrane. In addition, the membrane potential was changed in 10 mV increments, and the current was recorded to create an IV graph. The single-channel conductance estimated from the IV graph was 156.8 pS, which was in good agreement with the value obtained by the conventional method. Similarly, the current of porcine BK channels was also successfully measured. The single-channel conductance was 302 pS, which was in agreement with the value obtained by the conventional method. In addition, channel current measurements of RP-CNG channels, SthK channels, VDACs, etc. were also successfully performed, and the properties exhibited by these currents (single-channel conductance, ion species selectivity, etc.) were in good agreement with the values ​​measured by the conventional method.

[0065] (Evaluation test using an ion channel current measuring device: Embodiment 2) A method of extruding the recording solution was also implemented (the conditions for other beads, etc., were the same as in Embodiment 1 above). Specifically, as shown in Figure 14, after fixing the beads in the holes in the channel, the channel was filled with a lipid solution, and the recording solution was extruded and brought into contact with the beads. Similar to the method described above, a channel current was observed as soon as the beads came into contact with the recording solution.

[0066] Furthermore, two sets of the measurement system were installed in parallel, and simultaneous measurements were performed on two channels to confirm that the measurements could be carried out normally. [Explanation of symbols]

[0067] 1. Ion channel protein 2. Phospholipids 3 Lipid bilayer membrane 4 Lipid solution layer 5. Water-soluble solvent layer 6 First lipid layer 7 Gel Beads 8 Membrane vesicles 9 Lipids 10 Lipid bilayer membrane 11. Space for holding water-soluble solvent (space provided on the side opposite the gel beads) 12 Space for holding one or more gel beads 13. Suction port for securing gel beads

Claims

1. An ion channel protein evaluation model in which an ion channel protein is provided penetrating a lipid bilayer, and the ion channel protein is linked to gel beads.

2. An ion channel current measuring device comprising the ion channel protein evaluation model described in claim 1.

3. The ion channel current measuring device according to claim 2, comprising one or more suction ports for aspirating and fixing one or more gel beads, a space for holding the one or more gel beads, and a space for holding a water-soluble solvent on the side opposite to the one or more suction ports.

4. A step of bringing ion channel proteins immobilized on gel beads into contact with the surface of a lipid dissolution layer laminated on a water-soluble solvent layer, and A method for producing an ion channel protein evaluation model according to claim 1, comprising the step of inserting the ion channel protein to the interface between the water-soluble solvent layer and the lipid-soluble solution layer.

5. The manufacturing method according to claim 4, wherein the ion channel protein is contained in a membrane vesicle linked to the gel beads.

6. The manufacturing method according to claim 5, wherein the membrane vesicles are linked to the gel beads by a His-tag or a Biotin-tag.

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