Optogenetic probes for measuring membrane potential

• US 10,352,945 B2
• Filed: 10/13/2017
• Issued: 07/16/2019
• 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 Ezra, Kralj, Joel, Douglass, Adam D.
• Primary Examiner(s): Navarro, Albert M
• Application Number:
  US15/783,886
• Publication Number:
  US 20180031572A1
• Time in Patent Office: 641 Days
• 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 system for measuring membrane potential in a cell comprising:

a cell expressing a nucleic acid encoding a microbial rhodopsin protein fused to an additional fluorescent protein, wherein the cell is excited with light of at least one wavelength; and

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

2. The system of claim 1 further comprising a computer for analyzing the level of fluorescence emitted by the cell compared to a reference.

3. The system of claim 1, wherein the microbial rhodopsin protein is a modified microbial rhodopsin protein.

4. The system of claim 1, wherein the fluorescence emission of the additional fluorescent protein overlaps with the absorption of the microbial rhodopsin protein.

5. The system of claim 1, wherein the carboxylic amino acid on the third transmembrane helix of the microbial rhodopsin protein comprises a proton acceptor proximal to a Schiff base.

6. The system 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.

7. The system of claim 1, wherein the modified microbial rhodopsin protein is:

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

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

8. The system of claim 1, wherein the microbial rhodopsin protein is Proteorhodopsin Optical Proton Sensor, or a genetically encoded voltage indicator.

9. The system of claim 1, wherein the at least one wavelength is a wavelength between δ

=594-645 nm.

10. The system of claim 1, wherein the cell is selected from prokaryotic cells.

11. The system of claim 1, wherein the cell is selected from eukaryotic cells.

12. The system of claim 1, wherein the cell is selected from mammalian cells.

13. The system of claim 1, wherein the cell is selected from the group consisting of stem cells, pluripotent cells, progenitor cells, and induced pluripotent cells.

14. The system of claim 1, wherein the cell is selected from the group consisting of neurons and cardiomyocytes.

15. The system of claim 1, wherein the additional fluorescent protein is a protein that is capable of indicating ion concentration in the cell.

16. The system of claim 15, wherein the additional fluorescent protein that is capable of indicating ion concentration is a calcium indicator.

17. The system of claim 15, wherein the additional fluorescent protein that is capable of indicating ion concentration is a pH indicator.

18. The system of claim 4, wherein the additional fluorescent protein is capable of undergoing nonradiative fluorescence resonance energy transfer to the microbial rhodopsin protein, with a rate of energy transfer dependent on membrane potential.

19. The system of claim 1, wherein the additional fluorescent protein is selected from the group consisting of a green fluorescent protein, mOrange2, and eGFP.

20. The system of claim 1, wherein the microbial rhodopsin protein is a modified microbial rhodopsin protein selected from the group consisting of Ar1-D97N, Ar2-D96N, Ar3-D95N, and Ar4-D98N.

21. The system of claim 1, wherein the additional fluorescent protein is Venus, EYFP, or TagRFP.