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 that is permeable to thallium;   b) providing a loading buffer solution to the cell, the loading buffer solution comprising a 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 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 .- 28 . (canceled) 
     
     
         29 . The method of  claim 1  wherein the chloride ion concentration of the loading buffer solution is greater than about 2 mM. 
     
     
         30 .- 33 . (canceled) 
     
     
         34 . The method of  claim 1  wherein the thallium is in the form or 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 . The method  claim 1  wherein the stimulus buffer comprises thallium at a concentration of less than about 4.0 mM. 
     
     
         39 .- 41 . (canceled) 
     
     
         42 . The method of  claim 1 , further comprising quantifying the level of thallium. 
     
     
         43 . The method of  claim 43 , wherein quantifying the level of thallium comprises measuring at least one optical property of the thallium indicator in response to thallium influx. 
     
     
         44 . The method of  claim 43  wherein the at least one optical property of the thallium indicator is intensity, polarity, frequency, or optical density. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 1 , further comprising washing the cells after the loading buffer solution is provided to the cells. 
     
     
         47 . The method of  claim 1  wherein the method does not comprise washing the cells after the loading buffer is provided to the cells. 
     
     
         48 . The method of  claim 47 , further comprising adding a quencher to the loading buffer solution. 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 1 , further comprising stimulating the ion channel with a stimulus. 
     
     
         52 . The method of  claim 51  wherein the stimulus is a ligand that binds to the ion channel, a channel-linked receptor, or an electrical stimulus. 
     
     
         53 .- 61 . (canceled) 
     
     
         62 . The method of  claim 1 , wherein the ion channel is selected from ion channels that are linked to receptors, channel-linked receptors, ion transporters, ligand- or voltage-gated ion channels, stretch-activated cation channels, and selective or non-selective cation channels. 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . (canceled) 
     
     
         66 . The method of  claim 1 , wherein the cell comprises a sodium channel. 
     
     
         66 .- 71 . (canceled) 
     
     
         72 . A kit for detecting the activity of an ion channel that is permeable to thallium in a cell, the kit comprising:
 a) a loading buffer solution, wherein the loading buffer solution comprises a 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 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.   
     
     
         73 .- 79 . (canceled) 
     
     
         80 . The kit of  claim 72 , wherein the ion channel is selected from ion channels that are linked to receptors, channel-linked receptors, ion transporters, ligand- or voltage-gated ion channels, stretch-activated cation channels, and selective or non-selective cation channels.