JP2005083765A - Lipid substitution method in artificial lipid double film, artificial lipid double film obtained thereby, manufacturing apparatus of artificial lipid double film and ion permeation measuring instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lipid substitution method simply changing the lipid composition of an artificial lipid double film, and also to provide a manufacturing apparatus of the artificial lipid double film utilizing the lipid substitution method. <P>SOLUTION: This manufacturing apparatus 1 of the artificial lipid double film is equipped with an upper solution tank (first solution tank) 11 filled with an aqueous solution 13 and a lower solution tank (second solution tank) 12 and a partition 15 for partitioning the upper and lower solution tanks 11 and 12 is provided between two solution tanks. An opening part 16 is provided to the partition 15 and a first lipid solution is applied to the periphery of the opening part 16 to form the artificial lipid double film to the opening part 16. Further, in the manufacturing apparatus 1, a fine pipe 17 for substituting lipid is attached to the partition 15 of a part where the bulk phase of the artificial lipid double film is formed. The artificial lipid double film changed in its lipid composition is formed by adding a second lipid solution from the fine pipe 17. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a lipid replacement method for changing the lipid composition of an artificial lipid bilayer membrane in a lipid planar membrane method used for detection of minute currents via membrane proteins, peptides, etc., and this lipid replacement method. Artificial lipid bilayer membrane, an apparatus for producing the artificial lipid bilayer membrane, and an apparatus for measuring ion permeation in the lipid bilayer membrane, in the middle of measurement using the above lipid substitution The present invention relates to an ion permeation measuring device for changing a lipid composition in an artificial lipid bilayer membrane.

  In order for a living organism to maintain its vital activity, it is necessary for cells constituting the living organism to exchange ions with the outside through the cell membrane. Since the exchange of ions is performed by molecules such as membrane proteins called ion channels existing on the cell membrane, research on the action of ion channels on the cell membrane is important in basic research and application development of medicine and cell engineering. is important.

  This ion channel is composed of a pore that is a path for ions and a gate that controls the opening and closing of the channel, and the gate opens and closes by sensing a membrane potential or a physiologically active substance. This function can be observed by measuring the ionic current as ions pass through the ion channel. The patch clamp method is used as a method for measuring the ion current of a single ion channel, but it is used when it is necessary to conduct experiments in a simple reconstruction system to deepen the structure-function relationship study of the channel. Planar membrane method.

This lipid planar membrane method investigates the basic structure and detailed structure-function relationship of ion channels using minimal simple systems of ions, water, artificial lipid bilayer membranes, and ion channels ( Non-patent document 1). Further, as a device for conducting ion channel research using this lipid planar membrane method, a measuring device capable of simultaneously measuring the structure and function of ion channel molecules has been reported by the present inventors (Non-patent Document 2). reference).
“New Patch Clamp Experiment Method”, by Naoki Ogi, Yoshioka Shoten, 2001, pp. 208-215, “19. Lipid planar membrane method for channel research ” Ide, T., Takeuchi, U., Yanagida, T. Development of an Experimental Apparatus for Simultaneous Observation of Optical and Electrical Signals from Single Ion Cannels, Single Mol. 3 (2002) 1,33-42

  As described in Non-Patent Document 1 and Non-Patent Document 2, the lipid planar membrane method forms an artificial lipid bilayer membrane. Therefore, it is possible to investigate the influence of the lipid composition on the ion channel by forming lipid bilayer membranes having different lipid compositions.

  However, in the conventional lipid planar membrane method described above, once an artificial lipid bilayer membrane is produced, it is impossible to change the lipid composition during the measurement. Therefore, when investigating the relationship between the lipid composition and the ion channel, it is necessary to prepare lipid solutions having different compositions and form artificial lipid bilayers separately.

  Thus, in the conventional lipid planar membrane method, in order to change the lipid composition of the artificial lipid bilayer membrane, it is necessary to re-form the artificial membrane again, which is troublesome twice. In addition, since the function of the membrane protein that functions as an ion channel depends on the lipid composition of the membrane, changing the lipid composition while measuring ion permeation is highly useful for studying ion channels. However, in the conventional method described above, it is of course impossible to observe the relationship between the lipid composition and the ion channel over time, and depending on the lipid composition, it may be impossible to incorporate the ion channel itself.

  The present invention has been made in view of the above problems, and in the lipid planar membrane method, the lipid composition of the artificial lipid bilayer membrane can be easily changed without re-forming the artificial lipid bilayer membrane. Possible lipid substitution method, artificial lipid bilayer membrane obtained by using this lipid substitution method and apparatus for producing the same, and measurement of ion permeation while changing lipid composition of artificial lipid bilayer membrane using this lipid substitution method An object of the present invention is to provide an ion permeation measuring apparatus capable of performing the above.

  As a result of intensive studies on the above-mentioned problems, the present inventor has found that in an artificial lipid bilayer membrane formed by the lipid planar membrane method, a capillary tube is formed in the bulk phase formed around the region where the lipid bilayer is formed. When a lipid solution having a composition different from that of the lipid solution forming the artificial lipid bilayer membrane is added through this capillary tube, the composition of the lipid bilayer membrane can be changed by the diffusion of the lipid. The present invention has been completed.

  That is, in the method for replacing lipid in the artificial lipid bilayer membrane of the present invention, the first lipid substitution membrane is attached to the bulk phase of the artificial lipid bilayer membrane, and the artificial lipid bilayer membrane is formed from the capillary tube. A second lipid solution having a composition different from that of the lipid solution is added.

  Here, the “bulk phase” of the artificial lipid bilayer refers to a ring formed of a lipid solution formed so as to surround the lipid bilayer part in the artificial lipid bilayer formed by the lipid planar membrane method. Means the phase.

  In addition to the above-described features, the lipid replacement method of the present invention includes an artificial lipid bilayer formation step of forming a lipid bilayer using a first lipid solution, and the lipid replacement tubules as described above. A lipid solution injecting step of injecting a second lipid solution having a composition different from that of the lipid solution; a lipid in which the thin tube is brought into contact with the bulk phase of the lipid bilayer membrane, and the second lipid solution is added to the bulk phase It is characterized by comprising a solution addition step.

  According to the above lipid substitution method, an artificial lipid bilayer consisting of lipids in the second lipid solution can be obtained continuously from the artificial lipid bilayer formed by the lipids in the first lipid solution. it can. In other words, in order to change the lipid composition in the artificial lipid bilayer membrane until now, it was necessary to repeat the artificial lipid bilayer formation step and re-form the artificial lipid bilayer membrane from the beginning. Then, it is possible to change the lipid composition without destroying the already formed artificial lipid bilayer membrane.

  In addition, the lipid replacement method may further include a lipid solution suction step of sucking the excess first lipid solution using the capillary after the lipid solution addition step. In the lipid replacement method of the present invention, the artificial lipid bilayer formation step may be performed by a painting method or a folding method. In the lipid substitution method of the present invention, it is preferable that a channel molecule is incorporated in the artificial lipid bilayer membrane. In the lipid replacement method of the present invention, the first lipid solution does not contain ergosterol, the second lipid solution contains ergosterol, and the channel molecule is amphotericin. B may be sufficient.

  The artificial lipid bilayer membrane of the present invention is an artificial lipid bilayer membrane whose lipid composition has been changed using any one of the above-described artificial lipid bilayer replacement methods. Since this artificial lipid bilayer membrane can change the lipid composition after being formed, it can be effectively used for functional analysis of the ion channel to be incorporated.

  Moreover, the artificial lipid bilayer membrane production apparatus of the present invention includes two solution tanks filled with an aqueous solution, the first solution tank and the second solution tank, the first solution tank, and the second solution tank. A partition wall disposed between the solution tank and partitioning the two solution tanks, and by applying a first lipid solution around the opening provided in the partition wall, In the artificial lipid bilayer membrane production apparatus for forming a membrane, a thin tube for lipid replacement is attached to the partition wall in the vicinity of the opening, and a second composition having a composition different from that of the first lipid solution is formed from the thin tube. By injecting the lipid solution, an artificial lipid bilayer membrane containing the lipid in the second lipid solution as a component is formed. Moreover, in the artificial lipid bilayer membrane production apparatus of the present invention, it is preferable that the tubule is connected to a fine movement manipulator.

  Moreover, the ion permeation measuring apparatus of the present invention includes two solution tanks filled with an aqueous solution, a first solution tank and a second solution tank, and an electrode for detecting a current flowing in the aqueous solution. In the ion permeation measuring apparatus for measuring the permeation of ions in an ion channel incorporated in an artificial lipid bilayer membrane formed at the boundary between the first solution tank and the second solution tank, the artificial lipid bilayer membrane Is formed in an opening provided in a partition wall disposed between the first solution tank and the second solution tank, and in the partition wall in the vicinity of the opening, a capillary tube for lipid substitution is formed. It is characterized by being attached.

  Here, the above-mentioned capillary tube for lipid replacement is a lipid solution having a composition different from the lipid composition of the artificial lipid bilayer membrane formed in the opening of the partition wall, and the bulk phase of the lipid solution formed in the vicinity of the opening. It is intended for injection. According to the above ion permeation measuring apparatus, the state of ion permeation through the ion channel can be confirmed in the process of replacing the artificial lipid bilayer membrane with the lipid replacement method described above. Therefore, the above ion permeation measuring apparatus can investigate the influence of lipid molecules on the function of the ion channel, and can be used for the function analysis of the ion channel.

  In the ion permeation measuring apparatus, the artificial lipid bilayer membrane may be formed using a painting method or a folding method. Moreover, in the above ion permeation measuring apparatus, it is preferable that the thin tube is connected to a fine movement manipulator.

  As described above, according to the lipid substitution method in the artificial lipid bilayer membrane of the present invention, in order to change the lipid composition in the artificial lipid bilayer membrane so far, the artificial lipid bilayer formation step is performed again to perform artificial The composition of the lipid can be easily changed without destroying the artificial lipid bilayer that has already been formed, in which it was necessary to re-form the lipid bilayer from the beginning. Moreover, according to the artificial lipid bilayer membrane production apparatus of the present invention realized by using the above lipid substitution method, the artificial lipid bilayer capable of freely changing the lipid composition in the artificial lipid bilayer membrane A film can be made.

  Furthermore, according to the ion permeation measuring apparatus of the present invention, it is possible to confirm the state of ion permeation in the ion channel incorporated in the artificial lipid bilayer membrane to be lipid-substituted as described above. Therefore, the above ion permeation measuring apparatus can investigate the influence of lipid molecules on the function of the ion channel, and can be used for the function analysis of the ion channel.

  There are many types of ion channels, and since they play an important role in the survival of living organisms by being distributed to almost all cells, they are likely to cause diseases. For this reason, many drugs that target ion channels are needed. Since the present invention has a possibility of being used for screening and pharmacological tests in drug development targeting ion channels, it can be said that its usefulness is high.

  Embodiments of the present invention will be described below, but the present invention is not limited to this description.

(1) About Artificial Lipid Bilayer Prior to describing the embodiment of the present invention, a general artificial lipid bilayer will be described.

  Artificial lipid bilayers are artificially formed lipid bilayers, which are the basic components of biological membranes. For example, Teflon (registered trademark) with a small hole in a water tank filled with a monomolecular film of phospholipid. ) It can be formed by inserting a plate. Furthermore, by incorporating a membrane protein (channel molecule) that functions as an ion channel into the thus formed lipid bilayer membrane, an artificial lipid bilayer membrane imitating an actual biological membrane can be obtained. The artificial lipid bilayer membrane in which this ion channel is incorporated can be used, for example, for studying the function of an ion channel considered to be involved in a disease.

  The artificial lipid bilayer membrane can be produced by various conventionally known methods. Examples thereof include a painting method and a bonding method (also referred to as a folding method) described in Non-Patent Document 1.

  FIG. 2 (a) schematically shows a cross-sectional structure of the artificial lipid bilayer membrane. As shown in this figure, the artificial lipid bilayer membrane forms a bilayer structure in water (or in an aqueous solution) by regularly arranging amphiphilic phospholipid molecules 21. In this two-layer structure of phospholipid molecules, the hydrophilic portions composed of phosphoric acid and base of each phospholipid molecule are in contact with the external aqueous phase, and the hydrophobic portions composed of fatty acids are arranged to face each other.

  This artificial double lipid membrane is formed in the opening C of the support 23 such as a Teflon (registered trademark) plate or a plastic plate provided with an opening (small hole) C having a diameter of about 0.1 to 0.5 mm. ing. That is, this artificial lipid bilayer membrane is obtained by applying a phospholipid dissolved in an organic solvent (referred to as a lipid solution 22) to the periphery of the opening C so that the phospholipid that is an amphiphilic molecule is water. It is formed by being oriented at the interface between the organic solvent and becoming a monomolecular layer, and by eliminating the organic solvent from between the two monomolecular layers. Therefore, this artificial lipid bilayer membrane has a lipid bilayer membrane portion A and an annular bulk phase B composed of a lipid solution formed so as to surround it.

  Further, the artificial lipid bilayer membrane can incorporate the ion channel 24 by embedding the ion channel 24 between the lipid bilayer structures. The method for embedding the ion channel in the artificial lipid bilayer membrane can be a conventionally known method and is not particularly limited. Specifically, for example, there is a method in which a membrane fraction containing an ion channel is solubilized with a surfactant, reconstituted into a membrane vesicle, and fused to an artificial lipid bilayer membrane.

(2) Lipid Replacement Method in Artificial Lipid Bilayer Subsequently, the lipid replacement method in the artificial lipid bilayer membrane of the present invention will be described with reference to FIG.

  The lipid replacement method of the artificial lipid bilayer membrane of the present invention is the first lipid solution in which a capillary tube for lipid substitution is attached to the bulk phase of the artificial lipid bilayer membrane, and the artificial lipid bilayer membrane is formed from the capillary tube And adding a second lipid solution having a different composition.

  Specifically, the lipid replacement method includes an artificial lipid bilayer formation step in which a lipid bilayer is artificially formed using the first lipid solution, and the first lipid solution in a capillary tube for lipid replacement. A lipid solution injecting step of injecting a second lipid solution having a different lipid composition from the above, a capillary tube 17 for lipid substitution is brought into contact with the bulk phase B of the artificial lipid bilayer membrane, and the second lipid solution is added to the bulk phase. And a lipid solution adding step to be added.

  FIG. 2 (b) shows a state of the lipid solution addition step in which a lipid-displacement capillary 17 is brought into contact with the bulk phase B of the artificial lipid bilayer membrane. In this lipid solution addition step, the upper end portion of the thin tube 17 is added to an injection / injection means (not shown) such as a syringe, a dropper, or a microinjector that is a device for adding a drug into the microelectrode by the patch clamp method. 2), the second lipid solution can be easily added to the bulk phase B. In the lipid solution injecting step of injecting the second lipid solution into the thin tube 17, the second lipid solution can be easily injected into the thin tube 17 by using the above addition / injection means.

  The artificial lipid bilayer formation step is not particularly limited as long as it is performed by various conventionally known lipid bilayer formation methods as described in (1) above. As the lipid bilayer formation method, for example, a painting method or a folding method is preferably used. In other words, the lipid substitution method of the present invention can be applied to an artificial lipid bilayer membrane having a bulk phase, and particularly to an artificial lipid bilayer membrane formed by a painting method or a folding method. It is preferable.

  According to the above lipid replacement method, after the artificial lipid bilayer membrane is formed by the first lipid solution, the second lipid solution having a different lipid composition from the first lipid solution is added from the capillary tube 17. It can be changed to an artificial lipid bilayer membrane composed of lipids contained in the second lipid solution.

  That is, the lipid substitution method in the artificial lipid bilayer membrane of the present invention utilizes the fact that the bulk phase B is continuous with the phospholipid 11 constituting the lipid membrane formed so as to cover the support 13. is there. Since the lipid bilayer membrane part A has a very weak structure, it is impossible to contact a capillary directly with this part, but even if some mechanical stimulation is applied to the bulk phase B, the lipid bilayer membrane part A It will not destroy the structure. Therefore, in the lipid substitution method according to the present invention, a lipid solution is added to the bulk phase B through a thin tube, and the lipid composition of the lipid bilayer membrane part is changed for lipid diffusion.

  Therefore, if this method is used, an artificial lipid bilayer consisting of lipids in the second lipid solution can be obtained continuously from the artificial lipid bilayer formed by the lipids in the first lipid solution. . In other words, in order to change the lipid composition in the artificial lipid bilayer membrane until now, it was necessary to repeat the artificial lipid bilayer formation step and re-form the artificial lipid bilayer membrane from the beginning. Then, it is possible to change the lipid composition without destroying the already formed artificial lipid bilayer membrane.

  Here, as the lipid constituting the artificial lipid bilayer membrane of the present method, the above-mentioned phospholipid is preferably used, but is not particularly limited as long as it forms an artificial lipid bilayer membrane. Specific examples include phosphatidylcholine, diphytanoyl phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and the like. The two hydrocarbon chains of these phospholipids are preferably 10 to 24 carbons long. The hydrocarbon chain may be a saturated hydrocarbon or an unsaturated hydrocarbon. These lipids may be pure or may be a mixture of at least two types of lipids. Further, for example, cholesterol or the like may be added as necessary to maintain the activity of the ion channel.

  The lipid solution means a solution in which lipid is dissolved in an organic solvent. The organic solvent used in the lipid solution is not particularly limited as long as it is a nonpolar organic solvent. As a specific example, saturated hydrocarbons such as decane, hexadecane, and hexane, squalene, and the like are preferably used. The lipid concentration is preferably 5 to 40 mg / ml, and more preferably 15 to 20 mg / ml. This makes it possible to quickly form a stable artificial lipid bilayer membrane.

  The artificial lipid bilayer membrane may incorporate an ion channel made of a membrane protein as a channel molecule. Examples of the ion channel in which the ion channel is incorporated include all those that function as ion channels in biological membranes and artificial membranes. Examples of the ion channel include all types of biological channels such as antibiotics such as amphotericin B and valinomycin, channels formed by bacterial toxins such as hemolysin, electric-dependent Na channels, and acetylcholine receptor channels.

  This ion channel is a very important element for living organisms to maintain life activity, and is likely to cause various diseases. Therefore, functional analysis of this ion channel is important in basic research and application development of medicine and cell engineering. The function of membrane proteins that act as ion channels depends on the lipid composition of the membrane.

  Therefore, by using the lipid substitution method of the present invention, in the lipid planar membrane method for analyzing the function of the ion channel in the artificial lipid bilayer membrane, the lipid of the artificial lipid bilayer membrane is measured while measuring the ion permeation in the ion channel. The composition can be changed. That is, the relationship between the change in the lipid composition of the artificial lipid bilayer membrane and the ion permeation in the ion channel can be observed over time.

  Although the material of the said thin tube 17 will not be specifically limited if it is used as a thin tube by a chemical test, a biochemical test, etc., For example, glass, a plastics etc. are used suitably. Moreover, it is preferable that the opening diameter of the front-end | tip part which contacts the bulk phase B of the said thin tube 17 is 5 micrometers-20 micrometers. When glass is used as the material of the thin tube 17, for example, a commercially available glass tube having a diameter of 1 to 1.5 mm may be hot-stretched so that the tip diameter is 5 μm to 20 μm.

  In addition, in order to adjust a contact position with the bulk phase B of an artificial lipid bilayer membrane, it is preferable that the upper end part of the said thin tube 17 is connected with the fine movement manipulator. According to this, the capillary 17 can be brought into contact with the desired position of the bulk phase B, and the second lipid solution can be reliably injected into the bulk phase B, and the substitution of lipid in the artificial lipid bilayer membrane is ensured. Realized. When the addition / injection means is attached to the thin tube 17, the fine movement manipulator is connected to the addition / injection means.

  Furthermore, the lipid replacement method in the artificial lipid bilayer membrane of the present invention further includes a lipid solution suction step of sucking the excess first lipid solution using the capillary after the lipid solution addition step. Is preferred. The above-described addition / injection means can be connected to a capillary tube for use in suctioning excess lipid solution in the lipid solution suction step.

  By further including this lipid solution suction step, an effect that a lipid bilayer membrane can be formed quickly can be obtained.

  Specific examples of the lipid replacement of the artificial lipid bilayer membrane in the lipid replacement method according to the present invention include those that do not contain ergosterol as the first lipid solution, as described in the examples below. Examples include lipid solutions containing ergosterol and those incorporating amphotericin B as channel molecules.

  Amphotericin B is a kind of antibiotic, and enters the lipid bilayer membrane from the aqueous phase to form an ion channel. And this amphotericin B is considered to function as a channel molecule in a fungal cell membrane containing a large amount of ergosterol, but not to function in a human cell membrane containing only a small amount of ergosterol.

  Then, after forming the artificial lipid bilayer membrane which does not contain ergosterol using the lipid substitution method of the present invention, a second lipid solution containing ergosterol is added from the tubule to the bulk phase to perform lipid substitution. An artificial lipid-lipid bilayer membrane containing ergosterol is formed. If amphotericin B is added above the membrane during this lipid substitution, the change in the activity of amphotericin B accompanying the change in the lipid composition in the artificial lipid bilayer can be observed.

  In fact, as shown in Examples, it was confirmed that the activity of amphotericin B was generated by adding ergosterol to form an artificial lipid bilayer membrane containing ergosterol.

  Other than this, for example, a lipid having no net charge such as phosphatidylcholine is used as the first lipid solution, and a charged lipid such as phosphatidylethanolamine is used as the second lipid solution, and skeletal muscle muscle is used as the channel molecule. Using the endoplasmic reticulum K channel, the relationship between the lipid composition of the lipid bilayer membrane and the channel can be investigated.

  In addition, the artificial lipid bilayer membrane according to the present invention is obtained by using the artificial lipid bilayer membrane composed of lipids in the first lipid solution using the artificial lipid bilayer membrane replacement method described here, and then the second lipid solution. The composition of the lipid is changed to an artificial lipid bilayer membrane containing the lipid therein.

  The above artificial lipid bilayer membrane can be used not only effectively for functional analysis of ion channels incorporated therein, but can also be used to determine whether or not channel molecules can be incorporated according to the lipid composition of the artificial lipid bilayer membrane. .

(3) Apparatus for producing artificial lipid bilayer membrane Next, an apparatus for producing an artificial lipid bilayer membrane according to the present invention will be described.

  A schematic configuration of an example of an apparatus for producing an artificial lipid bilayer membrane of the present invention is shown in FIG. The artificial lipid bilayer membrane production apparatus of the present invention includes two solution tanks, an upper solution tank (first solution tank) 11 and a lower solution tank (second solution tank) 12 filled with an aqueous solution 13. . As shown in FIG. 1, in the manufacturing apparatus 1, the upper solution tank 11 having a smaller size than the lower solution tank 12 is disposed in the lower solution tank 12. In the manufacturing apparatus 1 shown in FIG. 1, “a partition wall arranged between the first solution tank and the second solution tank and partitioning the two solution tanks” refers to the bottom surface of the upper solution tank 11. Means. Therefore, the bottom surface of the upper solution tank 11 is referred to as a partition wall 15 here.

  The partition wall 15 is provided with an opening 16. In the manufacturing apparatus 1 described above, an artificial lipid bilayer membrane can be formed in the opening 16 by applying the first lipid solution around the opening 16. Here, the mechanism by which the artificial lipid bilayer membrane is formed is as described in (1) above.

  Further, in the production apparatus 1 of the present invention, the partition wall 15 in the vicinity of the position where the opening 16 is provided, that is, the partition wall 15 where the bulk phase of the artificial lipid bilayer membrane is formed is used for lipid substitution. A thin tube 17 is attached. The capillary tube 17 has the same function as that described in the above-mentioned section (2) of lipid replacement method, and is obtained by the same method as described in the section of (2) lipid replacement method. The description thereof is omitted here. By injecting a second lipid solution having a composition different from that of the first lipid solution from the capillary tube 17, an artificial lipid bilayer membrane containing the lipid in the second lipid solution as a component can be formed. . Further, it is preferable that the above-mentioned addition / injection means is connected to the narrow tube 17. Thereby, injection | pouring and addition of a 2nd lipid solution, aspiration of the excess 1st lipid solution, etc. can be performed easily.

  As described above, the production apparatus 1 first forms an artificial lipid bilayer with the lipid in the first lipid solution, and further passes the second lipid solution through the thin tube 17 to the bulk phase of the lipid bilayer. By adding, the lipid composition of the lipid bilayer is changed to form an artificial lipid bilayer composed of lipids in the second lipid solution.

  That is, it can be said that the artificial lipid bilayer membrane production apparatus of the present invention utilizes the lipid substitution method of (2) described above, and can produce the artificial lipid bilayer membrane according to the present invention. . Therefore, as for each element (lipid, lipid solution, channel molecule, etc.) necessary for forming this artificial lipid bilayer membrane, those described in the lipid replacement method of (2) above are used in the same manner. be able to.

  A fine movement manipulator 18 is connected to the upper end side of the thin tube 17 of the manufacturing apparatus 1. According to this, since the thin tube 17 can be brought into contact with a desired position of the bulk phase of the artificial lipid bilayer membrane and the second lipid solution can be surely injected into the bulk phase, the lipid in the artificial lipid bilayer membrane can be injected. The replacement is surely realized. When the addition / injection means is attached to the thin tube 17, the fine movement manipulator is connected to the addition / injection means. The fine movement manipulator 18 is not particularly limited and various conventionally known ones can be used. For example, a hydraulic type, a hydraulic type and a piezoelectric type can be preferably used.

  Although the shape of said two solution tanks 11 * 12 is not specifically limited, For example, a cylindrical thing can be used. Further, the material of the two solution tanks is not particularly limited except for the partition wall 15 that forms the bottom surface of the upper solution tank 11, and for example, glass, plastic, or the like is used. Although the material of the said partition 15 is not specifically limited, For example, plastics, such as a polypropylene, a polyvinyl chloride, a polystyrene, Teflon (trademark) etc. are used suitably. Further, the thickness of the partition wall 15 is preferably thick in order to reduce the floating electric capacity. However, if the opening 16 is thick, the film may move along the opening and becomes unstable. Therefore, the partition wall 15 is usually formed using a dielectric plate of about 0.1 to 0.3 mm, and is thinner than the above thickness (0.1 to 0.3 mm) only around the opening 16. It is preferable to process.

  The aqueous solution is not particularly limited as long as it does not contain a surfactant, an organic solvent, or the like. A preferable example of the aqueous solution is an aqueous solution in which a salt such as potassium chloride, sodium chloride, calcium chloride or the like is dissolved. The aqueous solution in the upper solution tank 11 and the aqueous solution in the lower solution tank 12 may be prepared to have the same composition and concentration, or may have different concentrations and compositions.

  In addition, the opening 16 is preferably provided in a circular shape at a substantially central portion of the partition wall 15. The diameter of the circular opening is preferably 0.05 to 0.5 mm, and the most stable film can be rapidly formed in the opening having this size.

(4) Ion permeation measuring device Moreover, in the apparatus for producing an artificial lipid bilayer membrane, if an electrode for detecting a current flowing through an aqueous solution in a solution tank is attached, it can be used as an ion permeation measuring device.

  That is, the ion permeation measuring apparatus of the present invention includes two solution tanks filled with the aqueous solution 13, which are an upper solution tank (first solution tank) 11 and a lower solution tank (second solution tank) 12, and the above And an electrode 14 for detecting a current flowing in the aqueous solution 13, and the artificial lipid 2 formed in the partition wall 15 at the boundary between the upper solution tank 11 and the lower solution tank 12 when the electrode 14 detects the current. It measures the permeation of ions through the ion channel incorporated in the double membrane.

  The artificial lipid bilayer membrane is formed in the opening 16 provided in the partition wall 15 disposed between the upper solution tank 11 and the lower solution tank 12, and is formed in the partition wall 15 in the vicinity of the opening 16. Is attached with a capillary tube 17 for lipid replacement.

  FIG. 3 shows a specific example of an ion transmission measuring device according to the present invention. The ion permeation measuring apparatus 2 shown in FIG. 3 includes two solution tanks, an upper solution tank (first solution tank) 11 and a lower solution tank (second solution tank) 12 filled with an aqueous solution 13. In the ion permeation measuring apparatus 2, the upper solution tank 11, which is smaller in size than the lower solution tank 12, is disposed in the lower solution tank 12 as in the artificial lipid bilayer membrane manufacturing apparatus 1 described above. In the ion permeation measuring apparatus 2, “the partition wall 15 arranged between the first solution tank and the second solution tank” means the bottom surface of the upper solution tank 11. Therefore, the bottom surface of the upper solution tank 11 is referred to as a partition wall 15 here.

  An opening 16 is provided in the partition wall 15, and an artificial lipid bilayer membrane can be formed in the opening 16 by applying the first lipid solution around the opening 16. Here, the mechanism by which the artificial lipid bilayer membrane is formed is as described in (1) above.

  In addition, the ion permeation measuring apparatus 2 includes an electrode 14 and measures the permeation of ions through the artificial lipid bilayer membrane formed in the opening 16 by measuring the current flowing in the aqueous solution. Can do. What is necessary is just to use what is used as this electrode 14 as an electrode of a conventionally well-known ion permeation | transmission measuring apparatus, It does not specifically limit. For this electrode 14, an Ag—AgCl electrode is often used.

  Furthermore, in the ion permeation measuring apparatus 2, the partition wall 15 in the vicinity of the position where the opening 16 is provided, that is, the partition wall 15 where the bulk phase of the artificial lipid bilayer membrane is formed is used for lipid substitution. The thin tube 17 is attached. The capillary tube 17 has the same function as that described in the above-mentioned section (2) of lipid replacement method, and is obtained by the same method as described in the section of (2) lipid replacement method. The description thereof is omitted here.

  In the ion permeation measuring apparatus 2 described above, an artificial fluid containing the lipid in the second lipid solution as a component by injecting a second lipid solution having a composition different from that of the first lipid solution from the capillary tube 17. A lipid bilayer can be formed. Further, it is preferable that the above-mentioned addition / injection means is connected to the narrow tube 17. Thereby, injection | pouring and addition of a 2nd lipid solution, aspiration of the excess 1st lipid solution, etc. can be performed easily.

  As described above, the ion permeation measuring apparatus 2 first forms an artificial lipid bilayer with the lipid in the first lipid solution, and then passes the second lipid solution through the thin tube 17 to the bulk phase of the lipid bilayer. Is added to the lipid bilayer to change the lipid composition of the lipid bilayer and form an artificial lipid bilayer composed of lipids in the second lipid solution. In the process of replacing the lipid composition of the artificial lipid bilayer membrane formed here with the lipid composition contained in the second lipid solution from the lipid composition in the first lipid solution, the electrode 14 The ion permeation through the artificial lipid bilayer can always be measured.

  That is, it can be said that the ion permeation measuring apparatus of the present invention can measure ion permeation while changing the lipid composition of the artificial lipid bilayer membrane using the lipid substitution method (2) described above. Therefore, for each element necessary for this lipid substitution, those described in the lipid substitution method of (2) above can be similarly applied. According to the above ion permeation measuring apparatus, the relationship between the change in the lipid composition of the artificial lipid bilayer membrane and the ion permeation in the ion channel can be observed over time. Therefore, it is very useful for functional analysis of ion channels.

  A fine movement manipulator 18 is connected to the upper end side of the thin tube 17 of the ion permeation measuring apparatus 2. According to this, since the thin tube 17 can be brought into contact with a desired position of the bulk phase of the artificial lipid bilayer membrane and the second lipid solution can be surely injected into the bulk phase, the lipid in the artificial lipid bilayer membrane can be injected. The replacement is surely realized. When the addition / injection means is attached to the thin tube 17, the fine movement manipulator is connected to the addition / injection means. The fine movement manipulator 18 is not particularly limited and various conventionally known ones can be used. For example, a hydraulic type, a hydraulic type and a piezoelectric type can be preferably used.

  In the ion transmission measurement device of the present invention, an optical measurement device may be provided at a position facing the bottom surface of the lower solution tank 12. As a result, the ion channel can be optically observed simultaneously with the measurement of the current flowing through the ion channel. Examples of such observation include observation of changes in the fluorescence intensity of a fluorescently-labeled ion channel accompanying the opening and closing of the gate, movement of the ion channel, and changes in the spectrum due to energy transfer between two fluorescent dyes. In addition, it is possible to observe the formation of an artificial lipid bilayer membrane and the state of lipid substitution in the artificial lipid bilayer membrane using the optical measurement apparatus. Furthermore, the movement of lipid molecules can be observed using an artificial lipid bilayer membrane using a fluorescently labeled lipid. Of course, the optical measurement is not limited to these, and any conventionally known method can be applied. Examples of the optical measuring device include a near-field light excitation fluorescence microscope, an optical microscope, and a spectrophotometer.

(5) Utilization method of the present invention If the lipid substitution method in the artificial lipid membrane of the present invention is utilized, the artificial lipid bilayer membrane having different lipid composition is obtained by directly replacing the artificial lipid bilayer membrane that has already been formed. Can be formed. Furthermore, if the ion permeation measuring apparatus of the present invention is used, it is possible to confirm the state of ion permeation in the ion channel incorporated in the artificial lipid bilayer membrane to be lipid-substituted as described above.

  Therefore, the present invention can be used, for example, to evaluate the influence of lipid molecules on the function of ion channels. That is, if the present invention is applied to the lipid planar membrane method for ion channel research, the influence of the lipid composition of the lipid bilayer membrane on various ion channels can be examined. Ion channels are often closely related to the lipid composition of the incorporated lipid bilayer membrane, and many have activity only on specific lipids. Therefore, investigating the relationship between the ion channel and the lipid composition of the lipid bilayer membrane is important in the functional analysis of the ion channel, and the usefulness of the present invention is high.

  Moreover, the ion permeation measuring apparatus of the present invention can be used for screening and pharmacological tests in drug discovery using ion channels involved in diseases. There are many types of membrane proteins that function as ion channels, and they are distributed in almost all cells, which are likely to cause diseases. Therefore, the development of many drugs targeting ion channels is expected in the future. In addition, among the drugs that have already been developed that target ion channels, there are many drugs that exhibit activity on specific lipids. Therefore, if the influence of a lipid molecule on the activity of an ion channel is evaluated using the present invention, the effect of a specific lipid can be directly confirmed in the pharmacological experiment of the above drug. In particular, many drugs that act on the nervous system, such as psychotropic drugs, work directly on channel proteins and can be suitably used for drug discovery in this field. On the contrary, in drug discovery of agricultural chemicals, it can be used to select those that do not act on human channels.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this.

  In this example, the first lipid solution does not contain ergosterol, the second lipid solution contains ergosterol, and amphotericin B is used as the channel molecule. The influence of lipid molecules on the artificial lipid bilayer membrane was evaluated.

  In this example, the influence of lipid on channel molecules was evaluated by measuring ion permeation in the artificial lipid bilayer formed in the opening 16 using the above-described ion permeation measuring apparatus (see FIG. 3). .

  First, as a first lipid solution not containing ergosterol, a lipid solution in which phosphatidylcholine (manufactured by Avanti) is dissolved in an organic solvent decane so as to have a concentration of 20 mg / ml is used according to the procedure described in the above embodiment. A self-standing horizontal membrane artificial lipid bilayer was formed. The aqueous solution filled in the upper solution tank 11 and the lower solution tank 12 was 3M KCl, 10 mM Hepes / Tris (pH 7.4).

Subsequently, amphotericin B (manufactured by Sigma) dissolved in DMSO (manufactured by Sigma) was added at about 10 ⁻⁷ M from above the formed lipid bilayer membrane. After addition of amphotericin, current was detected over time at the electrode 14 (see FIG. 4).

  Here, it was allowed to stand for about 20 minutes, but no change was observed in the current, and it was confirmed that there was no ion channel activity. Thereafter, a lipid solution prepared by dissolving ergosterol (manufactured by Nacalai Tesque) as a second lipid solution in an organic solvent decane to a concentration of 5 mg / ml is added from the capillary 11 to the bulk phase of the artificial lipid bilayer membrane. did. During this time, current was detected at the electrode 14 over time.

  FIG. 4 shows the results of detecting the current at the electrode 14 over time. In FIG. 4, the current detection results after about several minutes have elapsed from the addition of the ergosterol solution are shown from the top in the order of time passage. Although not shown in FIG. 4, in the phosphatidylcholine artificial membrane formed from the first lipid solution, no change was observed in the current even when amphotericin B was added, and amphotericin B did not show channel activity relative to phosphatidylcholine. I found out.

  However, as shown in FIG. 4, a few minutes after the addition of the ergosterol solution as the second lipid solution, a change in current was observed, confirming that amphotericin B exhibited channel activity against ergosterol. . From these results, it was confirmed that by adding the second lipid solution, the lipid composition in the artificial lipid bilayer changes, and amphotericin B exhibits a function as an ion channel.

  From the results of the above examples, it was shown that the lipid composition of the artificial lipid bilayer membrane of the present invention can easily change the lipid composition of the artificial lipid bilayer membrane. Moreover, it was shown that the effect with respect to the channel molecule of the specific lipid which forms an artificial lipid bilayer membrane can be confirmed with the ion-permeation measuring apparatus of this invention.

  The ion permeation measuring apparatus of the present invention can investigate the influence of lipid molecules on the function of ion channels, and can be used for functional analysis of ion channels. There are many types of ion channels, and since they play an important role in the survival of living organisms by being distributed to almost all cells, they are likely to cause diseases. Therefore, it is expected that drugs targeting ion channels will be required more and more in the future. Since the present invention has a possibility of being used for screening and pharmacological tests in drug development targeting ion channels, it can be said that its usefulness is high.

It is a schematic diagram which shows the structure of an example of the artificial lipid bilayer membrane manufacturing apparatus concerning this invention. (A) is a schematic diagram showing a cross section of an artificial lipid bilayer membrane in which channel molecules (ion channels) are incorporated. (B) is a schematic diagram for explaining a lipid substitution method in an artificial lipid bilayer membrane. It is a mimetic diagram showing the composition of an example of the ion penetration measuring device concerning the present invention. It is a graph which shows the result of having detected the electric current in a solution in time in the lipid substitution experiment of the artificial lipid bilayer membrane of a present Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Artificial lipid bilayer membrane manufacturing apparatus 2 Ion permeation measuring apparatus 11 Upper solution tank (1st solution tank)
12 Lower solution tank (second solution tank)
13 Aqueous solution 14 Electrode 15 Septum 16 Opening 17 Capillary 18 Fine movement manipulator 21 Phospholipid (lipid)
22 Lipid solutions (first and second lipid solutions)
23 Support 24 Ion channel (channel molecule)
A Lipid bilayer part B Bulk phase C Small pore (opening)

Claims (12)

  A lipid-displacement tubule is attached to the bulk phase of the artificial lipid bilayer membrane, and a second lipid solution having a composition different from that of the first lipid solution forming the artificial lipid bilayer membrane is added from the tubule. A method for replacing lipid in an artificial lipid bilayer membrane characterized by the following. An artificial lipid bilayer formation step of forming a lipid bilayer using the first lipid solution;
A lipid solution injection step of injecting a second lipid solution having a composition different from that of the first lipid solution into the lipid-displacement capillary;
The artificial lipid bilayer membrane according to claim 1, comprising a lipid solution addition step of bringing the capillary tube into contact with the bulk phase of the lipid bilayer membrane and adding the second lipid solution to the bulk phase. Lipid replacement method.   3. The lipid replacement method according to claim 2, wherein the lipid replacement method further includes a lipid solution suction step of sucking an excess of the first lipid solution using the capillary after the lipid solution addition step. Artificial lipid bilayer lipid replacement method.   The artificial lipid bilayer membrane replacement method according to claim 2 or 3, wherein the artificial lipid bilayer formation step is performed by a painting method or a folding method.   5. The artificial lipid bilayer lipid replacement method according to any one of claims 1 to 4, wherein a channel molecule is incorporated in the artificial lipid bilayer membrane.   The first lipid solution does not contain ergosterol, the second lipid solution contains ergosterol, and the channel molecule is amphotericin B. 6. The method for replacing an artificial lipid bilayer membrane according to 5.   An artificial lipid bilayer membrane in which the lipid composition is changed using the method for replacing an artificial lipid bilayer membrane according to any one of claims 1 to 6. Two solution tanks filled with an aqueous solution, the first solution tank and the second solution tank, and the two solution tanks disposed between the first solution tank and the second solution tank. A partition wall for partitioning,
In the artificial lipid bilayer membrane manufacturing apparatus for forming a lipid bilayer membrane in the opening by applying the first lipid solution around the opening provided in the partition wall,
A lipid replacement capillary is attached to the partition wall in the vicinity of the opening, and the second lipid solution is injected from the capillary by injecting a second lipid solution having a composition different from that of the first lipid solution. An artificial lipid bilayer production apparatus characterized by forming an artificial lipid bilayer membrane containing lipids in a solution as a component.   The artificial lipid bilayer membrane production apparatus according to claim 8, wherein the thin tube is connected to a fine movement manipulator. A first solution tank and a second solution tank, which are two solution tanks filled with an aqueous solution, and an electrode for detecting a current flowing in the aqueous solution, the first solution tank and the second solution tank; In an ion permeation measuring device that measures the permeation of ions in an ion channel incorporated in an artificial lipid bilayer membrane formed at the boundary with a solution tank,
The artificial lipid bilayer membrane is formed in an opening provided in a partition wall disposed between the first solution tank and the second solution tank,
An ion permeation measuring device, wherein a lipid replacement capillary is attached to the partition wall in the vicinity of the opening.   11. The ion permeation measuring device according to claim 10, wherein the artificial lipid bilayer membrane is formed by using a painting method or a folding method. 12. The ion permeation measuring apparatus according to claim 10, wherein the thin tube is connected to a fine movement manipulator.

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