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.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09 / 765,104, filed Jan. 16, 2001, issued Oct. 26, 2004, as U.S. Pat. No. 6,808,873; which claims the benefit of U.S. Provisional Application No. 60 / 176,384 filed Jan. 14, 2000, which are incorporated herein by reference in their entireties.TECHNICAL FIELD [0002] The invention relates generally to assays for screening for agents that affect mitochondrial activity. More specifically, the invention is directed to screening methods for use in identifying agents that alter mitochondrial regulation of intracellular calcium. An assay for the presence of extramitochondrial calcium and for factors that influence levels of intramitochondrial and / or extramitochondrial calcium, such as the calcium uniporter (CaUP), is provided herein. BACKGROUND OF THE INVENTION [0003] Mitochondria are organelles that are the main energy source in cells of higher organisms. These organelles prov...

AI Technical Summary

Benefits of technology

[0011] The present invention is directed in part to methods for identifying agents that alter mitochondrial regulation of intracellular calcium. Thus, in one aspect the invention provides a method of identifying an agent that alters mitochondrial function, comprising (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array, (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, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points; and (c) comparing the signal generated by the calcium indicator molecule at one or more of the time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of the time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function. In one embodiment the step of contacting is repeated at least once. In another embodiment the sample contains at least one compound that alters intracellular distribution of a calcium cation. In a further embodiment the compound that alters intracellular calcium cation distribution is thapsigargin or Ru360. In another embodiment the compound that alters intracellular calcium cation distribution is a calcium ionophore or a membrane permeable compound that alters intracellular calcium distribution. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the candidate agent is membrane permeable, and in another embodiment the calcium indicator molecule is membrane permeable. In another embodiment the source of calcium cations is exogenous to the cell. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the cell comprises at least one polypeptide that is a member of the Bcl-2 family. In another embodiment the cell expresses a gene encoding a polypeptide that regulates cytosolic calcium. In another embodiment the gene encodes a mitochondrial calcium uniporter. In another embodiment the gene is a transfected gene. In another embodiment the gene encodes a mitochondrial calcium uniporter. In another embodiment the cell is a permeabilized cell. In certain embodiments the cell adheres to a solid substrate and in certain other embodiments the cell is a non-adherent cell.

Problems solved by technology

These processes require the maintenance of a mitochondrial membrane electrochemical potential, and defects in such membrane potential can result in a variety of disorders.

When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during mitochondrial permeability transition (MPT) that accompanies certain diseases associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby uncoupling respiration (i.e., ETC activity) from ATP production.

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