Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

• the invention relates to devices and methods for the investigation of ion channels and receptors in membranes, in particular devices and methods for carrying out simultaneous electrophysiological measurements on a collective of biological cells using connexins or innexins.
• the voltage clamp method was established as a precise and reliable method for determining the activity of ion channels and receptors in the membrane of living cells [1, 2].
• the cell to be examined is pierced with two microelectrodes, i.e. pointed glass capillaries filled with saline.
• An electrode measures the potential inside the cell, i.e. the electrical voltage dropping across the cell membrane.
• the second electrode is used to generate an electrically regulated current flow through the cell membrane.
• this current flow is regulated in such a way that the potential across the cell membrane remains constant (hence the name "voltage clamp").
• the size of the current flowing through the membrane is then a direct and very precise and timely measure of the activity of the ion channels located in the cell membrane, which are activated directly or indirectly by receptors in the cell membrane.
• the current is set to a fixed value, which is often zero, and the membrane voltage, which is now freely set, is measured (for which purpose only one microelectrode is required for a currentless measurement).
• the value of the voltage reflects the activity of those in the cell Receptors and channels, this arrangement is not as meaningful and precise as the voltage clamp method, because the relationship between the activity of the receptors and the measured voltage signal in the current clamp arrangement is usually not linear, while the measured current signal and the on - The number of open ion channels in the voltage clamp arrangement are directly proportional.
• a disadvantage of the classic electrophysiological voltage clamp and current clamp methods is that they are connected to the puncture of a microelectrode in the cell and are therefore only for very large cells, such as. B. the octopus axon,
• Muscle cells, or frog egg cells are suitable.
• the majority of all cells that would be interesting for electrophysiological experiments are much smaller and therefore inaccessible to this method.
• a second disadvantage is that this method, like all electrophysiological methods, is very complex and has to be carried out manually by experienced specialists, so that only a few experiments can be carried out per day and an industrial active ingredient search ("High Throughput Screening or HTS) is eliminated.
• a disadvantage of the patch clamp method is again the time-consuming preparation of the measurements, which only allows experienced electrophysiologists to make around 20 measurements per day. This is far less than is required for modern high-throughput processes.
• the conventional patch clamp technology requires a great deal of experience and a great deal of sensitivity and can therefore only be automated to a limited extent.
• the patch clamp pipette is replaced by a planer or a microstructured substrate.
• it can be a membrane or thin film that has been provided with small ( ⁇ m) holes [5, 6, 7].
• ⁇ m small holes
• the idea is that cells attach themselves to the holes and form a seal there similar to the Giga-Seal in the patch pipette, so that a similar electrophysiological measurement of the electrical through the hole
• Receptors or ion channels of an automatic measurement are accessible. This enabled the throughput of measurements to be increased about ten times.
• this method is only restricted to large cells, such as Xenopus oocytes, and is not suitable for small cells, which represent the vast majority of the preparations.
• the throughput of this arrangement of measurements is comparable to the automated patch clamp methods, and the throughput required for a HTS cannot be achieved in this way. Abbott, Axon, and other companies are also developing such methods.
• Bilayer can also be stabilized on the substrate with suitable chain molecules ('tethered bilayers'), [13].
• Membrane is not yet reproducible and seems to be fundamentally impossible for many types of more complex membrane receptors. However, with some simpler membrane proteins (gramizidine, alamethizine, melittin, hemolysin), [10] some potassium channels and especially connexins [14] electrical measurements on such artificial membranes could be carried out.
• Connexins Biological protein molecules, so-called connexins, which play a special role in the communication between living cells, are known to the person skilled in the art. In the meantime, about fifteen different connexins are differentiated based on their amino acid sequence [15, 16]. Connexins occur in all vertebrates and are usually abbreviated, such as B. Cx26. The number indicates the chromatographic size of the connexins in kD. Connexins with a molecular weight between 26 and 56 kD are known to date.
• a connexon is a ring-shaped structure that crosses the cell membrane and is basically able to form a very wide, non-specific ion channel or a water-filled pore. However, these pores are usually closed as long as the connexon is in the membrane of a single, healthy cell. However, if two cells touch, each of which has connexons that are compatible with each other in their membrane, then there are two
• gap junction channel also known as an electrical synapse
• a gap junctions channel is usually formed in a few minutes on contact.
• the gap junction channel formed is a structure of generally 12 identical or different connexins, or of two connexons.
• the channel has a closable central pore with a diameter of about 1.5 to 2 nm.
• the main difference to other membrane channels is that gap junction channels run through two adjacent cell membranes and therefore not a connection between the cell interior with the external medium, but create a connection between the intracellular media of the two cells.
• Gap junction channels enable inorganic ions and small water-soluble molecules up to a molecular mass of approx. 1000 Daltons to pass directly from the cytoplasm of one cell into the cytoplasm of the other
• Gap junction channels belong to the epithelial cell-to-cell connections and can be found in almost all epithelia and many other types of tissue. As a rule, many gap junction channels are organized in the form of fields, these structures then being referred to as gap junctions in the true sense.
• the gap junction channels of connected cells are usually open and the connexins stretched. If a cell experiences a massive influx of calcium from the outside, for example due to an injury, the connection to neighboring cells is interrupted by the connexins twisting allosterically.
• Connexins can be made available by purifying cell membranes from cells containing connexins, e.g. B. eye lens, heart muscle, smooth muscles, or epithelial cells and by genetic expression of the connexins in bacteria, yeast or other cells. It is also known that connexins can be provided with a marker, such as a fluorescent protein fragment, so that their presence in a cell membrane can be demonstrated using simple optical methods [17].
• a marker such as a fluorescent protein fragment
• Connexone and gap junctions often have the same properties - e.g. Pore size, ion selectivity, electrical behavior - based on how in their natural environment. It is known that between two connexons, which are embedded in artificial membranes, a functional gap junction channel is formed when the membrane surfaces come into contact [18].
• invertebrates have a functionally similar class of membrane proteins called Innexins [19].
• the channels formed with this have a larger pore, which allows molecules up to a weight of 2000 Daltons to pass through.
• Ions and small molecules pass from cell to cell. In contrast to the Channels in animal organisms, however, the plasmodesmata are limited by the plasma membrane.
• the invention relates to devices and methods for performing electrical measurements on membrane bodies, preferably biological membrane bodies. These electrical measurements allow conclusions to be drawn about the state and behavior of membrane-bound biomolecules and their reaction to any effector molecules.
• Devices according to the invention contain at least one electrical measuring apparatus (1), but preferably two electrodes (2) and a membrane (3), in which biological molecules (4) are embedded, which have the same or similar properties as innexins, connexins or connexones , Innexins, connexins or connexones are preferably embedded in the membrane.
• Innexine, Connexine or Connexone of the same type or also Innexine, Connexine or Connexone of different types can be embedded in the membrane.
• an electrolytic liquid on both sides of the membrane, which preferably has buffer properties.
• a liquid which has the properties necessary for the survival of living cells is preferably used on one side of the membrane. This includes, for example, a suitable concentration and composition of salts, a physiologically tolerable pH, possibly also the presence of nutrients and / or a suitable concentration of oxygen.
• the electrodes are preferably arranged such that there is one electrode on each side of the membrane.
• the membrane with the embedded biomolecules is preferably designed such that it has a high electrical resistance in the absence of open ion channels.
• the device according to the invention can be used for the methods according to the invention for carrying out electrical measurements on membrane bodies.
• biological membrane bodies (5) are selected, the membrane of which also contains biomolecules that have the same or similar properties as Innexine, Connexine or Connexone.
• Particularly preferred membrane bodies in the sense of the invention are living cells. These cells preferentially express connexins or innexins. Cells that normally do not express connexins or innexins can be genetically modified by transfection with cDNA, mRNA or another form of suitable sequences, or by incorporating existing connexins or innexins in another way, in such a way that the desired connexins or Innexins are built into the membrane of the cells and preferably function there exactly like Connexins and Innexins in other cells. It is preferable to choose a stable one
• membrane bodies are now preferably superimposed Gap junctions (7) on the membrane.
• the gap junctions formed in this way represent an electrical access from the side of the membrane facing away from the membrane bodies to the interior of the attached membrane bodies.
• Functional gap junctions can be detected using electrical measurements (double voltage clamp) or optical observation of the transfer of dyes with low molecular weight (e.g. Lucifer yellow). The latter allows the coupling of an ensemble of cells to be estimated using image processing methods.
• the membrane bodies according to the invention preferably contain further membrane-bound biomolecules (8) (targets), the properties of which can be investigated by the methods according to the invention.
• targets are preferably ion channels or receptors or other biomolecules which can directly or indirectly influence charge movements through membranes.
• Charge movements and / or potential differences through the membrane of the attached membrane bodies can now preferably be derived and quantified via the two electrodes.
• a particularly preferred method according to the invention is the investigation of the effects that substances have on the membrane-specific biomolecules (targets) to be examined.
• modulators i.e. inhibitors and activators of the target and other substances which influence the expression of the target
• These substances are potential active substances for the treatment of diseases which are related to the function of the respective target.
• the invention further relates to the active substances found using the methods according to the invention and to processes for their preparation.
• Electrical signals in the sense of the invention, are physical quantities which are related to the distribution of electrical charges, ie electrons, protons, or ions, in the system under consideration. Electrical signals that can be recorded in devices according to the invention are, for example, the electrical current strength, the electrical capacitance, or the electrical potential difference and
• Membrane bodies in the sense of the invention are volume elements surrounded by a membrane and filled with a liquid.
• Membrane bodies according to the invention are preferably biological membrane bodies, such as e.g. living cells. These include cells that have been isolated from living tissues by dissociation (primary cultures). This also includes cells that are kept in culture as established cell lines, such as CHO cells, HEK cells, NIH3T3 cells, HeLa cells, but also transiently transfected cells or primary cells.
• Biological membrane bodies, in the sense of the invention are also artificially produced membrane bodies, in which e.g. a lipid bilayer encloses a limited volume of an aqueous medium (vesicle).
• membrane bodies then preferably contain at least one biological component, e.g. a polypeptide embedded in the lipid bilayer, a membrane-bound enzyme, an ion channel or a G protein-coupled receptor.
• Biological membrane bodies in the sense of the invention, can also be bacterial cells, fungal cells or cells of other unicellular or multicellular organisms.
• Biological membrane bodies within the meaning of the invention are e.g. also protoplasts of fungal cells and plant cells, which are achieved by removing external cell walls or similar structures.
• Biological membrane body in
• the invention furthermore also includes membrane bodies which, such as synaptosomes, are produced by cleavage or association from the membranes of living organisms or which have been obtained by combining such preparations with synthetic lipid vesicles.
• Electrical measuring apparatus in the sense of the invention, is a device that allows electrical signals to be recorded and, if necessary, quantified.
• membrane potential is the electrical potential difference between the opposite sides of a membrane.
• Active ingredients in the sense of the invention are substances that can influence the activity of biological molecules.
• Preferred active substances in the sense of the invention are those which specifically influence the activity of individual biological molecules or of groups of biological molecules. Particularly preferred
• Active substances are those which influence the activity of receptors and / or ion channels.
• “Supported bilayer” are membranes that are on the one hand in contact with or in the immediate vicinity of a suitable solid, porous or gel-like
• the present invention relates to 1. a measuring arrangement for measuring electrical signals on membrane bodies containing an electrical measuring apparatus (1), electrodes (2), a membrane (3) containing connexins or innexins (4), and a membrane body (5) also containing connexins or Innexine (6) characterized in that an electrically conductive access from the side of the membrane facing away from the membrane body to the inside of the membrane body through gap junction
• Affect receptors and / or ion channels (8) characterized in that i) at least one membrane body (5) containing said receptors and / or ion channels is brought into contact with at least one test substance, and ii) at least one electrical signal on the membrane body or the Membrane bodies are measured with a measuring arrangement according to point 1,
• test substances which influence the measured electrical signal are selected as active substances.
• a method for transporting substances into a membrane body or out of a membrane body characterized in that the substance through gap junction channels into or out of the membrane body
• Membrane body came out.
• the substance to be transported follows an electrical potential gradient, a concentration gradient, or a pressure gradient over the membrane of the arrangement according to the invention
• FIG. 1 shows a typical measuring arrangement in the sense of the invention with electrical measuring apparatus (1), electrodes (2), a membrane (3) containing connexins or innexins (4), a membrane body (5), gap junction channels (7) and targets ( 8th).
• a measuring arrangement for measuring electrical signals on membrane bodies is shown in Figure 1. It consists of an electrode (e.g. a gold electrode) at the bottom of a small chamber, e.g. a chamber in a microtiter plate.
• An electrically dense artificial membrane (3) is attached above the electrode, an electrolyte solution as an ion reservoir being located in the space between the membrane and the electrode.
• the measuring arrangement has a second electrode, which is located above the artificial membrane.
• Functional hemichannels (connexones) are introduced into the artificial membrane in such a way that they can diffuse freely in the membrane and that their normally extracellular domain is located above (trans) the membrane.
• suitable types of connexins are used. Possibly.
• connexones are built up from more than one connexin (heteromeric connexones).
• the suitable connexins are selected according to the requirements of the intended test. The procedure is such that an electrical signal which is as small as possible is measured without additions of the active substances to be investigated, and when the active substances interact with the ion channels and / or receptors (8) to be investigated, the observed signal increases as much as possible.
• a suspension with suitable cells is added to said chamber, which already contains the artificial membrane as described above.
• These cells (5) have at least one ion channel or receptor (8) to be examined in the cell membrane and additionally hemichannels (6), which form functional gap junctions (7) in a suitable manner with the hemichannels in the artificial membrane (4) of the measuring arrangement ,
• the hemichannels in the artificial membrane are initially closed as long as there is no cell in place. This is ensured by the fact that an electrical voltage is applied across the membrane.
• an electrical voltage is applied across the membrane.
• gap junctions are formed between neighboring cells, or that some of the cells only establish a conductive connection to the ion reservoir indirectly via other cells.
• this is not an obstacle to the use of this arrangement according to the invention. Rather, this can even result in an amplification of the observed signal, which further improves the sensitivity of the measuring arrangement.
• Connexons with a voltage behavior such that they are closed as a hemichannel or as a hemichannel are only particularly suitable for the measuring arrangement with a low potential difference (for example less than 20 mV) across the cell membrane and closed with a larger potential difference.
• the result of the measurement is therefore a current signal which corresponds to the total current flow through the cell membranes of all those cells which are in conductive connection with the ion reservoir via the built-in gap junctions.
• Suitable measuring arrangement the electrical behavior of ion channels and receptor to determine directly and immediately, with high precision and good temporal resolution and also to precisely demonstrate and evaluate the changes in this behavior, which are triggered, for example, by known or potential active ingredients.
• the time resolution of the measurement arrangement is determined by the electrical properties of the gap junctions, which in their natural function have a time resolution in the sub-millisecond range.
• the formation of the desired lead configuration in which the cells adjacent to the substrate establish an electrical connection with the ion reservoir by means of gap junctions, can also be monitored with electrical measurements.
• the electronic capacitance of the membrane and the membrane bodies connected to it via gap junctions can be determined using suitable electronic measurement methods known to the person skilled in the art.
• This method is suitable to determine the total membrane area of the system and thus to determine the number of attached cells connected to the membrane via gap junctions.
• This signal can also be used to determine the influence of test substances on this arrangement. In particular, the occurrence of exocytosis in the attached membrane bodies can be determined in this way.
• Molecular weight such as B. Lucifer Yellow, which can diffuse through gap junction channels.
• the measured signal can be normalized so that the results different experiments can be compared directly with similar but different experimental arrangements.
• Example 1 The structure described in Example 1 is modified in such a way that several or a larger number of the chambers described are set up next to one another, so that, for example, each chamber of a microtiter plate represents a measuring arrangement according to Example 1.
• the individual measuring chambers are read out sequentially, in groups or simultaneously.
• Multi-channel amplifier systems such as those known from MEA (multi-electrode array) technology or detectors in high-energy physics can be used for this.
• the microtiter plate For example, those with 96, 384, 1536 or any other number of chambers are used as the microtiter plate.
• the measuring arrangement is thus preferably designed such that it is mechanically and geometrically compatible with the HTS systems and systems already established in drug discovery, so that there are no obstacles to the technical application of the invention for practical drug searches. Existing pipetting and dispensing devices can then continue to be used. Only the detection system is expanded by a suitable readout head, which is able to read the electrical signals from the microtiter plates.

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• 2002
  • 2002-08-09 DE DE10236528A patent/DE10236528A1/de not_active Withdrawn
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