Methods to identify modulators of RyR calcium channels
Provided herein are methods for identifying a compound that modulates a Ryanodine receptor (Ryr). Fluorescence resonance energy transfer between an FKBP bound to an RyR and fluorescent derivatives of RyR binding partners (e.g., calmodulin) or domain-peptide biosensors is used to provide a readout dependent on the RyR functional state. The methods permit measurement of RyR present in a permeabilized cell or in a purified membrane.
1. A method for identifying a compound that modulates a ryanodine receptor (RyR) homotetrameric calcium channel comprising:
providing a complex comprising an RyR homotetrameric calcium channel (RyR molecule), an FK-binding protein (FKBP), and a modulatory protein, wherein the FKBP comprises a covalently attached donor probe, and wherein the modulatory protein comprises a covalently attached acceptor probe, and wherein the donor probe and the acceptor probe can be used for energy transfer;
contacting the complex with a test compound to form a mixture;
measuring the fluorescence lifetime or intensity of the donor probe, wherein measuring fluorescence lifetime comprises direct waveform recording detection.
2. The method of claim 1 wherein the molar concentration of the FKBP is at, or within one order of magnitude, of the dissociation constant (K d ) of the FKBP for the RyR molecule, and the molar concentration of the modulatory protein is at, or within one order of magnitude, of the K d of the modulatory protein for the RyR molecule.
3. The method of claim 1 wherein the modulatory protein is calmodulin, S100A1, or sorcin.
4. The method of claim 1 wherein the fluorescence lifetime or intensity of the donor probe is changed in the presence of the test compound.
5. The method of claim 4 wherein the change between the fluorescence lifetime in the presence of the test compound and the fluorescence lifetime in the absence of the test compound is a ΔE of greater than 3 times the ΔE standard deviation of a control.
6. The method of claim 1 wherein the measuring comprises high throughput screening.
7. The method of claim 1 wherein the RyR molecule is an RyR1molecule or an RyR2 molecule.
8. The method of claim 1 wherein the complex is present in a permeabilized cell.
9. The method of claim 1 wherein the complex is present in a purified membrane.
10. The method of claim 1 wherein the mixture comprises a compound selected from H 2 O 2 and oxidized glutathione.
11. The method of claim 1 wherein the complex further comprises a domain peptide.
12. The method of claim 11 wherein the domain peptide is DPc-10.
13. The method of claim 11 wherein the concentration of the domain peptide is at, or within one order of magnitude, of the K d of the domain peptide for the RyR molecule.
14. The method of claim 11 wherein the concentration of the FKBP is a saturating concentration, the concentration of the domain peptide is a saturating concentration, or the combination thereof.
15. The method of claim 1 wherein the FKBP is FKBP12 or FKBP12.6.