MX2008009035A - Method for the determination of the activity of the organic cation transporter - Google Patents

Abstract

The present invention refers to a method for determining the activity of the organic cation transporter (OCT), a method for determining the activity of or identifying a chemical compound that modulates the activity of OCT with the help of a cell free electrophysiological sensor chip containing a solid-supported sensor electrode and a lipid layer containing the OCT located in the immediate spatial vicinity to the sensor electrode, whereas the sensor electrode is electrically insulated relative to the solutions used and to the lipid layer, as well as to the sensor chip itself and a kit containing same.

Description

METHOD FOR THE DETERMINATION OF THE ACTIVITY OF THE CONVEYOR OF ORGANIC CATIONES The present invention relates to a method for determining the activity of the organic cation transporter (OCT), a method for determining the activity of or identifying a chemical compound that modulates the activity of OCT with the help of an electrophysiological sensor chip without cells containing an electrode of the solid support sensor and a lipid layer containing the OCT located in the immediate spatial proximity of the sensor electrode, while the sensor electrode is electrically isolated with respect to the solutions used and the lipid layer, as well as to the sensor chip itself and a kit that contains it.

The transport of human organic cations is an important mechanism for the transcellular transport of organic cations. Therefore, organic cation transporters (OCT) are not only potential pharmaceutical targets that allow a direct influence on disease-related abnormalities, but are also potential ADMET targets (Adsorption, Distribution, Metabolism, Excretion and Toxicity) that have account for alterations in bioavailability parameters of potential drugs.

OCT belongs to a superfamily that includes uniporters, symporters, and antiporters, such as proteins related to multidrug resistance, diffusion facilitating systems, and proton antiporters. They mediate the transport of small cations with different molecular structures independently of the sodium and proton gradients. Sodium-independent and substrate-specific transport mechanisms via human OCT (hOCT) have been described in liver, kidney, small intestine and nervous system (Pritchard JB and Miller DS (1993), Physiol Rev. 73 (4 765-796). The human organic cation transporter hOCTI has already been cloned in 1997 (Zhang.L., et al. (1997) Mol.Pharmacology 51 (6), 913-921).

The OCT changes the electrical charges when it passes through its transport cycle. This change can be caused by the movement of loaded substrates or by the movement of protein residues that present (partial) charges. The OCT activities can be controlled via radio waves and standard electrophysiology voltage clamp with two electrodes with the common methodological disadvantages and poor resolution time, low sensitivity, difficult discrimination between blocking and competitive substrates, false positives and negatives, etc. (Arndt et al. (2001) Am J Physiol Renal Physiol, 281, F454-F468).

In some cases, the currents related to the transporter can be monitored directly in a quite physiological environment by means of patch-clamp experiments or in artificial "black lipid membranes". In the latter case, a lipid bilayer is generated in a small gap between two buffer reservoirs, each containing an Ag / AgCl electrode. After the incorporation of the protein into the bilayer, the biological activity (for example, enzymatic activity) can be activated, for example, by photoactivation of ATP derivatives. Even so, due to its lack of stability, rapid buffer exchange experiments can not be performed in this system, which limits the system to photoactivatable substrates. The lack of stability can be overcome by immobilizing particles containing proteins on a sensor surface or on the sensor chip.

An electrophysiological sensor chip without cells is generally based on membrane fragments that contain the transporter or vesicles normally electrically coupled to a gold-coated biochip. The fragments of the membrane are normally adsorbed to the surface of the sensor chip which preferably carries a modified lipid layer on a thin gold film. The membrane fragments can generally form cavities that are capable of maintaining ionic gradients across the membranes. After activation with a suitable substrate, charged ions or substrates are transported through the membrane. Since the adsorbed fragments of the membrane and the surface of the coated electrode behave like electric capacitors, the ions in motion represent a variable current that becomes detectable if a reference electrode is placed in the surrounding solution.

The problem of the present invention concerns the question of whether OCT activity can be detected specifically and sensitively with such a sensor chip even if patch clamp experiments with hOCTL are not achieved Surprisingly it has been found that a cellless assay could be established that would show the sensitivity required to detect a specific signal under OCT activation. It was particularly surprising because the OCT functioned in the cell-free assay according to the present invention without the cell bottom line, ie, without intracellular substances, the cytoskeleton, etc. In particular, the assay of the present invention can be carried out over a wide range of pH and / or ionic concentrations, which is a particular advantage.

Accordingly, a first embodiment of the present invention relates to a method for determining OCT activity with the following consecutive steps: (a) providing an electrophysiological sensor chip without cells containing an electrode of the solid support sensor and a lipid layer containing the OCT located in the immediate spatial proximity of the sensor electrode, while the sensor electrode is electrically isolated from the electrode. solutions used and the lipid layer, (b) treat the sensor chip with a non-activating solution containing ions, (c) treating the sensor chip with an activating solution containing ions and substrate, and (d) measuring the electrical signal.

The OCT is selected, for example, from SLC22A1 (OCT1), SLC22A2 (OCT2, SLC22A3 (OCT3), SLC22A4 (OCTN1) and SLC22A5 (OCTN2). It usually has a mammalian origin, particularly from rat, mouse, rabbit, pig, guinea pig, drosophila melanogaster, caenorhabditis elegans or human. Preferably it is OCT1 human.

The electrode typically comprises a metallic material or an electrically conductive metal oxide, particularly gold, platinum, silver or tin and indium oxide.

The electrode of the solid support sensor is generally a sensor electrode supported on glass or a polymer, in particular a sensor electrode supported on Borofloat glass, particularly a Gold electrode supported on Borofloat glass. In a preferred reslization the lipid layer is attached to the electrode via a chemical bond, particularly via coupling with histidine tail or coupling with streptavidin-biotin, or by means of hydrophobic, hydrophilic or ionic forces.

The electrode is further electrically isolated, for example, by one or more insulating monolayer (s), particularly by one or more insulating amphiphilic organic compounds, more particularly by one or more insulating membrane monolayer (s), more particularly by a mercaptan layer, especially octadecyl thiol, as a lower layer facing the electrode and a membrane monolayer as a top layer oriented in the opposite direction from the electrode.

A sensor chip contains in particular a solid support carrying the sensor electrode and a coating plate with an orifice, which forms a well similar to those of the titration plates. Suitable glass and polymer plates can be used as a suitable support. In the case of a glass support, for example, a glass plate, the electrode preferably consists of a thin gold film structured lithographically, which has been chemically modified, for example, by means of a mercaptan, on its surface, while that with a modified thick film of polymeric support gold electrodes can also be used. Due to the range of suitable supports, individual sensor chips can be manufactured in this way as sensor strips or even sensor set plates with 96 or 384 sensors. In particular, polymer-based sensors have the potential for mass production at low cost.

Generally for all types of sensors the gold surface is transformed into a condenser after the modification of the surface has been made and the well has been filled with an aqueous solution. The properties of said capacitor can be determined by means of a reference current electrode, such as Pt / Pt or Ag / AgCl or tin and indium oxide or others, brought into contact with the solution. In addition, the surface of the sensor is preferably very hydrophilic, ie tacky for membrane fragments and vesicles. Consequently, the OCT maintained within its native or similar environment, ie sheets of biological membranes, vesicles or proteoliposomes, is easily adsorbed on the hydrophilic surface of the sensor, forming compartments whose interior space with its dissolution is electrically isolated from the gold surface and the dissolution of the environment within the well. If it is inserted in a cuvette, the well of the chip defines the interior volume of a flow cell, allowing a rapid exchange of the solution above the surface of the sensor.

An electrophysiological sensor chip without cells used for the present invention is, for example, described in WO02 / 074983, in particular in the claims and / or in Figures 1 and / or 2, including the description of the figures of said PCT application, which is incorporated herein by way of reference, and which in any case is described herein invention. It is also available at lonGate Biosciences GmbH, Frankfurt / Main, Germany sold under the name of the SURFE2R ONE® biosensor system The change of a solution that does not contain a substrate or activator of the OCT by a solution that does have it, induces a transient charge current that can be measured, which is typically in the range of 100 pA to 4 nA. From there, the substitution of the non-activating solution for the activating solution, ie, the solution containing the substrate will activate the OCT activity. The subsequent substitution of the solutions in reverse order returns the sensor chip to its initial state. According to the present invention, a particular advantage of solutions containing ions is that the artifacts are minimized, which leads to a specific and sensitive signal.

All the components necessary to carry out the solution exchange experiments are adapted to a PC, or are controlled in some way with a workstation. In the conventional system, the non-activating (i.e., substrate-free) solution as well as the activating solution are stored in glass bottles. The application of Air pressure in the bottles leads to dissolution through a system of electromechanically operated valves and through the flow cell. Alternatively, an autosampler can be used to process several solutions in an automatic way.

Before using the sensor chip, it is preferred to wash the electrode with a washing solution containing ions.

In any case, the solutions containing ions of the present invention preferably contain univalent and bivalent ions selected from Na +, K +, Mg2 + and / or Ca2 +.

The total concentration of the ions in the solutions containing ions is preferably from about 100 mM to about 1000 mM, particularly from about 200 mM to about 500 mM, more particularly from about 300 mM to about 500 mM, more particularly about 435 mM. The concentration of the univalent ions in the solutions containing ions is preferably from about 300 mM to about 400 mM and the concentration of the divalent ions in the solutions containing ions is preferably from about 2 mM to about 10 mM, particularly from about 5 mM to about 8 mM, more particularly about 5 mM.

In another preferred embodiment, solutions containing ions also contain a buffer, particularly a HEPES / NMG buffer, 30 ± 10 mM, pH 7.0 ± 1.0.

Examples of solutions containing ions are for (a) a washing solution: 30 ± 10 mM of a buffer, for example HEPES / NMG, pH 7.0 ± 1.0, 300 ± 100 mM of a univalent ion, for example NaCl, 4 ± 2 mM of a bivalent ion, for example MgCl2. (b) a non-activating solution: 30 ± 10 mM of a buffer, for example HEPES / NMG, pH 7.0 ± 1.0, 300 + 100 mM of a univalent ion, for example NaCl, 4 ± 2 mM a bivalent ion, for example MgCl 2, and 0.5-100 mM of a univalent ion, for example NaCl, which must be equimolar with the concentration of the substrate in the activating solution. (c) an activating solution: 30 ± 10 mM of a buffer, for example HEPES / NMG, pH 7.0 ± 1.0. 300 ± 100 mM of a univalent ion, for example NaCl, 4 ± 2 mM of a bivalent ion, for example MgCl 2, and 0.5-100 mM of a substrate, for example choline chloride.

The substrate of the activating solution is generally an organic cation, particularly a cationic drug, a cationic xenobiotic agent and / or a cationic vitamin, more particularly a primary, secondary, tertiary or quaternary amine, more particularly choline, acetylcholine, nicotine, N1- methylnitamide, morphine, 1-methyl-4-phenylpyridinium, procainamide, tetraethylammonium, tributylmethylammonium, debrisoquine or a biogenic amine such as epinephrine, norpenephrine or camitine or lipophilic compounds such as quinine, quinidine or spheroids such as corticosterone or organic anions such as para acid. hippuric amino, probenecid.

In general, the electrical signal is measured using amperometric and / or potentiometric means, and steps (b) to (d) are carried out at least 2 times, particularly 2 to 4 times.

The term "electrical signal" or the term "current" in the context of this invention means the peak current in response to the change of non-activating solution by activating solution, which includes the peak peak current but is not limited thereto. The current amplitude normally rises between 10 and 100 ms, followed by a slower drop in the next 2 seconds. The polarity of the current can be positive or negative, depending on the polarity of the transported ions and / or the polarity of the changed residues in the protein and the vector orientation of its transport or displacement along or within the membranes of the compartments. The currents resulting from the substitution of the activating solution by non-activating solution or the substitution of the non-activating solution by the washing solution are not generally taken into consideration with respect to the determination of the OCT activity. The flow rates and ranges are preferably chosen such that the current response with respect to the substitution of the non-activating solution for the activating solution is left unselected by the current responses elicited by the other substitution steps.

The method of the present invention can be carried out in the presence of a chemical compound, particularly a stimulator (activator) or an OCT inhibitor.

Therefore, the present invention also relates to a method for identifying a chemical compound that modulates OCT activity with the following consecutive steps: (a) carrying out the method of the present invention, and (b) identifying the compound chemical.

The chemical compound is generally an organic cation, particularly a cationic drug, a cationic xenobiotic agent and / or a cationic vitamin and / or biogenic amines, more particularly a primary, secondary, tertiary or quaternary amine, in which the chemical compound is usually a stimulator or an OCT inhibitor. The chemical compound can, for example, be present in a collection of chemical compounds.

Another subject matter of the present invention is the electrophysiological sensor chip itself without cells containing the OCT, as described above in detail. The OCT is attached to the sensor chip according to methods generally known to any person skilled in the art and / or as specifically described in the Example.

The sensor chip may further comprise a data acquisition device for acquiring the measurement data of the electrode, and optionally exchange and / or mixing means for performing the exchange and / or the available mixture of the solutions containing ions. The sensor chip may be in the form of a microplate or microtiter plate.

Another subject matter of the present invention is an apparatus that contains a sensor chip of the present invention, a reference electrode, a data acquisition device for acquiring electrode measurement data, exchange and / or mixing means for performing the exchange and / or the available mixture of solutions containing ions, a flow analysis device, a power source, a computer and an autosampler. The reference electrode is preferably a Pt / Pt electrode, Ag / AgCl or tin and indium oxide.

A further subject matter of the present invention is a kit containing: (a) an electrophysiological sensor chip without cells of the present invention or an apparatus of the present invention, (b) at least one solution containing ions as defined above , and optionally (c) a substrate as defined above.

The following Figures, Tables, Sequences and Examples will explain the present invention without limiting the scope of the invention.

DESCRIPTION OF THE FIGURES: Figure 1A shows the electrical responses of a typical sensor with immobilized membranes harboring rOCT2 (slc22a2) in response to the addition of activating solution (ColinaCI 30 mM) before (black trace) and after inhibition (gray trace) with 1 mM TBA .

Figure 1 B shows the electrical responses of a typical sensor with immobilized membranes harboring hOCT2 (SLC22A1) in response to the addition of activating solution (ColinaCI 30 mM) before (black trace) and after inhibition (gray trace) with TBA 1 mM. Figure 2A shows the dependence of the choline concentration of rOCT2 (slc22a2) (CHO cell membranes).

Figure 2B shows the dependence of the choline concentration of hOCT2 (SLC22A1) (CHO cell membranes).

Figure 3 shows the pH dependence of rOCT2 (slc22a2) and hOCT2 (SLC22A2) from insect cells.

Figure 4A shows. the IC50 of TBA of rOCT2 (slc22a2) (CHO cells). The IC50 was determined using 10 mM choline as a substrate.

Figure 4B shows the T50 IC50 of hOCT2 (SLC22A2) (CHO cells). The IC 50 was determined using 30 mM choline as a substrate.

Figure 5A shows the stably expressed rOCT2 electric current (slc22a2) in patch clamp experiments (CHO cells).

Figure 5B shows the electric current of stably expressed hOCT2 (slc22a2) in patch clamp experiments (CHO cells).

Figure 6A shows the IC50 of quinine from rOCT2 (slc22a2) (CHO cells). The IC50 was determined using 10 mM choline as a substrate.

Figure 6B shows the dependence of the acetylcholine concentration of rOCT2 (slc22a2) (CHO cells).

Figure 7 shows a nucleic acid sequence containing the coding region of human OCT2 (hOCT2, SLC22A2)). The initiation (ATG) and termination (TAA) sites of the gene are in bold and underlined. The Xhol / Xhol cloning sites (CTCGAG) are underlined.

Figure 8 shows a nucleic acid sequence containing the coding region of rat OCT2 (rOCT2; SLC22A2). The initiation (ATG) and termination (TGA) sites of the gene are in bold and underlined.

The cloning sites Kpnl (GGTACC) and BamHI (GGATCC) are underlined.

Figure 9 shows a nucleic acid sequence containing the coding region of human OCT1 (hOCTI; SLC22A1). The The initiation (ATG) and termination (TGA) sites of the gene are in bold and underlined. The cloning sites HINDIII (AAGCTT) and EcoRV (GATATC) are underlined.

Figure 10 shows a nucleic acid sequence containing the coding region of human OCT3 (hOCT3; SLC22A3). The initiation (ATG) and termination (TGA) sites of the gene are in bold and underlined.

Figure 11 shows a nucleic acid sequence containing the coding region of human OCTN1 (SLC22A4). The initiation (ATG) and termination (TGA) sites of the gene are in bold and underlined.

Figure 12 shows a nucleic acid sequence containing the coding region of human OCTN2 (SLC22A5). The initiation (ATG) and termination (TGA) sites of the gene are in bold and underlined.

DESCRIPTION OF THE SEQUENCES I KNOW THAT. ID. No. 1 shows a nucleic acid sequence containing the coding region of human OCT2 (rOCT2, SLC22A2).

I KNOW THAT. ID. No. 2 shows a nucleic acid sequence containing the coding region of rat OCT2 (rOCT2; slc22a2)).

I KNOW THAT. ID. No. 3 shows a nucleic acid sequence containing the coding region of human OCT3 (hOCT3, SLC22A3).

I KNOW THAT. ID. No. 4 shows a nucleic acid sequence containing the coding region of human OCT3 (hOCT3, SLC22A3).

I KNOW THAT. ID. No. 5 shows a nucleic acid sequence containing the coding region of human OCTN1 (SLC22A4).

I KNOW THAT. ID. No. 6 shows a nucleic acid sequence containing the coding region of human OCTN2 (SLC22A5).

EXAMPLES materials Washing solution (C): HEPES / 30 mM NMG, pH 7.4 NaCl 300 M MgCl2 5 mM Non-activating solution (B): HEPES / 30 mM NMG, pH 7.4 NaCl 400 mM mM MgCl2 Activating solution (A): 30 mM HEPES / NMG, pH 7.4 NaCl 300 mM Choline / 100 mM CI 5 mM MgCl2 In solution C, B and A ,. TBA or Quinine 10μM; respectively Procedure of Ensavo (a) Membrane Preparation After collecting the cells from a virally transfected Sf9 cell line or HighFive suspension or a stably transfected CHO cell line via centrifugation, aliquots of ca. 2 g of cells in wet weight were deep-frozen in liquid nitrogen and stored at -80 ° C for further preparations.

The cell pellet was thawed on ice and transferred to ice cold buffer (0.25 M sucrose, 5 mM Tris, pH 7.5, 2 mM DTT, one tablet of complete protease inhibitor mixture per 50 ml (Roche Diagnostics GmbH, Mannheim, Germany).

The membrane fragments were prepared by cell disruption. Cells were homogenized by the cell-disruption method in nitrogen using a Parr Cell Rupture Pump (Parr Instrument, Illinois, USA) or the Dounce homogenization method using a Dounce Homogenizer (7ml by Novodirect GmbH, Kehl / Rhein, Germany) and the suspension was centrifuged 10 min at 4 ° C and 680 g and 10 min at 4 ° C and 6100 g. The supernatants were recovered and centrifuged again for 1 h at 4 ° C and 100,000 g in a SW41 tiltrotator.

The pellets were suspended in approximately 2 ml of mM Tris, pH 7.5. With sucrose 87% (in Tris 5 mM) the suspension was adjusted to 56%. The sucrose gradient is increased starting in 2 mL of the 56% fraction in the lower part, followed by 3 mL of 45% sucrose, 3 mL of 35% and 2 mL of 9% sucrose.

After centrifugation for 2.5 h (or even more) at 4 ° C and 100000 g gradient bands were carefully aspirated with a pasteur pipette and pooled into new tubes together with 5 ml of 300 mM NaCl, 25 mM MgCl, 30 mM Hepes, pH 7.5 or 10 mM Tris / HC1, pH 7.5.

Another centrifugation step followed: 1 h at 150000 g, 4 ° C.

The resulting pellet was resuspended in 300 mM NaCl, 5 mM MgCl 2, 2 mM DTT, 30 mM Hepes, pH 7.5, 10% glycerol. (b) Preparation of Biosensors The biosensors were prepared according to the following protocol. 1. Addition of 30 μl of mercaptan solution (2% mercaptan in isopropanol) to biosensor 2. Incubation time: 15 min 3. Rinse with 3x70 μl isopropanol 4. Vacuum biosensor dried 5. Drying time: 30 min 6. Addition of 2 μl of the lipid (60 units (weight) 2-Difitanoyl-sn-Glycero-3-phosphocholine + 1 unit of octadecylamine dissolved in 800 units of n-decane) 7. Immediate addition of 30 μl of DTT buffer (1, 542 mg DTT / Buffer C 50 ml) 8. Incubation time: 20 min. 9. Addition of 20 μl of membrane preparation + 135 μl of DTT-Buffer C and Mix (for 6 sensors) . Sonication: 2x10 times (adjusted 0.5 s / 30%) with an ice pause of 30 s 11. Elimination of the biosensor buffer 12. Immediate addition of 25 μl of membrane solution to the biosensors (mix 3 times) 13. Storage overnight in the refrigerator (in Petri dish with high humidity) (c) Protocol for the Exchange of Dissolution For the determination of its activity, the OCT protein was treated consecutively with a washing solution, not activating and activating and the electric current was measured when it was changed from the charge treatment to the activation one. The substitution of the washing solution and the non-activating solution for the activating solution (solution containing the substrate) triggers the OCT activity. The subsequent substitution of solutions in reverse order returns the sensor chip to its initial state.

Cycle 1: 1 minute stop Cycle 2: Stop 5 minutes and add a compound to be analyzed Cycle 3: 1 minute stop Cycle 4: Stop 5 minutes and add the same compound to another concentration, or another compound, etc. The following adjustments were used for hOCT2 measurements: After the containers of buffer A, B and C of the biosensor system with "activating" buffer and "non-activating" buffer were filled, a simulation was mounted in the sensor holder and the system was rinsed with all the buffers to eliminate the bubbles of air of the entire fluid system. An empty or blind sensor was then replaced by a standard glass-based sensor preloaded with CHO membrane fragments containing hOCT2 (chemically modified gold surface 3 mm in diameter, LonGate Biosciences GmbH, Frankfurt / M., Germany). The transport of liquid through the flow system, incng the sensor flow cell, was achieved by applying pressurized air to the buffer containers.

Normally the measurements were carried out at an overpressure of 250 mbar, resulting in a flow rate of approximately 300 μL s "1. For the determination of its activity, the membranes harboring the OCT protein were treated consecutively with a" non-activating "solution. "and" activating. "Subsequent replacement of solutions in reverse order returns the sensor chip to its initial state.For the control software, a sequence was defined (see Figure 1), in which the" non-activating "buffer flowed. on the surface of the sensor, followed by the buffer "activating" and the buffer "not activating." During the complete sequence, the response of the current was saved (2,000 samples s "1) and saved in data files. For the dose response experiments, the inhibitors were dissolved in "non-activating" and "activating" buffer, respectively. All the chemicals were of analytical grade or higher.

Analysis of data High control: valley electric current after activation with choline / 100 mM CI before inhibition; Low control: valley electric current after activation with choline / CI 0 mM after inhibition; The results are calculated from the corrected raw data.

Inhibitor of the conveyor = 100 * íl - (sample - 8ntro1 ba¡0 ^ "i V (high control - low control) / Results 1. Figures 1A and 1B show the electrical responses with the addition of choline-containing activating solution to sensors with immobilized membranes harboring rOCT2 and hOCT2 respectively before (black trace) and after inhibition (gray trace). The amplitude of the peak is equivalent to the initial activity of the transporters; the decay has to be attributed to the charge of the capacitance of the sandwich structure of the biosensor. 2. Figures 2A and 2B show the influence of choline concentration on the amplitude of the electrical response (high control) in membranes containing rOCT2 and hOCT2 respectively.

According to the results of a titration of the concentration of choline a choline concentration of 100 mM was used in the following tests since this provided measurement signals with high amplitude. 3. The dependence of the measured pH showed the highest activity of the protein at pH 7.4, which was therefore used in subsequent tests (Fig. 3). For the inhibition experiments, the concentration of choline was decreased to 10 mM (in the range of the KM value to detect competitive inhibitory effects). The IC50 for a standard OCT inhibitor (TBA) was determined to be 3.5 μM for rOCT2 (Fig. 4A) and 2.9 μM for hOCT2 (Fig. 4B) respectively. 4. Using the parameters defined above, different membrane preparations were compared from recombinant cell lines. The best results were obtained with a CHO cell line. The insect cell preparations provided high quality signals, albeit with an unadjusted decrease for the determination of IC5o.

. The CHO cell line was also monitored via manual patch clamp electrophysiology, considered a gold standard for the investigation of ion transporters. For the electric currents of rOCT2 they were hardly undetectable for hOCT2 and the IC50 values could not be determined (Fig. 5A and 5B). 6. For another evaluation of the sensitivity of the signal, other substrates and inhibitors were analyzed. Figures 6A and 6B show these examples.

Along with the essays published in this document for the OCT2 were cloned and generated other members of the family, for example hOCTI or hOCT3, and constructs. The cell lines were generated using the Flpln- and T-REX System from Invitrogen (Cat. No. R758-07).

Claims (29)

1. CLAIMS 1.- A method to determine the activity of the organic cation transporter (OCT), said method comprising the consecutive steps of: (a) providing an electrophysiological sensor chip without cells containing a solid support sensor electrode and a lipid layer containing the OCT located in the immediate spatial proximity of the sensor electrode, while the sensor electrode is electrically isolated from the solutions used and the lipid layer, (b) treating the sensor chip with a non-activating solution containing ions, (c) treating the sensor chip with an activating solution containing ions and substrate, and (d) measuring the electrical signal.
2. 2. The method of claim 1, wherein the OCT is selected from OCT1 (SLC22A1), OCT2 (SLC22A2), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5).
3. 3. - The method of claim 1 or 2, wherein the OCT is of mammalian origin, particularly rat, mouse, rabbit, pig, guinea pig indians, drosophila melanogaster, caenorhabditis elegans or human, more particularly human OCT1 (SLC22A1).
4. 4. - The method according to any of claims 1-3, wherein the electrode comprises a metallic material or an electrically conductive metal oxide, particularly gold, platinum, silver or tin oxide and indium.
5. 5. - The method according to any of claims 1-4, wherein the solid support sensor electrode is a glass sensor electrode or supported on a polymer, particularly a sensor electrode supported on Borofloat glass, more particularly an electrode Gold supported in Borofloat glass.
6. 6. - The method according to any of the claims 1-5, in which the lipid layer is attached to the electrode via a chemical bond, particularly via coupling with histidine tail or streptavidin-biotin coupling, or via hydrophobic, hydrophilic or ionic forces.
7. 7. - The method according to any of the claims 1-6, wherein the electrode is electrically isolated by one or more insulating monolayer (s), particularly by one or more insulating amphiphilic organic compounds, more particularly by one or more monolayer (s) of insulating membrane (s), more particularly by a mercaptan layer, especially octadecyl-thiol, a lower layer facing the electrode and a membrane monolayer as a top layer oriented in the opposite direction from the electrode.
8. 8. - The method according to any of claims 1-7, wherein the electrode is first washed with a washing solution containing ions.
9. 9. - The method according to any of the claims 1-8, in which the solutions containing ions contain univalent and bivalent ions selected from Na +, K +, Mg2 + and / or Ca2 +.
10. 10. - The method according to any of claims 1-9, wherein the total concentration of the ions in the solutions containing ions is from about 100 mM to about 1000 mM, particularly from about 200 mM to about 500 mM, more particularly from about 300 mM to about 500 mM, more particularly about 435 mM.
11. 11. The method according to claim 9 or 10, wherein the concentration of the univalent ions in the solutions containing ions is from about 300 mM to about 400 mM.
12. 12. - The method according to any of the claims 9-11, wherein the concentration of the bivalent ions in the solutions containing ions is from about 2 mM to about 10 mM, particularly from about 5 mM to about 8 mM, more particularly about 5 mM.
13. 13. - The method according to any of claims 1-12, wherein the solutions containing ions further contain a buffer, particularly a HEPES / NMG buffer, 30 ± 10 mM, pH 7.0 ± 1.0.
14. 14. - The method according to any of claims 1-13, wherein the substrate of the activating solution is an organic cation, particularly a cationic drug, a cationic xenobiotic agent and / or a cationic vitamin, more particularly a primary amine, secondary, tertiary or quaternary, more particularly choline, acetylcholine, nicotine, N 1 -methylnicotinamide, morphine, 1-methyl-4-phenylpyridinium, procainamide, tetraethylammonium, tributylmethylammonium, debrisoquine or a biogenic amine such as epinephrine, norpenephrine or camitine or lipophilic compounds as quinine, quinidine or steroids such as corticosterone or organic anions such as para-amino hippuric acid, probenecid.
15. 15. - The method according to any of claims 1-14, wherein the electrical signal is measured using amperometric and / or potentiometric means.
16. 16. - The method according to any of the claims 1-15, wherein steps (b) to (d) are carried out at least 2 times, particularly 2 to 4 times.
17. 17. - The method according to any of claims 1-16, wherein the method is carried out in the presence of a chemical compound, particularly an OCT inhibitor.
18. 18. - A method for determining the activity of a chemical compound, said method comprising the consecutive steps of: (a) carrying out the method according to any of claims 1-17, and (b) determining the activity of the chemical compound.
19. 19. - The method of claim 18, wherein the method is carried out in the presence and / or in the absence of the substrate of the activating solution.
20. 20. - A method for identifying a chemical compound that modulates the activity of OCT, said method comprising the consecutive steps of: (a) carrying out the method according to any of claims 1-19, and (b) identifying the chemical compound .
21. 21. - The method of claim 20, wherein the chemical compound is an organic cation, particularly a cationic drug, a cationic xenobiotic agent and / or a cationic vitamin and / or biogenic amines, more particularly a primary, secondary, tertiary amine or Quaternary, lipophilic compounds, organic anions.
22. 22. - The method of claim 20, wherein the chemical compound is an OCT inhibitor.
23. 23. - The method according to any of claims 17-22, wherein the chemical compound is present in a collection of chemical compounds.
24. 24. - An electrophysiological sensor chip without cells as defined in any of claims 1-7 and 15.
25. 25. - The sensor chip according to claim 24 further comprising a data acquisition device for acquiring the measurement data of the electrode, and optionally exchange and / or mixing means for performing the exchange and / or the available mixture of the solutions that contain ions.
26. 26. - The sensor chip according to claim 24 or 25 in the form of a microplate or microtiter plate.
27. 27. - An apparatus containing a sensor chip according to claim 24 or 26, a reference electrode, a data acquisition device for acquiring the measurement data of the electrode, exchange and / or mixing means to perform the exchange and / or the available mixture of solutions containing ions, a flow analysis device, a power source, a computer and an autosampler.
28. 28. - The apparatus of claim 27, wherein the reference electrode is a Pt / Pt electrode, Ag / AgCl or tin oxide and indium.
29. 29. - A kit containing: (a) an electrophysiological sensor chip without cells according to any of claims 24-26 or an apparatus according to claim 27 or 28, (b) at least one solution containing ions as defined in any of claims 9-13, and optionally (d) a substrate as defined in claim 14.