Screening assays using intramitochondrial calcium

a technology of intramitochondrial calcium and screening assay, which is applied in the direction of instruments, biochemistry apparatus and processes, material analysis, etc., can solve the problems of compromising the integrity of the inner mitochondrial membrane, uncoupling respiration (i.e., etc. activity) from atp production

Inactive Publication Date: 2005-08-04
MIGENIX CORP
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  • Abstract
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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.

Method used

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  • Screening assays using intramitochondrial calcium
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  • Screening assays using intramitochondrial calcium

Examples

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example 1

General Assay Reagent, Other Components and Conditions

Calcium

[0151] Calcium chloride (CaCl2) is commercially available (Sigma, St. Louis, Mo.; C3881). In initial experiments, atomic absorption was done on the stock calcium chloride solution in order to precisely determine its concentration. Autoclaved stock solutions of 0.025 mol / L are available (Sigma, St. Louis, Mo.). The concentration of CaCl2 is important because a difference between 4 and 8 micromolar can be important with regard to inducing spontaneous Ca2+ release from mitochondria.

Calcium-Sensitive Detectable Reagents

[0152] Calcium-Green-5N (potassium salt) is commercially available (Molecular Probes, Eugene, Oreg.; C-3737). Calcium-Green-5N is a low affinity Ca2+ indicator (as is, for example, Oregon Green 488 BAPTA-5N). Low affinity indicators are preferred because of the Ca2+ concentrations used in the assays. High affinity dyes require a lower Ca2+ concentration and therefore a lower number of cells, and thus a low...

example 2

General Assay Protocols

[0177] A master mix solution (MM Solution) was prepared by the addition of 5 glutamate and malate to final concentrations of 5 millimolar each, MgCl2 to a final concentration of 1 millimolar, EGTA to a final concentrations of 0 to 8 micromolar, and Calcium-Green-5N to a final concentration of 0.1 to 1.0 micromolar, to a basic KCl-based respiratory media (“BReM”) that is described in Table 3. Alternative respiratory media can be prepared using sucrose and / or mannitol with or without phosphate buffer. The hexapotassiun salt Calcium-Green-5N (Molecular Probes, Eugene, Oreg.) in the MM Solution is a fluorescent dye that has low binding affinity to Ca2+.

[0178] Thapsigargin (Calbiochem, San Diego, Calif.), a Ca2+ uptake inhibitor of the endoplasmic reticulum (ER), was added to the MM Solution to a final concentration of 1 micromolar to yield a solution designated “MM-T” (ie., Master Mix with Thapsigargin).

[0179] The cell membrane permeabilizing agent digitonin (S...

example 3

Detection of Ca2+ Uniporter Activity

[0183] Assays were performed to optimize the Ca2+ concentration whereby the concentration allows for Ca2+ uniporter transport into mitochondria but is not high enough to induce permeability transition. Stock solutions (10×) of Ca2+ were prepared by addition of CaCl2 to MM-T at final concentrations of 0, 40, 80, 120, 160, and 200 micromolar Ca2+. The cells used in this experiment were a mixture of control cybrids (MixCon) from multiple normal individuals (see U.S. Pat. No. 5,888,498). Cells were trypsinized in Dulbecco's modified Eagle's medium with 10% heat inactivated fetal bovine serum (FBS) and added to wells of a 96-well Costar 3603 microplate at a concentration of about 6×104 cells in 100 microliters per well 48 hours prior to performing assays. Prior to use of the cells, the growth media was aspirated from the wells. One hundred microliters of MM-TD was added to each well to permeabilize the cells. The plate was placed into a Fluorometric I...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/50
CPCG01N33/5079
Inventor MURPHY, ANNE N.STOUT, AMY K.
Owner MIGENIX CORP
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