Composition and method for measuring thallium influx and efflux

Patent metadata

Field	Value
Publication number	US-9476892-B2
Application number	US-201414521346-A
Country	US
Kind code	B2
Filing date	Oct 22, 2014
Priority date	Oct 15, 2007
Publication date	Oct 25, 2016
Grant date	Oct 25, 2016

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.

First claim

What is claimed is:

1. A method for detecting the activity of potassium ion channel in a cell, the method comprising:
providing a loading buffer solution to the cell, the loading buffer solution comprising a thallium indicator and a physiological concentration of chloride ions, wherein the thallium indicator is an acetoxymethyl ester of a xanthene-based compound comprising a thallium ion-complexing moiety, wherein the thallium ion-complexing moiety is 2-methoxy-aniline-N,N-diacetic acid or a derivative thereof, and wherein the chloride ions are at a concentration of about 50 mM to about 150 mM;
providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium at a concentration of about 0.1 mM to about 5.0 mM, thereby causing thallium influx into the cell through the potassium ion channel; and
measuring a change in at least one optical property of the thallium indicator in response to thalium influx thereby detecting the activity of the potassium ion channel.

2. The method of claim 1 , wherein the thallium indicator associates with thallium within the cell.

3. The method of claim 1 , wherein the thallium indicator is a compound that exhibits an increase in fluorescence when associated with a thallium ion.

4. The method of claim 1 , wherein the thallium indicator is a compound that exhibits a change in the optical density inside the cell when associated with a thallium ion.

5. The method of claim 1 , wherein the thallium indicator exhibits a decrease in fluorescence intensity in the presence of thallium ions.

6. The method of claim 1 , wherein the thallium indicator exhibits an increase in fluorescence in the presence of thallium ions.

7. The method of claim 1 , wherein the thallium indicator is a fluorescent compound that is sensitive to monovalent or divalent cations.

8. The method of claim 7 , wherein the thallium indicator is a Zn 2+ or Ca 2+ indicator.

9. The method of claim 1 , wherein the thallium indicator is FluoZin 1, FluoZin 2, or a derivative or salt thereof.

10. The method of claim 1 , wherein the thallium is in the form or a salt.

11. The method of claim 10 , wherein the thallium salt is soluble in the loading buffer solution.

12. The method of claim 10 , wherein the thallium salt is selected from Tl 2 SO 4 , Tl 2 CO 3 , TlCl, TlOH, TlOAc, and TlNO 3 .

13. The method of claim 1 , further comprising quantifying the level of thallium, or washing the cells after the loading buffer solution is provided to the cells.

14. The method of claim 1 , wherein the at least one optical property of the thallium indicator is intensity, polarity, frequency, or optical density.

15. The method of claim 1 , wherein the change in the at least one optical property of the thallium indicator is measured by light microscopy, confocal microscopy, fluorescence microscopy or spectrophotometry.

16. The method of claim 1 , further comprising adding a quencher to the loading buffer solution.

17. The method of claim 16 , wherein the quencher is substantially not cell permeant.

18. The method of claim 17 , wherein the quencher is tartrazine, amaranth, acid red 37, congo red, trypan blue, brilliant black, or a combination thereof.

19. The method of claim 1 , further comprising stimulating the ion channel with a stimulus.

20. The method of claim 19 , wherein the stimulus is a ligand that binds to the ion channel, a channel-linked receptor, or an electrical stimulus.

21. The method of claim 19 , wherein the stimulus is a G-protein coupled receptor agonist capable of activating activate GIRK potassium ion channels to allow thallium influx.

22. The method of claim 19 , wherein the stimulus is nicotine, acetylchloline, muscarine, carbamyline, or a GIRK potassium ion channel activator.

23. The method of claim 19 , wherein the stimulus comprises a composition that causes depolarization of the ion channel.

24. The method of claim 23 , wherein the composition comprises ionophores, valinocmycin, or potassium salts.

25. The method of claim 23 , wherein the composition comprises channel rhodopsin, halorhodopsin, or quantum dot nanocrystals.

26. The method of claim 1 , wherein the stimulus buffer comprises thallium at a concentration of about 0.1 mM to about 4.0 mM.

27. The method of claim 1 , wherein the method does not comprise washing the cells after the loading buffer is provided to the cells.

28. The method of claim 1 , wherein the cell is selected from the group consisting of bacterial, yeast, plant, and animal cells.

29. The method of claim 28 , wherein the animal cells is a mammalian cell selected from the group consisting of a neuron, cardiac cell, cancer cell, smooth muscle cell and an immortalized cell.

30. The method of claim 1 , wherein the xanthene-based compound is substituted on one or more aromatic carbons by fluorine.

Assignees

• Life Technologies Corp

Inventors

• Beacham Daniel
• Gee Kyle

Classifications

• G01N33/6872 Primary

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• C12Q1/02

  involving viable microorganisms · CPC title

• G01N33/84 Primary

  involving inorganic compounds or pH · CPC title

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title