Optogenetic probes for measuring membrane potential

• US 9,057,734 B2
• Filed: 08/23/2011
• Issued: 06/16/2015
• Est. Priority Date: 08/23/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Cohen, Adam E., Kralj, Joel M., Douglass, Adam D.
• Primary Examiner(s): NAVARRO, ALBERT MARK
• Application Number:
  US13/818,432
• Publication Number:
  US 20130224756A1
• Time in Patent Office: 1,393 Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2810/00 Vectors comprising a targeting moiety
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

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.

Claims

1. A method for measuring membrane potential in a cell expressing a nucleic acid encoding a microbial rhodopsin protein, the method comprising the steps of:

a. exciting, in vitro, at least one cell comprising a nucleic acid encoding a microbial rhodopsin protein with light of at least one wave length; and

b. detecting, in vitro, at least one optical signal from the at least one cell, wherein the level of fluorescence emitted by the at least one cell compared to a reference is indicative of the membrane potential of the cell.

2. The method of claim 1, wherein the microbial rhodopsin protein comprises:

a mutation to a carboxylic amino acid on a third transmembrane helix of the microbial rhodopsin protein; and

reduced ion pumping activity compared to a natural microbial rhodopsin protein from which it is derived.

3. The method of claim 2, wherein the carboxylic amino acid on the third transmembrane helix of the natural microbial rhodopsin comprises a proton acceptor proximal to a Schiff base.

4. The method of claim 1, wherein the microbial rhodopsin protein is a member of the proteorhodopsin family of proteins or a member of the archaerhodopsin family of proteins.

5. The method of claim 1, wherein the at least one wavelength is a wavelength between λ

=594-645 nm.

6. The method of claim 1, wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a mammalian cell, a stem cell or a pluripotent or a progenitor cell, an induced pluripotent cell, a neuron, and a cardiomyocyte.

7. The method of claim 1 further comprising a step of transfecting, in vitro, the at least one cell with a vector comprising the nucleic acid encoding the microbial rhodop sin protein.

8. The method of claim 1, wherein the nucleic acid encoding the microbial rhodopsin protein is operably linked to a cell-type specific promoter.

9. The method of claim 1, wherein the nucleic acid encoding the microbial rhodopsin protein is operably linked to a membrane-targeting nucleic acid sequence.

10. The method of claim 9, wherein the membrane-targeting nucleic acid is a plasma membrane targeting nucleic acid sequence or a subcellular compartment-targeting nucleic acid sequence.

11. The method of claim 1, wherein the nucleic acid encoding a microbial rhodopsin protein is operably linked to a nucleic acid encoding at least one additional fluorescent protein or a chromophore.

12. The method of claim 11, wherein the at least one additional fluorescent protein is selected from the group consisting of:

a green fluorescent protein, mOrange2, and eGFP.

13. The method of claim 11 further comprising steps of exciting, in vitro, the at least one cell with light of at least a first and a second wavelength;

and detecting, in vitro, the at least first and the second optical signal resulting from the excitation with the at least the first and the second wavelength from the at least one cell.

14. The method of claim 13, wherein the at least second wave length is between λ

=447-594 nm.

15. The method of claim 1, further comprising a step of calculating the ratio of the fluorescence emission from the microbial rhodopsin to the fluorescence emission of the at least one additional fluorescent protein to obtain a measurement of membrane potential independent of variations in expression level.

16. The method of claim 1 further comprising the step of exposing, in vitro, the at least one cell to a stimulus capable of, or suspected to be capable of changing membrane potential.

17. The method of claim 1 further comprising the step of measuring, in vitro, the at least one optical signal at a first and at least at a second time point.

18. The method of claim 17, wherein the first time point is before exposing the at least one cell to a stimulus and the at least second time point is after exposing the at least one cell to the stimulus.

19. A method for measuring membrane potential in a cell expressing a nucleic acid encoding a microbial rhodopsin protein, the method comprising the steps of:

a. exciting at least one cell comprising a nucleic acid encoding a microbial rhodopsin protein with light of at least one wave length; and

b. detecting at least one optical signal from the at least one cell, wherein the level of fluorescence emitted by the at least one cell compared to a reference is indicative of the membrane potential of the cell.

20. The method of claim 19, wherein the microbial rhodop sin protein comprises:

a mutation to a carboxylic amino acid on a third transmembrane helix of the microbial rhodopsin protein; and

reduced ion pumping activity compared to a natural microbial rhodopsin protein from which it is derived.

21. The method of claim 20, wherein the carboxylic amino acid on the third transmembrane helix of the natural microbial rhodopsin comprises a proton acceptor proximal to a Schiff Base.

22. The method of claim 19, wherein the microbial rhodopsin protein is a member of the proteorhodop sin family of proteins or a member of the archaerhodop sin family of proteins.

23. The method of claim 19, wherein the at least one wave length is a wave length between λ

=594-645 nm.

24. The method of claim 19, wherein the cell is a eukaryotic cell.

25. The method of claim 24, wherein the eukaryotic cell is a mammalian cell.

26. The method of claim 24, wherein the eukaryotic cell is selected from a stem cell, a pluripotent cell, a progenitor cell, an induced pluripotent cell, a neuronal cell, and a cardiomyocyte.

27. The method of claim 19, wherein the nucleic acid encoding a microbial rhodopsin protein is operably linked to a nucleic acid encoding at least one additional fluorescent protein or a chromophore.

28. The method of claim 27, wherein the at least one additional fluorescent protein is a fluorescent protein capable for indicating the ion concentration in the cell.

29. The method of claim 27, wherein the at least one additional fluorescent protein is selected from the group consisting of:

a green fluorescent protein, mOrange2, and eGFP.

30. The method of claim 27 further comprising steps of exciting the at least one cell with light of at least a first and a second wavelength;

and detecting the at least first and the second optical signal resulting from the excitation with the at least the first and the second wavelength from the at least one cell.

31. The method of claim 30, wherein the at least second wave length is between λ

=447-594 nm.

32. The method of claim 27, further comprising a step of calculating the ratio of the fluorescence emission from the microbial rhodopsin to the fluorescence emission of the at least one additional fluorescent protein to obtain a measurement of membrane potential independent of variations in expression level.

33. The method of claim 19 further comprising the step of exposing the at least one cell to a stimulus capable of, or suspected to be capable of changing membrane potential.

34. The method of claim 19, further comprising the step of measuring, in vitro, the at least one optical signal at a first and at least at a second time point.

35. The method of claim 34, wherein the first time point is before exposing the at least one cell to a stimulus and the at least second time point is after exposing the at least one cell to the stimulus.