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:
 providing a cell with an ion channel;   providing a loading buffer solution to the cell, the loading buffer solution comprising a thallium indicator and a concentration of chloride ions that is greater than about 2 mM to 150 mM;   providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium at a concentration of about 0.1 to about 5.0 mM, and wherein providing the stimulus buffer causes thallium efflux or influx into the cell through the ion channel; and   detecting the thallium indicator.   
     
     
         2 - 3 . (canceled) 
     
     
         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; or   the thallium indicator is a compound that exhibits a change in optical density inside the cell when associated with a thallium ion.   
     
     
         5 - 7 . (canceled) 
     
     
         8 . The method of  claim 1  wherein the thallium indicator comprises an acetoxymethyl ester moiety. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the thallium indicator associates with thallium to form a precipitate or a colored product. 
     
     
         12 . The method of  claim 1 , wherein the thallium indicator is an ion selected from iodide, bromide, and chromate. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the thallium indicator exhibits a decrease in fluorescence intensity in the presence of thallium ions and is selected from ANTS, Fluo-4, Fluo-3, PBFI, Phen Green, APTRA-BTC or Mag-Fura Red or a derivative or salt thereof. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1  wherein the thallium indicator exhibits an increase in fluorescence in the presence of thallium ions and is selected from 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 a 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 indicator comprises a coumarin moiety. 
     
     
         23 . The method of  claim 22  wherein the thallium indicator is BTC or APTRA-BTC or a derivative or salt thereof. 
     
     
         24 . The method of  claim 1  wherein the thallium indicator comprises a benzofuran moiety. 
     
     
         25 . The method of  claim 24  wherein the thallium indicator is Mag-Fura Red or a derivative or salt thereof. 
     
     
         26 . The method of  claim 1  wherein the thallium indicator comprises a naphthalene moiety. 
     
     
         27 . The method of  claim 26  wherein the thallium indicator comprises a crown ether moiety. 
     
     
         28 - 32 . (canceled) 
     
     
         33 . The method of  claim 1  wherein the chloride ions are provided by NaCl or KCl. 
     
     
         34 . The method of  claim 1  wherein the thallium is a salt. 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 34  wherein the thallium salt is selected from Tl 2 SO 4 , Tl 2 CO 3 , TlCl, TlOH, TlOAc, and TlNO 3 . 
     
     
         37 - 39 . (canceled) 
     
     
         40 . The method of  claim 1  wherein the stimulus buffer comprises thallium at a concentration of about 0.1 mM to about 2.0 mM and the loading buffer comprises chloride ions at a concentration of about 50 mM to about 150 mM. 
     
     
         41 . The method of  claim 1  wherein the stimulus buffer comprises thallium at a concentration of about 2.0 mM to about 5.0 mM and the loading buffer comprises chloride ions at a concentration of about 50 mM to about 150 mM. 
     
     
         42 . The method of  claim 1 , further comprising quantifying the level of thallium by measuring at least one optical property of the thallium indicator in response to thallium influx wherein the at least one optical property of the thallium indicator is intensity, polarity, frequency, or optical density. 
     
     
         43 - 44 . (canceled) 
     
     
         45 . The method of  claim 1  wherein the thallium indicator is detected by light microscopy, confocal microscopy, fluorescence microscopy or spectrophotometry. 
     
     
         46 - 47 . (canceled) 
     
     
         48 . The method of  claim 1 , further comprising adding a quencher to the loading buffer solution. 
     
     
         49 . The method of  claim 48  wherein the quencher is substantially cell impermeant. 
     
     
         50 . The method of  claim 48  wherein the quencher is tartrazine, amaranth, acid red 37, congo red, trypan blue, brilliant black, or a combination thereof. 
     
     
         51 . The method of  claim 1 , further comprising stimulating the ion channel with a stimulus. 
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 51  wherein the stimulus is a G-protein coupled receptor agonist capable of activating activate GIRK potassium ion channels to allow thallium influx. 
     
     
         54 . The method of  claim 51  wherein the stimulus is nicotine, acetylchloline, muscarine, carbamyline, or a GIRK potassium ion channel activator. 
     
     
         55 . The method of  claim 51  wherein the stimulus comprises a composition that causes depolarization of the ion channel. 
     
     
         56 . The method of  claim 55  wherein the composition comprises ionophores, quantum dot nanocrystals, or potassium salts. 
     
     
         57 . The method of  claim 55  wherein the composition comprises channel rhodopsin, halorhodopsin, or valinomycin. 
     
     
         58 - 61 . (canceled) 
     
     
         62 . The method of  claim 1  wherein the ion channel is selected from potassium ion channels, ion channels that are linked to receptors, channel-linked receptors, and ion transporters. 
     
     
         63 . The method of  claim 1  wherein the ion channel is a ligand- or voltage-gated ion channel, a stretch-activated cation channel, or a selective or non-selective cation channel. 
     
     
         64 . The method of  claim 1  wherein the cell is selected from the group consisting of bacterial, yeast, plant, and animal cells. 
     
     
         65 . The method of  claim 1  wherein the cell is a mammalian cell. 
     
     
         66 . (canceled) 
     
     
         67 . The method of  claim 1  wherein the cell is a neuron, cardiac cell, cancer cell, immortalized cell, or smooth muscle cell. 
     
     
         68 . (canceled) 
     
     
         69 . The method of  claim 1  wherein the ion channel is permeable to thallium ions. 
     
     
         70 . (canceled) 
     
     
         71 . The method of  claim 1  wherein the thallium indicator is an acetoxymethyl (AM) ester derivative of a compound selected from the group consisting of ANTS, Fluo-4, Fluo-3, PB FI, Phen Green, Magnesium Green, Mag-Fura Red, Fluo-4FF, FluoZin-1, RhodZin-3 and FluoZin-2 or a salt thereof. 
     
     
         72 . A kit for detecting the activity of an ion channel in a cell, the kit comprising:
 a loading buffer solution, wherein the loading buffer solution comprises a thallium indicator and a concentration of chloride ions that is greater than about 2 mM to 150 mM; and   a stimulus buffer, wherein the stimulus buffer comprises thallium at a concentration of about 0.1 to about 5.0 mM, and wherein providing the stimulus buffer causes thallium efflux or influx into the cell through the ion channel.   
     
     
         73 . (canceled) 
     
     
         74 . A solution for detecting the activity of an ion channel in a cell comprising:
 a thallium indicator;   a concentration of chloride ions that is greater than about 2 mM to 150 mM; and   thallium at a concentration of about 0.1 to about 5.0 mM.   
     
     
         75 - 76 . (canceled) 
     
     
         77 . The solution of  claim 74  wherein the thallium concentration in the solution is about 0.1 mM to about 2 mM. 
     
     
         78 - 79 . (canceled) 
     
     
         80 . A method for detecting the activity of an ion channel in a cell, the method comprising:
 providing a cell with an ion channel;   contacting the cell with a loading buffer solution comprising (a) a thallium indicator selected from iodide, bromide, chromate, ANTS, Fluo-4, Fluo-3, PBFI, Phen Green, APTRA-BTC or Mag-Fura Red, Magnesium Green, Fluo-4FF, FluoZin-1 or FluoZin-2, RhodZin-3, or a derivative or salt thereof, (b) a concentration of chloride ions that is greater than about 2 mM to 150 mM, and (c) a quencher selected from tartrazine, amaranth, acid red 37, congo red, trypan blue, brilliant black, or a combination thereof;   washing the cells;   contacting the cell with a stimulus buffer comprising a thallium salt at a concentration of about 0.1 to about 5.0 mM to cause thallium efflux or influx into the cell through the ion channel, wherein the thallium salt is selected from Tl 2 SO 4 , Tl 2 CO 3 , TlCl, TlOH, TlOAc, and TlNO 3 ; and   detecting the thallium indicator by light microscopy, confocal microscopy, fluorescence microscopy or spectrophotometry.