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Publication numberUS20020104757 A1
Publication typeApplication
Application numberUS 09/952,461
Publication dateAug 8, 2002
Filing dateSep 14, 2001
Priority dateDec 17, 1997
Also published asCA2316966A1, CA2316966C, DE59801410D1, EP1040349A1, EP1040349B1, EP1040349B2, US6758961, US20090153153, WO1999031503A1
Publication number09952461, 952461, US 2002/0104757 A1, US 2002/104757 A1, US 20020104757 A1, US 20020104757A1, US 2002104757 A1, US 2002104757A1, US-A1-20020104757, US-A1-2002104757, US2002/0104757A1, US2002/104757A1, US20020104757 A1, US20020104757A1, US2002104757 A1, US2002104757A1
InventorsChristian Schmidt
Original AssigneeChristian Schmidt
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Efficient methods for the analysis of ion channel proteins
US 20020104757 A1
Abstract
Methods combining the creation of small bilayer membranes with the fusion of native cell membrane vesicles into those bilayer membranes. By placing a voltage gradient across the lipid bilayer and recording the transmembrane current, the activity of some membrane proteins, such as ion channels, can be analyzed. The methods permit access to a large variety of membrane proteins. The native vesicles may be derived from a variety of cells and intracellular organelles, and as the native cell membrane vesicles may be stored at low temperatures over long periods, the methods reduce the need to maintain cell cultures or obtain primary cells. Fluorescence measurements also may be performed on the bilayer membranes.
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Claims(18)
I claim:
1. A method of analyzing membrane proteins, comprising:
positioning a vesicle on a substantially insulating carrier;
forming a lipid membrane on the carrier;
forming one or more smaller vesicles that contain at least one membrane protein, where the smaller vesicles are derived from a cell membrane or organelle membrane;
incorporating the membrane protein in the lipid membrane by fusing the small vesicles with the lipid membrane; and
analyzing the membrane protein.
2. The method of claim 1, where the step of analyzing comprises electrical analysis of the membrane protein.
3. The method of claim 1, where the step of analyzing comprises optical analysis of the membrane protein.
4. The method of claim 1, where the carrier separates two fluid compartments, and includes a small aperture between the two fluid compartments.
5. The method of claim 4, where the vesicle is positioned on the carrier across the aperture by generating a voltage difference between the fluid compartments.
6. The method of claim 1, where the carrier is in contact on one side with at least one fluid compartment that is confined to such a small area that the lipid membrane will form at a particular position.
7. The method of claim 1, where one carrier comprises a plurality of distinct sites for analyzing membrane proteins.
8. The method of claim 1, where a surface of the carrier is chemically or physically modified so as to enhance the formation of a high electrical seal with the lipid bilayer.
9. The method of claim 1, where the membrane protein is analyzed using voltage-clamp techniques.
10. The method of claim 1, where the carrier further comprises a conductive material, and where the step of analyzing comprises analyzing the membrane protein using impedance spectroscopy.
11. The method of claim 1, farther comprising orienting the lipid bilayer in the focal plane of an objective lens.
12. The method of claim 3, where the optical analysis comprises confocal observation.
13. The method of claim 1, where the smaller vesicles are derived by endocytosis, exocytosis, or synaptic vesicle release.
14. The method of claim 1, where the smaller vesicles are derived by mechanical destruction of a cell or chemical destruction of a cytoskeleton.
15. The method of claim 1, further comprising sorting the smaller vesicles according to origin and membrane protein content prior to incorporating them into the lipid bilayer.
16. The method of claim 1, where the step of incorporating is promoted by the presence of polyvalent cations, fusogenic proteins, or both.
17. The method of claim 1, where the step of incorporating is promoted by the application of osmotic stress on the lipid bilayer or the smaller vesicles or both.
18. A method of analyzing membrane proteins, comprising:
positioning a vesicle on a substantially insulating carrier;
forming a lipid membrane on the carrier;
forming one or more membrane fractions that contain at least one membrane protein, where the membrane fractions are derived by solubilization of a cell membrane or organelle membrane;
incorporating the membrane protein in the lipid membrane by integrating the membrane fractions into the lipid membrane; and
analyzing the membrane protein.
Description
    CROSS-REFERENCES
  • [0001]
    This application is a continuation of U.S. patent application Ser. No. 09/581,837, filed Jun. 16, 2000, which claims priority from PCT Patent Application Serial No. PCT/IB98/01150, filed Jul. 28, 1998, which claims priority from Swiss Patent Application Serial No. 2903/97, filed Dec. 17, 1997, each of which is incorporated by reference herein in its entirety for all purposes.
  • [0002]
    This application is based upon and claims the benefit under 35 U.S.C. 119 of the following U.S. provisional patent applications, which are incorporated herein by reference: Serial No. 60/232,365, filed Sep. 14, 2000, titled EFFICIENT METHOD FOR THE ANALYSIS OF ION CHANNEL PROTEINS, and naming Christian Schmidt as inventor; Serial No. 60/233,800, filed Sep. 19, 2000, titled DESIGN OF HIGHLY INTEGRATED PHARMACEUTICAL SCREENING CHIPS, and naming Christian Schmidt as inventor; and Serial No. ______, filed Sep. 13, 2001, titled HIGH-THROUGHPUT PATCH CLAMP SYSTEM, and naming Christian Schmidt as inventor.
  • [0003]
    This application incorporates by reference in their entirety for all purposes the following U.S. Pat. No. 5,355,215, issued Oct. 11, 1994; and U.S. Pat. No. 6,097,025, issued Aug. 1, 2000.
  • [0004]
    This application incorporates by reference in their entirety for all purposes the following patent applications: U.S. patent application Ser. No. 09/581,837, filed Jul. 28, 1998; U.S. Provisional Patent Application Serial No. 60/232,365, filed Sep. 14, 2000; U.S. Provisional Patent Application Serial No. 60/233,800, filed Sep. 19, 2000; U.S. patent application Ser. No. 90/708,905, filed Nov. 8, 2000; PCT Patent Application Serial No. PCT/IB00/00095, filed Jan. 26, 2001; and PCT Patent Application Serial No. PCT/IB00/00097, filed Jan. 26, 2001.
  • [0005]
    This application incorporates by reference in their entirety for all purposes the following U.S. patent applications: Ser. No. 09/337,623, filed Jun. 21, 1999; Ser. No. 09/349,733, filed Jul. 8, 1999; Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No. 09/596,444, filed Jun. 19, 2000; Ser. No. 09/710,061, filed Nov. 10, 2000; Ser. No. 09/722,247, filed Nov. 24, 2000; Ser. No. 09/759,711, filed Jan. 12, 2001; Ser. No. 09/765,869, filed Jan. 19, 2001; Ser. No. 09/765,874, filed Jan. 19, 2001; Ser. No. 09/766,131, filed Jan. 19, 2001; Ser. No. 09/767,434, filed Jan. 22, 2001; Ser. No. 09/767,579, filed Jan. 22, 2001; Ser. No. 09/767,583, filed Jan. 22, 2001; Ser. No. 09/768,661, filed Jan. 23, 2001; Ser. No. 09/768,765, filed Jan. 23, 2001; Ser. No. 09/770,720, filed Jan. 25, 2001; Ser. No. 09/770,724, filed Jan. 25, 2001; Ser. No. 09/777,343, filed Feb. 5, 2001; Ser. No. 09/813,107, filed Mar. 19, 2001; Ser. No. 09/815,932, filed Mar. 23, 2001; and Ser. No. 09/836,575, filed Apr. 16, 2001; and Ser. No. ______, filed Aug. 20, 2001, titled APPARATUS AND METHODS FOR THE GENERATION OF ELECTRIC FIELDS WITHIN MICROPLATES, and naming James M. Hamilton as inventor.
  • [0006]
    This application incorporates by reference in their entirety for all purposes the following U.S. Provisional Patent Applications: Serial No. 60/223,642, filed Aug. 8, 2000; Serial No. 60/244,012, filed Oct. 27, 2000; Serial No. 60/267,639, filed Feb. 10, 2001; Serial No. 60/287,697, filed Apr. 30, 2001; Serial No. ______, filed Aug. 2, 2001, titled pH PROBES FOR CELL-BASED FLUORESCENCE ASSAYS, and naming Zhenjun Diwu, Jesse J. Twu, Guoliang Yi, Luke D. Lavis, and Yen-Wen Chen as inventors; and Serial No. ______, filed Aug. 31, 2001, titled KINETIC ASSAY FOR DETERMINING CALCEIN RETENTION IN CELLS, and naming Kelly J. Cassutt, Jesse J. Twu, and Anne T. Ferguson as inventors.
  • [0007]
    This application incorporates by reference in its entirety for all purposes the following publications: Richard P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6th ed. 1996); and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2nd Ed. 1999).
  • [0008]
    This application incorporates by reference in their entirety for all purposes all of the patents, patent applications, publications, and other materials cited below.
  • FIELD OF THE INVENTION
  • [0009]
    This invention relates to methods for the functional analysis of membrane proteins. In particular, the invention relates to methods for the reconstitution of membrane proteins derived from biological cells into artificial membranes so as to facilitate their analysis using electrical and optical techniques.
  • BACKGROUND OF THE INVENTION
  • [0010]
    All biological cells have cell membranes. These cell membranes contain a variety of different classes of membrane proteins that are vital for the proper functioning of the cell. Membrane proteins play key roles in absorbing nutrients, secreting wastes, controlling cell volume, and communicating with the outside environment. Important classes of membrane proteins include ion channel proteins that control the ionic flux across the membrane. The analysis of both the electric currents controlled by these proteins (referred to as functional analysis or functional screening) and the fluorescence of species adapted to report on these proteins and other related substances is important to provide a basic understanding of cellular processes in biology and medicine, as well to aid in the development of new drugs.
  • [0011]
    Standard methods used for the electrophysiological analysis of ion channels and biological membranes, such as standard patch clamp techniques (B. Sakmann and E. Neher, Eds., Single-Channel Recording, Plenum, 1983), black lipid membrane techniques (“BLM”: W. R. Schlue and W. Hanke, Planar Lipid Bilayers, Academic Press, 1993), and voltage clamp techniques (J. G. Nicholls, From Brain to Neuron, Sinauer Association, Sunderland, 1992) are limited in performance due to the slow and typically manual handling of the membranes. Recent methods permit the automated positioning of biological and artificial membranes (e.g., PCT Publication WO 99/31503) on structured carriers in such a way that electrical and optical measurements on them are possible. These methods also permit integration and miniaturization of several such systems on one carrier.
  • [0012]
    However, the analysis of cell membranes directly still requires the maintenance of cell lines in culture, or the availability of donors for primary cells. Additionally, some cells are not compatible with such new techniques due to unfavorable surface properties (e.g., they lack surface charge or have an extracellular matrix) or due to general inaccessibility (e.g., because they are bound to tissue, or possess an unfavorable morphology). In other cases, it would be helpful to study intracellular membranes that are not normally directly accessible.
  • [0013]
    Attempts to circumvent these problems by purifying the protein of interest and subsequently reconstituting it into an artificial bilayer positioned on a carrier may fail due to problems associated with finding the right purification/reconstitution protocols for each protein. Moreover, such manipulation risks the loss of possible membrane-bound cofactors or native lipids that are required for proper function of the membrane protein of interest (W. R. Schlue and W. Hanke, Planar Lipid Bilayers, Academic Press, 1993).
  • SUMMARY OF THE INVENTION
  • [0014]
    The invention provides methods for analyzing membrane proteins. In one embodiment, the methods involve the fusion of small vesicles derived from the cell membrane, or the membrane of an intracellular organelle, into lipid membranes that have been formed by autopositioning giant vesicles on a mostly insulating carrier, followed by the subsequent electrical and/or optical analysis of the membrane proteins. The methods provide a powerful tool for the analysis of membrane proteins, with applications to pharmacology, biosensors, and other scientific fields.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • [0015]
    [0015]FIG. 1 is a schematic view of a measurement system for the fusion and electrical analysis of cell-derived vesicles.
  • [0016]
    [0016]FIG. 2 is a schematic view of a multiaperture measurement system having several recording setups on one chip.
  • [0017]
    [0017]FIG. 3 is a flow chart showing the steps leading from a biological cell to a reconstituted membrane protein that can be analyzed.
  • DETAILED DESCRIPTION
  • [0018]
    The invention provides methods for the electrical and/or optical analysis of membrane proteins. These methods may partially or completely circumvent the need for preparing and/or maintaining primary cells or cell cultures, as well as the difficulties attending the complicated protein reconstitution processes associated with such analyses. The methods are based on the formation of lipid bilayers that are tightly bound to a carrier material, for example, by the “autopositioning” of giant vesicles (e.g., PCT Publication WO 99/31503) and the fusion of small, cell-derived vesicles into this bilayer. The methods of the invention may be used for the direct electrophysiological analysis of ionotropic membrane proteins, as well as for the analysis of various associated proteins and factors. The methods additionally permit the optical analysis of such proteins.
  • [0019]
    The term “autopositioning,” as used here, refers to any and all methods that lead to a positioning of vesicles and cells at a predetermined position as a result of preexisting or imposed constraints (e.g., electrical and magnetic fields, and/or shape or geometry of the setup) during the positioning process. These constraints do not necessarily require manual or user-intervention during the positioning process.
  • [0020]
    For the analysis of membrane proteins, such as ion channel proteins, small vesicles containing the protein of interest (i.e., proteoliposomes) are derived, typically directly, from biological cells. The vesicle-containing suspension then is added to one or both sides of a small lipid bilayer (diameter <20 μm) that has been formed by autopositioning (for example, by electrophoretic positioning as described in PCT Publication WO 99/31503) and subsequent tight adhesion of a large unilamellar vesicle across a small opening positioned in a mostly insulating carrier. Both sides of the bilayer are in contact with a small fluid volume (usually 0.1-100 μL) that is itself in contact with electrodes suitable for voltage-clamp recordings. Experimental conditions are chosen so that the proteoliposomes approach the bilayer membrane, attach to the membrane, and eventually fuse to the membrane. To permit subsequent fluorescence and/or confocal optical measurements of the lipid bilayers, the carrier may be designed in such a manner that the bilayer may be readily placed in the focus of a lens. For example, the carrier may be designed so as to have a generally planar conformation.
  • [0021]
    The small cell-derived vesicles (or proteoliposomes), generally having a diameter less than about 500 nm, may be obtained by various procedures. Depending on the particular procedure used, other proteins and factors that are important for the functioning of the membrane protein of interest may be contained in the vesicle, or attached to its membrane, so that upon vesicle fusion these additional proteins and factors are located in or near the preformed bilayer membrane.
  • [0022]
    The small size of the preformed bilayer membrane also permits another very efficient reconstitution method. The stability of the bilayer increases if the diameter of the aperture is reduced, as the diameter of the bilayer also is reduced. The diameter of the bilayers is less that about 20 μm, and typically less than about 5 μm, as described in PCT Publication WO 99/31503. Consequently, the bilayers are significantly more stable than typical black lipid membrane bilayers. Under these conditions, detergent-solubilized membrane proteins, such as the nicotinic acetylcholine receptor or CIC-O channel from Torpedo marmorai, may be added directly to one of the buffer compartments in contact with the preformed bilayer. For the solubilization of the membrane protein, a purification step typically is not required. In most cases, it is sufficient to solubilize the original membrane (e.g., plasma membrane, endoplasmic reticulum membrane (ER), mitochondrial membranes) in detergent (e.g., CHAPS: 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) and to add a sufficient volume of solubilized membrane to one or both sides of the bilayer. After an optional washing step of the respective compartment, the membrane proteins may be analyzed.
  • [0023]
    The proteins of interest may be analyzed in a variety of ways, once they are reconstituted into the lipid bilayer. Most commonly, a voltage is applied across the bilayer via two redox electrodes that are immersed in the fluid compartments located on one or both sides of the bilayer membrane. The current registered using these electrodes serves as a direct measure of membrane protein activity. By placing the bilayer membrane within the focal spot of an objective, the (optionally confocal) optical observation of the bilayer also may provide information about binding properties and activity of the membrane protein of interest.
  • [0024]
    In a particular embodiment, the invention permits the measurement of the current through any ionotropic membrane protein expressed in the membrane of a biological cell, including the plasma membrane, the endoplasmic reticulum, the tonoplast and thylakoid membranes of plant cells, the inner and outer membranes of mitochondria, and the inner and outer membranes of bacteria. Fluorescent labeling of a particular membrane protein or another protein attached to it (e.g., G-protein) or a ligand (e.g., in case of the nicotinic acetylcholine receptor (nAChR), the fluorescent agonist (1-(5-dimethylaminonaphthalene)-sulfonamidol)-n-hexanoic acid-beta-N-trimerylammonium bromide ethyl ester (Dns-C6-Cho)) or other factor allows as well the (confocal) optical observation and optical analysis of such protein. The method achieves such broad applicability to membrane proteins by forming small vesicles of the original membrane and fusing these vesicles into a preformed lipid bilayer.
  • EXAMPLES
  • [0025]
    The method of the invention is readily adapted to incorporate modifications intended to permit additional specific applications, as described below. These applications are included for illustration and should not be interpreted so as to restrict, limit, or define the entire scope of the invention.
  • Example 1 Preparation of a Suitable Carrier
  • [0026]
    A carrier suited for electrophoretical positioning of large liposomes may be prepared as described in PCT Publication WO 99/31503. A suitable carrier is generally insulating, but contains a small hole typically having a diameter less than about 20 μm. The carrier generally separates two fluid compartments in such a way that the hole in the carrier is in contact with both compartments. Upon application of a voltage between the compartments, mediated by redox electrodes immersed in each compartment, a strongly inhomogeneous field around the aperture is created such that vesicles, cells and other charged objects are propelled towards the aperture.
  • [0027]
    A schematic depiction of a setup useful for the invention is shown in FIG. 1. A carrier (11, 12, 13, 14) is sandwiched between two fluid compartments (18, 19). Both compartments are confined on the carrier surface by a hydrophobic material (15) attached/bound to the surface. Redox electrodes (16, 17) used both for the application or recordation of voltage are immersed in the fluid compartments. The redox electrodes may be directly attached to the carrier (e.g., by sputtering or printing) or to a container that itself contains the carrier.
  • [0028]
    In one embodiment of the invention, the carrier is a silicon chip (11) containing a groove that is covered by a thin silicon nitride/silicon oxide diaphragm (13, 14), where the diaphragm itself contains a small aperture (diameter usually <20 μm). The chip is otherwise be surrounded by an insulating layer (12), such as thermally grown silicon oxide, to reduce the capacitance of the setup.
  • [0029]
    Preferably, the surface of the carrier is either selected, or is modified, so that lipid bilayers adhere to the surface tightly. For example, the surface may be modified by the physisorption of poly-L-lysine (molecular mass usually >15 000), by chemical modification with 4-aminobutyl-dimethyl-methoxysilane, or derivatization with molecules that bind (specifically or non-specifically) to cell surfaces (for example, some lectins).
  • [0030]
    In some embodiments, a silicon chip can be used as a carrier material, for example, as described in WO 99/31503. The carrier contains a small opening (aperture) of about 0.3 to 20 μm on which vesicles are positioned and subsequently a lipid membrane is created. On both sides of the carrier is a small buffer compartment located so that it may be free-standing (e.g., confined to hydrophilic carrier spots surrounded by hydrophobic areas) or physically confined (i.e., by grooves put into the carrier). The buffer compartments are usually between 0.1 to 40 μL. Redox electrodes are generally H made of Ag/AgCl or Platinum, and may be directly attached to the recording carrier or a cartridge in which the carrier is packaged or attached to a holder that is not in direct contact with the chip. For measurements that refer only to optical or impedance spectroscopic analysis, it may be sufficient to use carriers that do not require either apertures or electric fields for positioning. In this embodiment, only one buffer compartment is required.
  • [0031]
    In another embodiment of the invention, several recording setups are integrated on a single chip that contains several apertures. In this manner, all fluids and electrodes on one side of the carrier can be unified. On the other side of the carrier, however, fluid compartments and electrodes are necessarily separated to allow independent recordings. Separation is made possible by utilizing hydrophilic/hydrophobic surface patterning, or by producing small compartment wells on the carrier surface, for example by laminating a thin polydimethylsiloxane (or PDMS) layer containing small holes adjacent to the aperture to the carrier surface.
  • [0032]
    A schematic depiction of a carrier having multiple recording sites is shown in FIG. 2. The carrier (17, 18, 19) contains on one side a patterned surface to separate fluid compartments (16) physically and permit multiple independent recordings. Patterning is done by attachment of hydrophilic substances or materials (14, 15). These fluid compartments are accessed with independent electrodes for every compartment (1,2,3, . . . ) that are independently connected to voltage clamp circuits. On the other side, while each compartment can be separated as shown for the first side, it is however more simple to unify the compartments (19) and bring them in contact with only one electrode (20) (typically the ground electrode). In one embodiment, the carrier is a silicon chip (18) containing grooves that are closed by a silicon nitride/silicon oxide diaphragm (17) containing a small aperture (diameter usually <20 μm). The chip is otherwise typically surrounded by an insulating layer, e.g., thermally grown silicon oxide, to reduce the capacitance of the setup.
  • Example 2 Positioning a Vesicle Across the Aperture
  • [0033]
    To place the large, preferably unilamellar vesicle across the aperture, a voltage is applied between the two carrier compartments. The voltage is typically between −1 V and +1 V, and the vesicles added to one compartment are (typically electrophoretically) attracted towards the aperture and permitted to adhere to the carrier surface surrounding the aperture. Vesicles will usually break apart upon surface adhesion and leave a membrane patch that tightly seals the aperture, typically with a seal resistance greater than 100 MOhm. It may necessary to rinse both compartments with an osmotically appropriate solution after positioning, to match the osmolarity of the small vesicle to be fused in step 4 (as discussed below).
  • [0034]
    Large vesicles that are appropriate for the purposes of the method of the invention may consist of various combinations of particular lipids in particular relative amounts. In one embodiment of the invention, the large vesicle is composed of 70% asolectin, 25% 1-palmitoyl 2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), 5% cholesterol or 45% POPE, 25% cholesterol, 22.5% 1 palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 7.5% POPG. Vesicles are optionally prepared by rehydration of a thin and dried lipid film for several hours (usually more than 2 hours) with subsequent size purification to remove vesicles with diameters less than 5 μm. A film that is suitable for such rehydration is a film of 1.25 mg total lipid in 10 ml deionized water containing 200-1000 mM sorbitol. The formation of large vesicles by rehydration can be supported by the application of an AC electrical field (frequency about 10 Hz, E about 1000 V/m) as described in, for example, Mathivet L., Cribier S., and Devaux, P. F., Biophys J 1996 70(3):1112-21, Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. The resulting vesicles may be separated, for example by dialysis of the vesicle containing solution across a 10-μm nylon net.
  • [0035]
    The vesicles of the method may be positioned using various methods, including the applied constraints that focus the movement of the vesicles towards the recording site (e.g., aperture) or that only allow the attachment of vesicles at that place, as discussed above. Examples of such constraints include, without limitation, electric and dielectric forces, hydrophilic-hydrophobic surface patterning, and physical constraints of the setup, such as a cone-shaped compartment and/or liquid streams. For positioning, a small volume (usually 0.1 to 10 μL) of a giant vesicle-containing solution is added to the fluid compartment. The utilization of inhomogeneous electrical fields is particularly useful, as described in PCT Publication WO 99/31503. In this method, a voltage of about 10 to 200 mV (absolute value) is applied between the two fluid compartments located on both sides of the recording aperture of the carrier. As the voltage drop between the electrodes peaks near and within the aperture, there is a resulting very high field strength in this area that attracts charged vesicles to the aperture, where the field strength is highest. After vesicle positioning, any remaining giant vesicles may be washed away.
  • [0036]
    To make electrical recordings, a very tight adhesion of the giant vesicles and its remaining membrane with the carrier is required (a so-called “Giga-Seal”). The seal resistance between carrier and membrane should be >100 MΩ. Such resistances are promoted by smooth carrier surfaces and strong interaction (electrostatic forces, molecular recognition/binding) between carrier and membrane.
  • Example 3 Manufacturing of Small Vesicles Directly Derived from Cellular Membranes
  • [0037]
    Suitable small vesicles may be prepared using any of a variety of different methods, including for example:
  • [0038]
    Destruction of the cytoskeleton, for example using cytochalasin.
  • [0039]
    Application of mechanical shear-forces to the cell or cell fragments (e.g., by pressing cells through a filter membrane as described by Regueiro P., Monreal J., Diaz R. S., and Sierra F., J Neurochem 1996 67(5):2146-54, Preparation of giant myelin vesicles and proteoliposomes to register ionic channels.).
  • [0040]
    Solubilization of the membrane and subsequent detergent removal (by dialysis).
  • [0041]
    Purification of vesicles produced by osmotically driven shrinking of cells (Kubitscheck U., Homann U., and Thiel G., Planta 2000 210(3):423-3, Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm).
  • [0042]
    Isolation of vesicles produced by other endocytotic processes (e.g., Sattsangi S., and Wonderlin W. F., Methods Enzymol 1999 294:339-50, Isolation of transport vesicles that deliver ion channels to the cell surface).
  • [0043]
    Isolation of synaptic vesicles (Kelly M. L., and Woodbury D. J., Biophys J 1996 70(6):2593-9, Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers).
  • [0044]
    Ultrasonification of cells.
  • [0045]
    Alternatively, suitable small vesicles may be obtained directly from a variety of native sources, including:
  • [0046]
    Cytoplasmatic droplets of Chara corallina, made by cutting an internodal cell in 1M NaCl solution. The resulting droplets are surrounded by tonoplast, while larger vesicles may also be directly positioned across the recording aperture (Bertl A., J Membrane Biol 1989 109:9-19, Current-voltage relationships of a sodium sensitive potassium channel in the tonoplast of Chara corallina).
  • [0047]
    Cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica (Kelly M. L., and Woodbury D. J., Biophys J 1996 70(6):2593-99, Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers).
  • [0048]
    Transport vesicles isolated from cultured cells (e.g., N1E-115 cells: Sattsangi S., and Wonderlin W. F., Methods Enzymol 1999 294:339-50, Isolation of transport vesicles that deliver ion channels to the cell surface).
  • [0049]
    It generally is important that the vesicles used in the method be small, typically less than 500 mm in diameter, preferably even less than 150 mm in diameter, to encourage an effective fusion of vesicles to the lipid bilayer. In some embodiments, this vesicle suspension may be purified, for example by centrifugation (removal of supernatant solution and addition of clean buffer) or by dialysis.
  • Example 4 Adhesion and Fusion of Vesicles
  • [0050]
    Upon the addition of a suspension of small vesicles to one of the buffer compartments, the small vesicles will be in constant motion, due to turbulences, thermal movement, gravity, and other forces, and the vesicles will eventually touch the bilayer. To promote the adhesion and fusion of the vesicles to the lipid bilayer, the addition of calcium ions, zinc ions, polyethylene glycol (PEG) or any combination thereof to the buffer may be appropriate. Calcium ions are typically added to a concentration of about 40 mM, and zinc ions are added to a concentration of about 200 μM).
  • [0051]
    Fusion of the adhering vesicles is also strongly promoted by imposing mechanical stresses to them, or to the bilayer membrane to which they are fused (and which covers the aperture). Such stress may be induced by osmotic swelling of the vesicles, for example by reduction of the osmolarity of the buffer medium (e.g., to about 50% of its original value). Suitable stress to promote fusion may also be created by strongly increasing the osmolarity of the solution on the vesicle containing side of the aperture with respect to the solution on the other side of the aperture (Cohen F. S., Zimmerberg J., et al., J Gen Physiol 1980 75(3):251-70, Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of vesicular membrane market into the planar membrane). A sophisticated variant of this method involves the semi-permeabilization of the membrane (Woodbury D. J., Methods Enzymol 1999 294:319-39, Nystatin/ergosterol method for reconstituting ion channels into planar lipid bilayers).
  • Example 5 Addition of Solubilized Membrane Proteins
  • [0052]
    As an alternative to the methods of Example 4, detergent solubilized membrane proteins may be added to one compartment, and integration of those proteins into the bilayer will take place automatically without the need for further process steps.
  • [0053]
    The addition of solubilized membrane proteins requires that such proteins remain functional (i.e., are not denatured) upon solubilization with the addition of detergent. Examples of appropriate solubilization procedures include the solubilization of the ClC chloride channel with the addition of CHAPS to membrane preparations of Torpedo marmoraia (solubilized in 85 mM KCl, 4 mM NACl, 1 mM HEPES pH 7.4 and 59 mM CHAPS with 2 mg/ml lipids (85% asolectin, 8% cholesterol and 7% POPG). The suspension is 200-fold diluted in buffer immediately before addition to the sample compartment (final channel protein concentration of <10 pg/μL)).
  • [0054]
    The use of solubilized membrane proteins does not require the isolation or production of proteoliposomes, nor does it require the utilization of osmotic stress to integrate the membrane protein into the bilayer.
  • Example 6 Post-integration Analysis
  • [0055]
    After membrane protein integration, the compartment may be rinsed to remove excess vesicles or detergent traces. Rinsing may also be needed to change the buffer composition, if needed. Membrane proteins may then be analyzed by monitoring the membrane current at a given voltage, with the addition if necessary addition of ligands, co-factors, etc. Alternatively, the membrane proteins may be analyzed by confocal observation of the lipid bilayer patch (and consequently reconstituted membrane protein) covering the carrier aperture.
  • [0056]
    For electrical recordings, a voltage is typically applied across the bilayer membrane via the redox electrodes immersed in the buffer compartments (in case of WO 99/31503. It can be the same electrodes as used for positioning). The resulting current represents a direct measure of membrane protein activity.
  • [0057]
    For optical measurements, the membrane in which the proteins have been integrated is located within the focal point of an objective lens or the beam path of a laser. The membrane is typically illuminated at an appropriate excitation wavelength, and the resulting fluorescence is monitored. Typically, the fluorescence intensity and time-course are monitored. A variety of fluorescence-based techniques are available, including the binding of fluorescently labeled compounds to the membrane, or monitoring energy transfer between fluorescent parts of the membrane (including appropriately fluorescently labeled compounds). To increase the signal to noise ratio of the fluorescence signal, all light received from non-confocal areas can be eliminated before reaching the detector by e.g., placing an aperture within the confocal plane.
  • [0058]
    The method of the invention is also useful for screening the interactions between the membrane proteins and a variety of compounds, by observing the effect on protein activity, binding affinity, or binding modulation of the membrane proteins.
  • [0059]
    The method of the invention optionally may be performed using apparatus, methods, and/or compositions described in the various patents, patent applications, and other material listed above under Cross-References and incorporated herein by reference. The apparatus include planar electrophysiology substrates, electrical positioning and measurement devices, luminescence detectors, and sample holders such as microplates, among others. The methods include electrophysiology methods, such as patch clamp and voltage clamp methods, among others, and photoluminescence methods, such as fluorescence intensity, polarization, and energy transfer methods, among others. The compositions include photoluminescent probes, and precursors and partners thereof, such as polarization probes and energy transfer probes, including donors and acceptors, particularly for measuring membrane potentials and/or the presence or concentration of selected ions, including Na+, K+, Cl, and/or Ca2+, among others.
  • [0060]
    An overview of the key process steps of the instantly described new method of membrane protein analysis is given in FIG. 3.
  • [0061]
    The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in applications claiming priority from this or a related application. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.
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Classifications
U.S. Classification204/418
International ClassificationG01N37/00, G01N33/48, G01N33/483, C12M1/34, G01N33/487
Cooperative ClassificationG01N33/48728
European ClassificationG01N33/487B6, C12M1/34B
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