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 .- 71 . (canceled) 
     
     
         72 . A kit for detecting thallium influx through a potassium ion channel in a cell, comprising:
 a) a loading buffer solution, wherein the loading buffer solution comprises 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, wherein the loading buffer loads the thallium indicator into the cell; and   b) a stimulus buffer, wherein the stimulus buffer comprises thallium, wherein the thallium concentration in the stimulus buffer is about 0.1 mM to about 5.0 mM, and wherein the stimulus buffer causes thallium influx into the cell through the potassium ion channel.   
     
     
         73 - 79 . (canceled) 
     
     
         80 . The kit of  claim 72 , wherein the thallium in the stimulus buffer is a thallium ion. 
     
     
         81 . The kit of  claim 72 , wherein the thallium in the stimulus buffer is in the form of a salt. 
     
     
         82 . The kit of  claim 72 , wherein the physiological concentration of chloride ions in the loading buffer is about 10 mM to about 150 mM. 
     
     
         83 . The kit of  claim 72 , wherein the stimulus buffer further comprises a stimulus that activates the potassium ion channel. 
     
     
         84 . The kit of  claim 72 , further comprising a potassium ion or a ligand that activates the potassium ion channel. 
     
     
         85 . A kit for loading a cell with a thallium ion and a thallium indicator, comprising:
 a loading buffer for loading the cell with the thallium indicator, 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   a thallium loading solution for loading the cell with the thallium ion, wherein the thallium loading solution comprises thallium ions at a concentration of about 0.1 mM to about 5.0 mM, wherein the loading buffer and/or thallium loading solution comprises a physiological concentration of chloride ions.   
     
     
         86 . A method of loading a cell with thallium ions and a thallium indicator, comprising:
 contacting a cell with a loading buffer, wherein the loading buffer comprises 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, to provide a thallium indicator loaded cell;   contacting the thallium indicator loaded cell with a thallium loading solution, wherein the thallium loading solution comprises thallium ions, thereby loading the thallium indicator loaded cell with the thallium ions.   
     
     
         87 . The method of  claim 86 , wherein the concentration of the thallium ions in the thallium loading solution is about 0.1 mM to about 5.0 mM. 
     
     
         88 . The method of  claim 86 , wherein the cell further comprises a potassium ion channel, wherein the thallium ion enters the cell through the potassium ion channel. 
     
     
         89 . The method of  claim 86 , wherein the thallium indicator loaded cell further comprises a cytosol, wherein the thallium indicator is trapped within the cytosol of the thallium indicator loaded cell. 
     
     
         90 . The method of  claim 86 , wherein the thallium indicator is associated with a thallium ion within the thallium indicator loaded cell. 
     
     
         91 . The method of  claim 90 , wherein the thallium indicator exhibits fluorescence when associated with the thallium ion within the thallium indicator loaded cell. 
     
     
         92 . The method of  claim 91 , wherein the thallium indicator exhibits an increase in fluorescence intensity when associated with the thallium ion in the cell. 
     
     
         93 . The method of  claim 90 , further comprising detecting the intensity of the fluorescence exhibited by the thallium indicator, wherein the fluorescence intensity of the thallium indicator is a measure of the concentration of the thallium ions in the cell. 
     
     
         94 . The method of  claim 93 , wherein the intensity of the fluorescence exhibited by the thallium indicator is a measure of the activity of a potassium ion channel. 
     
     
         95 . The method of  claim 90 , further comprising detecting the thallium ions in the thallium indicator loaded cell, wherein the detection of the thallium ions in the cell is a measure of the activity of a potassium ion channel. 
     
     
         96 . The method of  claim 90 , further comprising quantifying the level of the thallium ion in the thallium indicator loaded cell.