# US 9,518,103 B2 · Optogenetic probes for measuring membrane potential > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,518,103 B2 - Document ID: US9518103B2 - Status: Granted - Kind: B2 - Application number: 14/742,648 - Filed: 2015-06-17 - Published: 2015-12-24 - Granted: 2016-12-13 - Earliest claimed: 2014-06-18 - Assignees: President and Fellows of Harvard College; The Governors of the University of Alberta - Applicants: President and Fellows of Harvard College; The Governors of the University of Alberta - Inventors: Adam E. Cohen; Daniel Hochbaum; Peng Zou; Samouil Leon Farhi; Robert Earl Campbell; Yongxin Zhao; Daniel Jed Harrison - Web page: https://patentgrep.com/patent/US9518103B2 ## CPC - C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} - C07K 14/215: from Halobacteriaceae (F) - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - 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} - G01N 2021/6441: {with two or more labels} - G01N 2333/726: G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Provided herein are variants of an archaerhodopsin useful for application such as optical measurement of membrane potential. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, cells comprising the polynucleotides, and cells comprising the polypeptides; and methods of using the variants. ## Claims ### Claim 1 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises at least one substitution mutation selected from the group consisting of P60S, T80S, D95H, D95Q, D106H, and F161V, wherein the position of the at least one substitution mutation in the amino acid sequence corresponds to SEQ ID NO:1. ### Claim 2 (depends on 1) The polypeptide of claim 1, wherein the amino acid sequence comprises at least two mutations selected from the group consisting of P60S, T80S, D95N, D95H, D95Q, D106H, and F161V. ### Claim 3 (depends on 1) The polypeptide of claim 1 comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. ### Claim 4 (depends on 1) A FRET pair comprising the polypeptide of claim 1 serving as a fluorescence acceptor and a fluorescent protein serving as a fluorescence donor. ### Claim 5 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises a membrane-targeting sequence. ### Claim 6 (depends on 5) The polypeptide of claim 5, wherein the membrane-targeting sequence is a subcellular compartment-targeting nucleic acid sequence. ### Claim 7 (depends on 1) The polypeptide of claim 1, wherein the polypeptide is linked to at least one additional protein or a chromophore. ### Claim 8 (depends on 1) The polypeptide of claim 1, wherein the variant polypeptide is fluorescent and has reduced ion pumping activity compared to a natural member of the archaerhodopsin family of proteins from which the polypeptide is derived. ### Claim 9 (depends on 1) A method for measuring a change in membrane potential in a cell expressing a variant microbial rhodopsin polypeptide, the method comprising the steps of: irradiating, in vitro, at least one cell expressing the polypeptide of claim 1 with light having a wave length suitable to produce fluorescence signal from the variant polypeptide; detecting, in vitro, a fluorescent 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; exposing, in vitro, the at least one cell to a stimulus capable of changing the membrane potential of the at least one cell; irradiating, in vitro, the at least one cell expressing the polypeptide of claim 1 with light having a wave length suitable to produce fluorescence signal from the variant polypeptide; detecting, in vitro, a second fluorescent 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; and comparing the first fluorescent signal with the second fluorescent signal to determine the change in membrane potential of the cell. ### Claim 10 (depends on 9) The method of claim 9, wherein the cell is irradiated with yellow light, red light, or combinations thereof. ### Claim 11 A polynucleotide comprising a nucleic acid sequence encoding a variant polypeptide having at least 80% identity to SEQ ID NO: 1, wherein the variant polypeptide comprises at least one substitution mutation selected from the group consisting of P60S, T80S, D95H, D95Q, D106H, and F161V, wherein the position of the at least one substitution mutation in the amino acid sequence corresponds to SEQ ID NO: 1. ### Claim 12 A polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. ### Claim 13 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a first substitution mutation at position 95, and at least one second substitution mutation selected from the group consisting of P60S, T80S, D106H, and F161V, wherein the positions of the substitution mutations in the amino acid sequence corresponds to SEQ ID NO:1. ### Claim 14 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a substitution mutation at position 95, a D106H mutation, and at least one substitution mutation selected from the group consisting of P60S, T80S, and F161V, wherein the positions of the substitution mutations in the amino acid sequence corresponds to SEQ ID NO:1. ### Claim 15 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a substitution mutation at position 95 of D to Q, C, or N, and at least one substitution mutation selected from the group consisting of P60S, T80S, and F161V; wherein the positions of the substitution mutations in the amino acid sequence corresponds to SEQ ID NO:1. ### Claim 16 A polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. ### Claim 17 A fusion protein comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and an amino acid sequence of a second fluorescent protein. ### Claim 18 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a D to H or D to Q substitution mutation at position 95, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO: 1. ### Claim 19 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a P to S substitution mutation at position 60, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO: 1. ### Claim 20 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a T to S substitution mutation at position 80, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO: 1. ### Claim 21 A polypeptide comprising a variant having at least 80% identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises a D to H substitution mutation at position 106, wherein the position of the substitution mutation in the amino acid sequence corresponds to SEQ ID NO: 1.