Composition and method for measuring thallium influx and efflux

Abstract

The present invention relates to methods for detecting the activity of an ion channel in a cell. The methods comprise providing a loading buffer solution to a cell that has an ion channel. The loading buffer comprises at least one thallium indicator (e.g., an environmentally sensitive, luminescent dye) and a physiological concentration of chloride ions. The methods further comprise providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium (e.g., thallium ions). Providing the stimulus buffer causes thallium influx into the cell through the ion channel. After providing the stimulus buffer, the luminescence (e.g., fluorescence) of the dye in the cell is detected. The luminescence of the dye can change in the presence or absence of thallium. The methods may be used to measure influx or efflux of thallium through an ion channel.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 60 / 979,958, filed Oct. 15, 2007, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods for detecting the activity of an ion channel in a cell. The methods comprise providing a loading buffer solution to the cell, with the loading buffer comprises at least one environmentally sensitive thallium indicator (e.g., luminescent dye) and chloride ions, and providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium. Providing the stimulus buffer causes thallium influx into the cell through the ion channel. After providing the stimulus buffer, the luminescence (e.g., fluorescence) of the thallium indicator in the cell is detected, where the luminescence of the dye can change in the presence or absence of thallium.

[0004] 2. Back...

Examples

example 1

Thallium Influx Assay

[0133]The following is in example of the components that may be used in some of the methods of the present invention.

[0134]Component A:

Per Vial:FluoZin-1 AM ester (Invitrogen) 1-20 μgLyophilized and stored in a single use 15 mL vial

[0135]Component B: 5× Assay Buffer: 1×25 mL

Per Vial:50% Vol 10X HBSS12.5 mL10% Vol 1 M HEPES 2.5 mL40% Vol H2O  10 mL

[0136]Component C: 5× Chloride Free Stimulus Buffer: 1×25 mL

Per vial: 700 mM Na Gluconate3.82 g12.5 mM K Gluconate  73 mg  9 mM Ca Gluconate96.7 mg  5 mM Mg Gluconate51.7 mg 100 mM HEPES 2.5 mL 1MWater22.5 mL

[0137]Component D: Concentrated (125 mM) K2SO4 Solution: 1×20 mL

Per vial125 mM Potassium Sulfate435 mgH2O 20 mL

[0138]Component E: Concentrated (50 mM) Tl2SO4 Solution: 1×20 mL

Per vial50 mM Thallium Sulfate505 mgH2O 20 mL

[0139]Component F: 100×: 1×1 mL

Per vialPLURONIC F12720-100mgPLURONIC P855-20mgH2O1mL

[0140]Cells comprising an ion channel are cultured to about 75% confluence and in log-phase growth. The cells are t...

example 2

Assay of Cells Transfected with hERG Ion Channels

[0147]Cells transfected with the hERG ion channels were assayed using the methods of the present invention. Cells were loaded in assay buffer containing 0.5 uM FluoZin-1 AM ester and stimulated with 1× dilution of stimulus buffer (Component C, containing 25 mM K2SO4 and 10 mM Tl2SO4). “Control” were tet-stimulated hERG 293 T Rex cells, and “no tet” traces were cells which were not stimulated to express hERG. The stimulus was delivered to the cells in a volume of 25 uL, added to 100 uL for a 5× dilution. In this particular example, the final concentration of thallium on the cells was about 4 mM. FIG. 1 demonstrates the absorbance curves of cells (plotted as dF / F as a function of time) transfected with the hERG ion channel and assayed using the methods of the present invention. FIG. 2 depicts hERG activity cells transfected with hERG ion channels (tetracycline induced and non-tetracycline induced) measured in a 96 well plate with the FL...

example 3

Thallium Sensitivity Assay

[0148]An assay is provided to evaluate a compound's sensitivity to thallium (I). Compounds with sufficient sensitivity to thallium (I) may be used as thallium indicators for measuring thallium influx and efflux through ion channels. A 10 micromolar solution of a fluorescent compound is prepared in 10 mM HEPES buffer, pH 7.4. The maximum fluorescence emission intensity is measured by excitation at the compound's excitation maximum wavelength. The measurement is performed in a cuvette in spectrofluorimeter or in a microplate well in a fluorescence microplate reader. Increasing amounts of a stock aqueous solution of thallium sulfate are added to the fluorescent compound solution, such that the final concentration of thallium (I) is 0, 5, 10, 50, 100, 500, 1000, and 2000 micromolar. After each addition of thallium sulfate solution, the fluorescence intensity is measured and compared with the intensity obtained in the presence of zero thallium (I). An overall fl...

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 60 / 979,958, filed Oct. 15, 2007, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods for detecting the activity of an ion channel in a cell. The methods comprise providing a loading buffer solution to the cell, with the loading buffer comprises at least one environmentally sensitive thallium indicator (e.g., luminescent dye) and chloride ions, and providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium. Providing the stimulus buffer causes thallium influx into the cell through the ion channel. After providing the stimulus buffer, the luminescence (e.g., fluorescence) of the thallium indicator in the cell is detected, where the luminescence of the dye can change in the presence or absence of thallium.

[0004] 2. Back...

Examples

example 1

Thallium Influx Assay

[0133]The following is in example of the components that may be used in some of the methods of the present invention.

[0134]Component A:

Per Vial:FluoZin-1 AM ester (Invitrogen) 1-20 μgLyophilized and stored in a single use 15 mL vial

[0135]Component B: 5× Assay Buffer: 1×25 mL

Per Vial:50% Vol 10X HBSS12.5 mL10% Vol 1 M HEPES 2.5 mL40% Vol H2O  10 mL

[0136]Component C: 5× Chloride Free Stimulus Buffer: 1×25 mL

Per vial: 700 mM Na Gluconate3.82 g12.5 mM K Gluconate  73 mg  9 mM Ca Gluconate96.7 mg  5 mM Mg Gluconate51.7 mg 100 mM HEPES 2.5 mL 1MWater22.5 mL

[0137]Component D: Concentrated (125 mM) K2SO4 Solution: 1×20 mL

Per vial125 mM Potassium Sulfate435 mgH2O 20 mL

[0138]Component E: Concentrated (50 mM) Tl2SO4 Solution: 1×20 mL

Per vial50 mM Thallium Sulfate505 mgH2O 20 mL

[0139]Component F: 100×: 1×1 mL

Per vialPLURONIC F12720-100mgPLURONIC P855-20mgH2O1mL

[0140]Cells comprising an ion channel are cultured to about 75% confluence and in log-phase growth. The cells are t...

example 2

Assay of Cells Transfected with hERG Ion Channels

[0147]Cells transfected with the hERG ion channels were assayed using the methods of the present invention. Cells were loaded in assay buffer containing 0.5 uM FluoZin-1 AM ester and stimulated with 1× dilution of stimulus buffer (Component C, containing 25 mM K2SO4 and 10 mM Tl2SO4). “Control” were tet-stimulated hERG 293 T Rex cells, and “no tet” traces were cells which were not stimulated to express hERG. The stimulus was delivered to the cells in a volume of 25 uL, added to 100 uL for a 5× dilution. In this particular example, the final concentration of thallium on the cells was about 4 mM. FIG. 1 demonstrates the absorbance curves of cells (plotted as dF / F as a function of time) transfected with the hERG ion channel and assayed using the methods of the present invention. FIG. 2 depicts hERG activity cells transfected with hERG ion channels (tetracycline induced and non-tetracycline induced) measured in a 96 well plate with the FL...

example 3

Thallium Sensitivity Assay

[0148]An assay is provided to evaluate a compound's sensitivity to thallium (I). Compounds with sufficient sensitivity to thallium (I) may be used as thallium indicators for measuring thallium influx and efflux through ion channels. A 10 micromolar solution of a fluorescent compound is prepared in 10 mM HEPES buffer, pH 7.4. The maximum fluorescence emission intensity is measured by excitation at the compound's excitation maximum wavelength. The measurement is performed in a cuvette in spectrofluorimeter or in a microplate well in a fluorescence microplate reader. Increasing amounts of a stock aqueous solution of thallium sulfate are added to the fluorescent compound solution, such that the final concentration of thallium (I) is 0, 5, 10, 50, 100, 500, 1000, and 2000 micromolar. After each addition of thallium sulfate solution, the fluorescence intensity is measured and compared with the intensity obtained in the presence of zero thallium (I). An overall fl...

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