Voltage indicators

Patent metadata

Field	Value
Publication number	US-11598792-B2
Application number	US-202016872188-A
Country	US
Kind code	B2
Filing date	May 11, 2020
Priority date	May 9, 2019
Publication date	Mar 7, 2023
Grant date	Mar 7, 2023

Abstract

A voltage indicator includes a polypeptide sequence comprising a voltage-sensitive opsin domain and a capture protein domain arranged and disposed to capture a fluorescent dye ligand. When the fluorescent dye ligand is captured and the voltage indicator is bound to a cell membrane, an increase in voltage across the cell membrane causes an increase in fluorescent emission.

First claim

What is claimed is:

1. A voltage indicator, comprising: a voltage-sensitive microbial rhodopsin domain comprising the amino acid sequence of SEQ ID NO: 9 with an amino acid mutation at one or more of residue 81, 92, and 199; and a capture protein that covalently or noncovalently binds a fluorescent dye ligand that is (i) a fluorescent protein; or (ii) a fluorescent dye, wherein the capture protein is provided together with the voltage-sensitive microbial rhodopsin domain in a fusion protein.

2. A voltage indicator, comprising an amino acid sequence selected from the group of amino acid sequences of SEQ ID NOS: 2, 4, 6, and 8.

3. The voltage indicator of claim 1 , wherein the capture protein is selected from the group consisting of biotin-avidin, a self-labeling protein tag, or a combination thereof.

4. The voltage indicator of claim 1 , wherein the capture protein domain is a self-labeling protein tag.

5. The voltage indicator of claim 1 , wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are removed from the junction between the rhodopsin domain and the capture protein.

6. The voltage indicator of claim 1 , and further comprising a targeting sequence.

7. The voltage indicator of claim 6 , wherein the targeting sequence is a soma targeting sequence.

8. The voltage indicator of claim 6 , wherein the capture protein is positioned at the c-terminal end of the voltage-sensitive microbial rhodopsin domain.

9. The voltage indicator of claim 1 , wherein the fluorescent dye ligand is an azetidine-containing fluorescent dye.

10. The voltage indicator of claim 1 , wherein the fluorescent dye ligand is a fluorescent protein.

11. A method of measuring voltage, the method comprising contacting the voltage indicator of claim 1 and a fluorescent dye ligand with a cell, and determining changes in fluorescence of the fluorescent dye ligand when the fluorescent dye ligand is captured by the voltage indicator.

12. The method of claim 11 , wherein the cell is a neuron.

13. The method of claim 11 , and further comprising observing changes in fluorescence with a microscope.

14. The method of claim 11 , wherein the voltage indicator further comprises a linker between the voltage-sensitive domain and the capture protein.

15. The method of claim 11 , further comprising modifying a length of the linker.

16. The method of claim 11 , wherein an increase in membrane potential lead to an increase in fluorescence.

17. A voltage indicator, comprising the amino acid sequence of SEQ ID NO: 20.

18. A method of measuring voltage, the method comprising contacting the voltage indicator of claim 17 and a fluorescent dye ligand with a cell, and determining changes in fluorescence when the fluorescent dye ligand is captured by the voltage indicator.

Assignees

• Hughes Howard Med Inst

Inventors

• Schreiter Eric R
• Abdelfattah Ahmed

Classifications

• C07K14/195

  from bacteria · CPC title

• G01R15/22 Primary

  using light-emitting devices, e.g. LED, optocouplers {(G01R31/31901 takes precedence)} · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• C07K2319/33

  fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies · CPC title