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.

1 . A method for detecting the activity of an ion channel in a cell, the method comprising:
 a) providing a cell with an ion channel;   b) providing a loading buffer solution to the cell, the loading buffer solution comprising a thallium indicator and a physiological concentration of chloride ions;   c) providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium, and wherein providing the stimulus buffer causes thallium influx into the cell through the ion channel; and   d) detecting the thallium indicator.   
     
     
         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 has an optical property that changes in the presence or absence of thallium. 
     
     
         4 . The method of  claim 1  wherein the thallium indicator is a compound that exhibits an increase in fluorescence when associated with a thallium ion. 
     
     
         5 . 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. 
     
     
         6 . The method of  claim 1  wherein the thallium indicator is hydrophobic. 
     
     
         7 . The method of  claim 1  wherein the thallium indicator is hydrophilic. 
     
     
         8 . The method of  claim 1  wherein the thallium indicator comprises an acetoxymethyl ester moiety. 
     
     
         9 . The method of  claim 1  wherein the thallium indicator is an environmentally sensitive fluorescent dye. 
     
     
         10 . The method of  claim 1  wherein the thallium indicator is a non-fluorescent compound. 
     
     
         11 . The method of  claim 10  wherein the thallium indicator associates with thallium to form a precipitate or a colored product. 
     
     
         12 . The method of  claim 10  wherein the thallium indicator is an ion selected from iodide, bromide, and chromate. 
     
     
         13 . The method of  claim 1  wherein the thallium indicator exhibits a decrease in fluorescence intensity in the presence of thallium ions. 
     
     
         14 . The method of  claim 13  wherein the thallium indicator is ANTS, Fluo-4, Fluo-3, PBFI, Phen Green, APTRA-BTC or Mag-Fura Red or a derivative or salt thereof. 
     
     
         15 . The method of  claim 1  wherein the thallium indicator exhibits an increase in fluorescence in the presence of thallium ions. 
     
     
         16 . The method of  claim 15  wherein the thallium indicator is Magnesium Green, Fluo-4FF, FluoZin-1 or FluoZin-2, RhodZin-3, or a derivative or salt thereof. 
     
     
         17 . The method of  claim 1  wherein the thallium indicator is a fluorescent compound that exhibits a fluorescence intensity change of 25% or greater in the Thallium Sensitivity Assay. 
     
     
         18 . The method of  claim 17  wherein the thallium indicator is a fluorescent compound that is sensitive to monovalent or divalent cations. 
     
     
         19 . The method of  claim 18  wherein the thallium indicator is a Zn 2+  or Ca 2+  indicator, 
     
     
         20 . The method of  claim 1  wherein the thallium indicator comprises a xanthene moiety. 
     
     
         21 . The method of  claim 20  wherein the thallium indicator is FluoZin 1, FluoZin 2, FluoZin 3, FluoZin 4, Red Fluo-4FF, Magnesium Green, Phen Green, RhodZin-3, or a derivative or salt thereof. 
     
     
         22 . The method of  claim 1  wherein the thallium is in the form or a salt. 
     
     
         23 . The method of  claim 22  wherein the thallium salt is soluble in the loading buffer solution. 
     
     
         24 . The method of  claim 22  wherein the thallium salt is selected from Tl 2 SO 4 , Tl 2 CO 3 , TlCl, TlOH, TlOAc, and TlNO 3 . 
     
     
         25 . The method of  claim 1  further comprising quantifying the level of thallium. 
     
     
         26 . The method of  claim 25  wherein quantifying the level of thallium comprises measuring at least one optical property of the thallium indicator in response to thallium influx. 
     
     
         27 . The method of  claim 26  wherein the at least one optical property of the thallium indicator is intensity, polarity, frequency, or optical density. 
     
     
         28 . The method of  claim 1  wherein the thallium indicator is detected by light microscopy, confocal microscopy, fluorescence microscopy or spectrophotometry. 
     
     
         29 . The method of  claim 1 , further comprising adding a quencher to the loading buffer solution. 
     
     
         30 . The method of  claim 29 , wherein the quencher is substantially not cell permeant. 
     
     
         31 . The method of  claim 29  wherein the quencher is tartrazine, amaranth, acid red 37, congo red, trypan blue, brilliant black, or a combination thereof. 
     
     
         32 . The method of  claim 1 , further comprising stimulating the ion channel with a stimulus. 
     
     
         33 . The method of  claim 32  wherein the stimulus is a ligand that binds to the ion channel, a channel-linked receptor, or an electrical stimulus. 
     
     
         34 . The method of  claim 32  wherein the stimulus is a G-protein coupled receptor agonist capable of activating activate GIRK potassium ion channels to allow thallium influx. 
     
     
         35 . The method of  claim 32  wherein the stimulus is nicotine, acetylcholine, muscarine, carbamyline, or a GIRK potassium ion channel activator. 
     
     
         36 . The method of  claim 32  wherein the stimulus comprises a composition that causes depolarization of the ion channel. 
     
     
         37 . The method of  claim 36  wherein the composition comprises ionophores, valinocmycin, or potassium salts. 
     
     
         38 . The method of  claim 36  wherein the composition comprises channel rhodopsin, halorhodopsin, or quantum dot nanocrystals. 
     
     
         39 . A kit for detecting the activity of an ion channel in a cell, the kit comprising:
 a) a loading buffer solution, wherein the loading buffer solution comprises a thallium indicator and a physiological concentration of chloride ions; and   b) a stimulus buffer, wherein the stimulus buffer comprises thallium, and wherein the stimulus buffer causes thallium influx into the cell through the ion channel.   
     
     
         40 . A kit for detecting the activity of an ion channel in a cell, the kit comprising:
 a) a thallium loading solution, wherein the solution comprises thallium; and   b) a loading buffer solution, wherein the loading buffer solution comprises a thallium indicator and a physiological concentration of chloride ions.   
     
     
         41 . A solution for detecting the activity of an ion channel in a cell comprising:
 a) a thallium indicator;   b) a physiological concentration of chloride ions; and   c) thallium.   
     
     
         42 . The solution of  claim 41  wherein the thallium concentration in the solution is about 0.1 mM to about 4 mM.