Screening assays using intramitochondrial calcium

Abstract

The invention provides methods for screening for agents that modulate mitochondrial function and in particular mitochondrial regulation of intracellular calcium. The methods may be used to detect agents that bind to a mitochondrial calcium uniporter and may also detect inhibitors or uncouplers of mitochondrial respiration. Agents identified using the screens provided herein have application in the prevention and treatment of a variety of diseases associated with abnormal mitochondrial function.

1 . A method of identifying an agent that uncouples oxidative phosphorylation from ATP production, comprising: 
 (a) contacting 
 (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with  
 (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell;  
   (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points;    (c) repeating steps (a) and (b) at least once; and    (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the absence of the candidate agent to (ii) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that uncouples oxidative phosphorylation from ATP production.    
     
     
         2 . A method of identifying an agent that is a respiratory inhibitor, comprising: 
 (a) contacting 
 (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with  
 (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell;  
   (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points;    (c) repeating steps (a) and (b) at least once; and    (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the absence of the candidate agent to (ii) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that is a respiratory inhibitor.    
     
     
         3 . A method of identifying an agent that alters a mitochondrial calcium uniporter, comprising: 
 (a) contacting 
 (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with  
 (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell;  
   (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points;    (c) repeating steps (a) and (b) at least once; and    (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the absence of the candidate agent to (ii) the signal generated by the calcium indicator molecule at one or more of said time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point following a contacting step in the presence of the agent, compared to the level of calcium in the cytosol following a contacting step in the absence of the agent, indicates that the agent alters a mitochondrial calcium uniporter.