Optogenetic probes for measuring membrane potential
The invention provides methods, cells and constructs for optical measurement of membrane potential. These methods can be used in cells that are not accessible to presently available methods using electrodes. The methods can be directed to, for example, high-throughput drug screening assays to determine agents that can affect membrane potential of a target cell.
1. An isolated and purified nucleic acid encoding Archaerhodopsin 3 with a mutation of D95 to reduce ion pumping activity compared to Arch 3 WT, the isolated and purified nucleic acid being operably linked to a nucleic acid encoding at least one additional fluorescent protein capable of indicating ion concentration in a cell.
2. The isolated and purified nucleic acid of claim 1 , wherein the mutation comprises D95N.
3. The isolated and purified nucleic acid of claim 1 operably linked to a nucleic acid sequence encoding a membrane-targeting nucleic acid sequence.
4. The isolated and purified nucleic acid of claim 3 , wherein the membrane-targeting nucleic acid sequence is a plasma membrane targeting nucleic acid sequence.
5. The isolated and purified nucleic acid of claim 3 , wherein the membrane-targeting nucleic acid sequence is a subcellular membrane-targeting nucleic acid sequence.
6. The isolated and purified nucleic acid of claim 5 , wherein the subcellular membrane is a mitochondrial membrane, an endoplasmic reticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome or a phagosome.
7. The isolated and purified nucleic acid of claim 1 operably linked to a cell-type specific promoter.
8. The isolated and purified nucleic acid of claim 1 , wherein the at least one additional fluorescent protein is a green fluorescent protein or a homolog thereof.
9. The isolated and purified nucleic acid of claim 1 , wherein the fluorescent protein capable for indicating ion concentration is a calcium indicator.
10. The isolated and purified nucleic acid of claim 1 , wherein the fluorescent protein capable for indicating ion concentration is a pH indicator.
11. The isolated and purified nucleic acid of claim 1 , further comprising a vector.
12. The isolated and purified nucleic acid of claim 11 , wherein the vector is a viral vector.
13. The isolated and purified nucleic acid of claim 12 , wherein the viral vector is a lentiviral vector.
14. The isolated and purified nucleic acid of claim 12 , wherein the viral vector is an adeno-associated viral vector.
15. An isolated cell comprising a nucleic acid encoding Archaerhodopsin 3 with a mutation at D95 to reduce ion pumping activity compared to Arch 3 WT and at least one additional fluorescent protein capable of indicating an ion concentration in the cell.
16. An isolated and purified nucleic acid encoding Archaerhodopsin 3 with a mutation of D95 to reduce ion pumping activity compared to Arch 3 WT, the isolated and purified nucleic acid being operably linked to a nucleic acid encoding at least one green fluorescent protein or a homolog thereof.
17. The isolated and purified nucleic acid of claim 16 , wherein the mutation comprises D95N.
18. The isolated and purified nucleic acid of claim 16 operably linked to a nucleic acid sequence encoding a membrane-targeting nucleic acid sequence.
19. The isolated and purified nucleic acid of claim 17 , wherein the membrane-targeting nucleic acid sequence is a plasma membrane targeting nucleic acid sequence.
20. The isolated and purified nucleic acid of claim 17 , wherein the membrane-targeting nucleic acid sequence is a subcellular membrane-targeting nucleic acid sequence, wherein the subcellular membrane is selected from the group consisting of a mitochondrial membrane, an endoplasmic reticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome, and a phagosome.
21. The isolated and purified nucleic acid of claim 16 operably linked to a cell-type specific promoter.
22. The isolated and purified nucleic acid of claim 16 , wherein the green fluorescent protein is capable of undergoing nonradiative fluorescence resonance energy transfer to the microbial rhodopsin, with a rate of energy transfer dependent on membrane potential.