Patent Application
20250115656
A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN-1931WO_SEQ_LIST” created on Jan. 20, 2023, and having a size of 39,000 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
ChRmine, which is a pump-like cation-conducting channelrhodopsin, exhibits puzzling properties, such as large photocurrents, red-shifted spectrum, and extreme light-sensitivity. ChRmine and its homologs function as ion channels, but by primary sequence more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family are unknown.
This disclosure provides the 2.0-Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an unprecedented opening at the trimer interface. Based on the structure of ChRmine, three types of proteins were designed that have desirable characteristics in optogenetics: for example, rsChRmine and hsChRmine, having further red-shifted and high-speed properties respectively; and frChRmine, having faster and more red-shifted performance. These proteins can be used in neuroscience research, particularly, using optogenetics.
Accordingly, certain embodiments of the disclosure provide a high-speed variant ChRmine protein having faster kinetic properties compared to a parent ChRmine protein, wherein the high-speed variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein. Certain embodiments of the disclosure also provide a red-shifted variant ChRmine protein having a red-shifted spectrum compared to a parent ChRmine protein, wherein the red-shifted variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein. Further embodiments of the disclosure provide a nucleic acid encoding for a variant ChRmine protein disclosed herein as well as a genetically modified cell comprising such nucleic acid. Even further embodiments of the disclosure provide an optogenetic method comprising: genetically modifying a subject to express in the subject's brain cells the variant ChRmine protein disclosed herein, applying stimulating light to the subject's brain, and imaging the subject's brain.
Additional embodiments of the disclosure provide methods comprising: genetically modifying a subject to express in a cell and/or organ the variant ChRmine protein disclosed herein. The methods can further comprise applying stimulating light to the modified cell and/or organ, and imaging the subject's cell and/or organ. The cell and/or organ can belong to the cardiovascular system, the gastrointestinal system, the urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic/endocrine system.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Reference to color in the brief description of drawings refers to color drawings, which may be provided based on the jurisdiction.
Provided are pump-like cation-conducting channelrhodopsins. Three types of proteins were designed that have desirable characteristics in optogenetics. Examples of these proteins include: rsChRmine and hsChRmine, having further red-shifted and high-speed properties respectively; and frChRmine, having faster/accelerated kinetics and greater red-shifted performance compared to rsChRmine. These proteins can be used in neuroscience research, particularly, using optogenetics.
Before the present invention is described in greater detail, it is to be understood that this invention is not limited to embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a protein” includes a plurality of such proteins and reference to “a mutation” includes reference to one or more discrete mutations, and so forth. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent the definition or usage of any term herein conflicts with a definition or usage of a term in an application or reference incorporated by reference herein, the instant application shall control.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
The terms “kinetics” or “kinetic property/properties” as used herein in reference to a ChRmine protein refer to the rates of opening and closing of channelrhodopsin ion channels of the ChRmine protein. A ChRmine protein having higher rates of opening and closing of channelrhodopsin ion channels compared to another ChRmine protein is said to have faster or accelerated kinetics or kinetic property/properties compared to the other ChRmine protein.
“A high-speed variant ChRmine protein” has accelerated kinetic property/properties, i.e., faster kinetic property/properties, compared to a parent ChRmine protein used to produce the high-speed variant ChRmine protein. A high-speed variant ChRmine protein can have faster kinetic properties compared to a parent ChRmine protein used to produce the high-speed variant ChRmine protein by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 80% or more.
ChRmine kinetics can be expressed as “rise time,” tau off (τoff), or a combination of both.
“Rise time” (tpeak) is the time-to-peak from the cessation of the light stimulus to the time point at which maximal-amplitude fluorescence was reached. For example, a wild-type ChRmine protein can have the time-to-peak of between 15-20 ms, whereas the corresponding high-speed variant ChRmine protein can have the time-to-peak of 5-10 ms. Thus, compared to a parent ChRmine protein used to produce a high-speed variant ChRmine protein, the high-speed variant ChRmine protein can have time-to-peak reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
Also, as an example, a wild-type ChRmine protein can have τoff between 50-150 ms, whereas the corresponding high-speed variant ChRmine protein can have τoff between 20-50 ms. Thus, compared to a parent ChRmine protein used to produce a high-speed variant ChRmine protein, the high-speed variant ChRmine protein can have τoff reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
“Red-shifted spectrum” as used herein in reference to a ChRmine protein refers to red-shifted absorption by a ChRmine protein, for example, a variant ChRmine protein, compared to another ChRmine protein, for example, a parent ChRmine protein. A ChRmine protein having red-shifted absorption compared to a parent ChRmine protein is referenced herein as “red-shifted variant.”
The wavelength eliciting maximum photocurrent is the same for both opsin proteins but the maximum photocurrent is different for all other wavelengths in a parent ChRmine protein as compared to the corresponding red-shifted variant ChRmine protein. For example, photocurrents can be lower for a red-shifted variant ChRmine protein to the corresponding parent ChRmine protein at 380, 440, 480 nm, this indicating photocurrent reduction at blue wavelengths, which represents red shifting, and photocurrents are higher at 650 nm, which also represents red shifting.
For example, a parent ChRmine protein can have the following maximum photocurrents: 380 nm: 0.49, 440 nm: 0.78, 480 nm: 0.94, 513 nm: 1, 580 nm: 0.82, 650 nm: 0.18. These absorption values are normalized to the maximum photocurrent, which is at 513 nm. A corresponding red-shifted variant ChRmine protein can have the following maximum photocurrents: 380 nm: 0.41, 440 nm: 0.50, 480 nm: 0.80, 513 nm: 1, 580 nm: 0.81, and 650 nm: 0.31. Again, these absorption values are normalized to the maximum photocurrent, which is at 513 nm.
Thus, at wavelengths lower than the wavelength that provides maximum photocurrent, compared to the maximum photocurrents of a parent ChRmine protein, a red-shifted variant ChRmine protein can have the maximum photocurrent reduced by 10% or more, 20% or more, 30% or more, 40% or more. On the other hand, at wavelengths higher than the wavelength that provides maximum photocurrent, compared to the maximum photocurrents of a parent ChRmine protein, a red-shifted variant ChRmine protein can have the maximum photocurrent increased by 10% or more, 20% or more, 30% or more, 40% or more.
A “parent ChRmine protein” as used herein refers to a wild-type or naturally occurring ChRmine protein. In some instances, a parent ChRmine protein can be mutated to produce a variant ChRmine protein. A parent protein can be a wild-type or naturally occurring ChRmine protein or a homolog thereof. Non-limiting examples of such parent ChRmine proteins are provided in
“A homologous protein or a protein homolog” of a protein is another protein having similar or identical function and a similar primary, secondary, and/or tertiary structures. Typically, homologous proteins or protein homologs have substantial sequence similarity, for example, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity. Certain non-limiting examples of ChRmine protein homologs are provided as SED ID NOs: 1 to 29. Sequence alignment of some of these proteins is provided in
The phrase “a corresponding residue in a homolog of ChRmine protein” refers to a residue in a homolog of ChRmine protein that aligns with a reference residue in a ChRmine protein, for example, as shown in
Provided are variants of ChRmine protein, which is a pump-like cation-conducting channelrhodopsin.
In certain embodiments, a variant ChRmine protein is a high-speed variant ChRmine protein having faster kinetic properties compared to a parent ChRmine protein, wherein the high-speed variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein.
In some cases, a high-speed variant ChRmine protein can have one or more amino acid substitutions in Schiff base counterion. Certain such amino acids are identified in
A high-speed variant ChRmine protein can also have one or more amino acid substitutions that alter the pore electrostatic potential of a parent protein. Certain such amino acid substitutions include substitutions in one or more of: 33rd histidine or a corresponding position; 92nd aspartate or a corresponding position; 154th glutamate or a corresponding position; 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position. Each of these positions can be substituted with any other amino acid. Substitutions at the 33rd histidine or a corresponding position can be with a histidine (when the corresponding amino acid is not histidine), arginine, or lysine. Substitution in the 92nd aspartate or a corresponding position; 154th glutamate or a corresponding position; 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position can be with aspartate, glutamate, asparagine, or glutamine.
For example, substitution at the 33rd histidine or a corresponding position can be with arginine. Substitution at the 33rd histidine or a corresponding position can also be with lysine. When the amino acid corresponding to the 33rd histidine is not histidine, such amino acid can be substituted with histidine.
Substitution in the 92nd aspartate or a corresponding position can be with aspartate. Substitution in the 92nd aspartate or a corresponding position can also be with glutamate. Substitution in the 92nd aspartate or a corresponding position can be with asparagine. Substitution in the 92nd aspartate or a corresponding position can also be with glutamine.
Substitution in the 154th glutamate or a corresponding position can be with aspartate. Substitution in the 154th glutamate or a corresponding position can also be with glutamate. Substitution in the 154th glutamate or a corresponding position can be with asparagine. Substitution in the 154th glutamate or a corresponding position can also be with glutamine.
Substitution in the 158th glutamate or a corresponding position can be with aspartate. Substitution in the 158th glutamate or a corresponding position can also be with glutamate. Substitution in the 158th glutamate or a corresponding position can be with asparagine. Substitution in the 158th glutamate or a corresponding position can also be with glutamine.
Substitution in the 242nd aspartate or a corresponding position can be with aspartate. Substitution in the 242nd aspartate or a corresponding position can also be with glutamate. Substitution in the 242nd aspartate or a corresponding position can be with asparagine. Substitution in the 242nd aspartate or a corresponding position can also be with glutamine.
Substitution in the 246th glutamate or a corresponding position can be with aspartate. Substitution in the 246th glutamate or a corresponding position can also be with glutamate. Substitution in the 246th glutamate or a corresponding position can be with asparagine. Substitution in the 246th glutamate or a corresponding position can also be with glutamine.
A high-speed variant ChRmine protein can be produced from a parent ChRmine protein selected from the proteins provided in
A parent ChRmine protein can have a sequence selected from SEQ ID NOs: 1 to 29 or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29. A sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29 can have conservative amino acid substitutions as compared a sequence from which it is derived. For example,
In one embodiment, compared to the parent ChRmine protein, a high-speed variant ChRmine protein has a substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein. The histidine amino acid can be substituted with any other amino acid, for example, histidine (when the corresponding amino acid is not histidine), arginine or lysine, i.e., a basic amino acid.
For example, substitution at the 33rd histidine or a corresponding position can be with arginine. Substitution at the 33rd histidine or a corresponding position can also be with lysine. When the amino acid corresponding to the 33rd histidine is not histidine, such amino acid can be substituted with histidine.
In certain embodiments, a high-speed variant ChRmine protein has an arginine substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein. Accordingly, a high-speed variant ChRmine protein can have a sequence of SEQ ID NO: 30 or a sequence having at least 80% sequence identity, least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 30, wherein the variations in the sequence having at least 80% sequence identity, least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 30 exclude the amino acid substitution used to produce the high-speed variant ChRmine protein. In a specific embodiment, a high-speed variant ChRmine protein has the sequence of SEQ ID NO: 30.
Additional embodiments of the disclosure provide a red-shifted variant ChRmine protein having a red-shifted spectrum compared to a parent ChRmine protein, wherein the red-shifted variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein.
A red-shifted variant ChRmine protein can have one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. Certain such amino acid substitutions include substitutions in one or more of: 146th isoleucine or a corresponding position; 174th glycine or a corresponding position; 178th phenylalanine or a corresponding position. Each of these positions can be substituted with any other amino acid. Substitutions at the 146th isoleucine or a corresponding position can be with a serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur/selenium-containing amino acid. Substitutions at the 174th glycine or a corresponding position can be with a serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur/selenium-containing amino acid. Substitutions at the 178th phenylalanine or a corresponding position can be with phenylalanine (when the corresponding amino acid is not phenylalanine), tyrosine, or Tryptophan, i.e., an aromatic amino acid.
For example, substitution at the 146th isoleucine or a corresponding position can be with serine. Substitution at the 146th isoleucine or a corresponding position can also be with cysteine. Substitution at the 146th isoleucine or a corresponding position can be with threonine. Substitution at the 146th isoleucine or a corresponding position can also be with methionine.
Substitution at the 174th glycine or a corresponding position can be with serine. Substitution at the 174th glycine or a corresponding position can also be with cysteine. Substitution at the 174th glycine or a corresponding position can be with threonine. Substitution at the 174th glycine or a corresponding position can also be with methionine.
Substitution at the 178th phenylalanine or a corresponding position can be with tyrosine. Substitution at the 178th phenylalanine or a corresponding position can also be with tryptophan. When the corresponding amino acid at the 178th phenylalanine is not phenylalanine, it can be substituted with phenylalanine.
A red-shifted variant ChRmine protein can be produced from a parent ChRmine protein selected from the proteins provided in
A parent ChRmine protein can have a sequence selected from SEQ ID NOs: 1 to 29 or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29. A sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29 can have conservative amino acid substitutions as compared a sequence from which it is derived.
In certain embodiments, a red-shifted variant ChRmine protein has one or both of: i) a substitution at the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and ii) a substitution at the glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. The isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein can be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., hydroxyl or sulfur/selenium-containing amino acid. The glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein can be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., hydroxyl or sulfur/selenium-containing amino acid.
For example, the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein can be substituted with serine. The isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein can also be substituted with cysteine. The isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein can be substituted with threonine. The isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein can also be substituted with methionine.
The glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein can be substituted with serine. The glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein can also be substituted with cysteine. The glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein can be substituted with threonine. The glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein can also be substituted with methionine.
Accordingly, a red-shifted variant ChRmine protein can have a sequence of SEQ ID NO: 31 or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 31, wherein the variations in the sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 31 exclude the amino acid substitution used to produce the red-shifted variant ChRmine protein. In a specific embodiment, a red-shifted variant ChRmine protein has a sequence of SEQ ID NO: 31.
Further embodiments of the disclosure provide a high-speed and red-shifted variant ChRmine protein having faster kinetics and red-shifted spectrum compared to a parent ChRmine protein, wherein the high-speed and red-shifted variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein.
A high-speed and red-shifted variant ChRmine protein can comprise: i) one or more amino acid substitutions in Schiff base counterion of the parent ChRmine protein or one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. In certain cases, a high-speed and red-shifted variant ChRmine protein comprises: i) one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein and ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.
The one or more amino acid substitutions that alter the pore electrostatic potential can be at: 33rd histidine or a corresponding position; 92nd aspartate or a corresponding position; 154th glutamate or a corresponding position; 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, or 246th glutamate or a corresponding position. The 33rd histidine or a corresponding position can be substituted with histidine, when the corresponding amino acid is not histidine, arginine, or lysine. Each of the 92nd aspartate or a corresponding position; 154th glutamate or a corresponding position; 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position can be independently substituted with aspartate, glutamate, asparagine, or glutamine.
For example, substitution at the 33rd histidine or a corresponding position can be with arginine. Substitution at the 33rd histidine or a corresponding position can also be with lysine. When the amino acid corresponding to the 33rd histidine is not histidine, such amino acid can be substituted with histidine.
Substitution in the 92nd aspartate or a corresponding position can be with aspartate. Substitution in the 92nd aspartate or a corresponding position can also be with glutamate. Substitution in the 92nd aspartate or a corresponding position can be with asparagine. Substitution in the 92nd aspartate or a corresponding position can also be with glutamine.
Substitution in the 154th glutamate or a corresponding position can be with aspartate. Substitution in the 154th glutamate or a corresponding position can also be with glutamate. Substitution in the 154th glutamate or a corresponding position can be with asparagine. Substitution in the 154th glutamate or a corresponding position can also be with glutamine.
Substitution in the 158th glutamate or a corresponding position can be with aspartate. Substitution in the 158th glutamate or a corresponding position can also be with glutamate. Substitution in the 158th glutamate or a corresponding position can be with asparagine. Substitution in the 158th glutamate or a corresponding position can also be with glutamine.
Substitution in the 242nd aspartate or a corresponding position can be with aspartate. Substitution in the 242nd aspartate or a corresponding position can also be with glutamate. Substitution in the 242th aspartate or a corresponding position can be with asparagine. Substitution in the 242nd aspartate or a corresponding position can also be with glutamine.
Substitution in the 246th glutamate or a corresponding position can be with aspartate. Substitution in the 246th glutamate or a corresponding position can also be with glutamate. Substitution in the 246th glutamate or a corresponding position can be with asparagine. Substitution in the 246th glutamate or a corresponding position can also be with glutamine.
The one or more amino acid substitutions in the RBP of the parent ChRmine protein can comprise substitutions in one or more of: 146th isoleucine or a corresponding position; 174th glycine or a corresponding position; 178th phenylalanine or a corresponding position. The substitution at the 146th isoleucine or a corresponding position can be with a serine, cysteine, threonine, or methionine. The substitution at the 174th glycine or a corresponding position can be with a serine, cysteine, threonine, or methionine. The substitution at the 178th phenylalanine or a corresponding position can be with phenylalanine, when the corresponding amino acid is not phenylalanine, tyrosine, or Tryptophan. Any combinations of these substitutions can be produced.
For example, substitution at the 146th isoleucine or a corresponding position can be with serine. Substitution at the 146th isoleucine or a corresponding position can also be with cysteine. Substitution at the 146th isoleucine or a corresponding position can be with threonine. Substitution at the 146th isoleucine or a corresponding position can also be with methionine.
Substitution at the 174th glycine or a corresponding position can be with serine. Substitution at the 174th glycine or a corresponding position can also be with cysteine. Substitution at the 174th glycine or a corresponding position can be with threonine. Substitution at the 174th glycine or a corresponding position can also be with methionine.
Substitution at the 178th phenylalanine or a corresponding position can be with tyrosine. Substitution at the 178th phenylalanine or a corresponding position can also be with tryptophan. When the corresponding amino acid at the 178th phenylalanine is not phenylalanine, it can be substituted with phenylalanine.
A high-speed and red-shifted variant ChRmine can be produced from a parent ChRmine protein selected from the proteins provided in
A parent ChRmine protein can have a sequence selected from SEQ ID NOs: 1 to 29 or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29. A sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29 can have conservative amino acid substitutions as compared a sequence from which it is derived.
In certain embodiments, a high-speed and red-shifted variant ChRmine protein has one or more of: i) a substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein, ii) a substitution at the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and iii) a substitution at the glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. The histidine amino acid in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein can be substituted with any other amino acid, for example, histidine (when the corresponding amino acid is not histidine), arginine or lysine, i.e., a basic amino acid. The isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein can be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., hydroxyl or sulfur/selenium-containing amino acid. The glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein can be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., hydroxyl or sulfur/selenium-containing amino acid.
For example, substitution at the 33rd histidine or a corresponding position can be with arginine. Substitution at the 33rd histidine or a corresponding position can also be with lysine. When the amino acid corresponding to the 33rd histidine is not histidine, such amino acid can be substituted with histidine.
Also, substitution at the 146th isoleucine or a corresponding position can be with serine. Substitution at the 146th isoleucine or a corresponding position can also be with cysteine. Substitution at the 146th isoleucine or a corresponding position can be with threonine. Substitution at the 146th isoleucine or a corresponding position can also be with methionine.
Substitution at the 174th glycine or a corresponding position can be with serine. Substitution at the 174th glycine or a corresponding position can also be with cysteine. Substitution at the 174th glycine or a corresponding position can be with threonine. Substitution at the 174th glycine or a corresponding position can also be with methionine.
Accordingly, a high-speed and red-shifted variant ChRmine protein can have a sequence of SEQ ID NO: 32 or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 32, wherein the variations in the sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 32 exclude the amino acid substitution used to produce the high-speed and red-shifted variant ChRmine protein. In a specific embodiment, a high-speed and red-shifted variant ChRmine protein has a sequence of SEQ ID NO: 32. Further embodiments of the disclosure provide a nucleic acid encoding for a variant ChRmine protein disclosed herein. Based on the sequence of a variant ChRmine protein and known codon usage, a person of ordinary skill in the art can design a nucleic acid encoding a specific variant ChRmine protein. A nucleic acid can be optimized for expression in a particular cell, for example, a mammalian cell or an insect cell. Methods of such codon-optimization are well-known in the art and are within the purview of this disclosure.
The nucleic acid encoding such variant ChRmine protein can be incorporated in an expression cassette, for example, an expression vector, for expressing the variant ChRmine protein in a cell. Non-limiting examples of a cell include a bacterial cell, a fungal cell, an insect cell, a plant cell, or a mammalian cell.
Accordingly, further embodiments of the disclosure provide a genetically modified cell comprising a nucleic acid encoding a variant ChRmine protein. Methods of introducing a nucleic acid, for example, a nucleic acid in an expression construct, into a target cell and expressing and purifying the proteins are well-known in the art and such embodiments are within the purview of the disclosure.
Optogenetics include genetic modification to the neurons followed by contacting the genetically modified neurons with light. The genetic modification causes the neurons to express light-sensitive ion channels, and contacting the neurons with light activates these channels, influencing the activation of the neuron.
As discussed above, certain embodiments of the disclosure provide variant ChRmine proteins that exhibit faster kinetics and/or red-shifted spectra compared to a parent ChRmine proteins. When used in optogenetic methods, such variant ChRmine proteins provide certain benefits over parent ChRmine proteins.
Accordingly, certain embodiments of the disclosure provide an optogenetic method comprising: genetically modifying a subject to express in the subject's brain cells the variant ChRmine protein disclosed herein, applying stimulating light to the subject's brain, and imaging the subject's brain.
A subject can be a human, a non-human primate, a bovine, a porcine, a feline, or a canine animal.
The details of the optogenetic methods are well known in the art and generally applying such methods using the variant ChRmine proteins disclosed herein is within the purview of the disclosure.
For example, in some cases the method involves electrical stimulation of the brain region using one or more electrodes. These electrodes can be positioned, either temporarily or permanently, at the brain region.
In some cases, the brain region which is genetically modified for an optogenetic method is selected from the group consisting of: hippocampus, septo-hippocampus, anterior cingulate cortex (ACC), basolateral amygdala (BLA), midline thalamus, insulate regions, medial septum, fimbria fornix. In some cases, the brain region is the hippocampus. In some cases, the brain region is the septo-hippocampus. In some cases, the brain region is the ACC. In some case, the brain region is the BLA. In some cases, the brain region is the medial septum. In some cases, the brain region is the fimbria fornix. In some cases, two or more of the listed brain regions are genetically modified.
Additional embodiments of the disclosure provide methods comprising: genetically modifying a subject to express in a cell and/or organ the variant ChRmine protein disclosed herein. The methods can further comprise applying stimulating light to the modified cell and/or organ, and imaging the subject's cell and/or organ. The cell and/or organ can belong to the cardiovascular system, the gastrointestinal system, the urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic/endocrine system.
Notwithstanding the appended claims, the disclosure is also defined by the following Embodiments:
Embodiment 1. A high-speed variant ChRmine protein having faster kinetic properties compared to a parent ChRmine protein, wherein the high-speed variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine.
Embodiment 2. The high-speed variant ChRmine protein according to Embodiment 1, comprising one or more amino acid substitutions in the Schiff base counterion of the parent ChRmine protein.
Embodiment 3. The high-speed variant ChRmine protein according to Embodiment 1, comprising one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein.
Embodiment 4. The high-speed variant ChRmine protein according to Embodiment 3, wherein the one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein are selected from: 33rd histidine or a corresponding position; 92nd aspartate or a corresponding position; 154th glutamate or a corresponding position; 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position.
Embodiment 5. The high-speed variant ChRmine protein according to Embodiment 4, wherein: the 33rd histidine or a corresponding position is substituted with histidine, when the corresponding amino acid is not histidine, arginine, or lysine.
Embodiment 6. The high-speed variant of ChRmine protein according to Embodiment 4, wherein: each of the 92nd aspartate or a corresponding position, 154th glutamate or a corresponding position, 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position is substituted independently of each other with aspartate, glutamate, asparagine, or glutamine.
Embodiment 7. The high-speed variant ChRmine protein according to any one of Embodiments 1 to 6, wherein the parent ChRmine protein has a sequence selected from SEQ ID NOs: 1 to 29 or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29.
Embodiment 8. The high-speed variant ChRmine protein according to any one of Embodiments 1 to 7, wherein, compared to the parent ChRmine protein, the high-speed variant ChRmine protein has a substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein.
Embodiment 9. The high-speed variant ChRmine protein according to Embodiment 8, wherein the high-speed variant ChRmine protein has an arginine substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein.
Embodiment 10. The high-speed variant ChRmine protein according to any one of Embodiments 1 to 9, having the sequence of SEQ ID NO: 30 or a sequence having at least 80% sequence identity to SEQ ID NO: 30, wherein the variations in the sequence having at least 80% sequence identity to SEQ ID NO: 30 exclude the amino acid substitution used to produce the high-speed variant ChRmine protein.
Embodiment 11. The high-speed variant ChRmine protein according to any one of Embodiments 1 to 10, having the sequence of SEQ ID NO: 30.
Embodiment 12. A red-shifted variant ChRmine protein having a red-shifted spectrum compared to a parent ChRmine protein, wherein the red-shifted variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein.
Embodiment 13. The red-shifted variant ChRmine protein according to Embodiment 12, comprising one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.
Embodiment 14. The red-shifted variant ChRmine protein according to Embodiment 13, wherein the one or more amino acid substitutions in the RBP of the parent ChRmine protein comprise substitutions in one or more of: 146th isoleucine or a corresponding position; 174th glycine or a corresponding position; 178th phenylalanine or a corresponding position.
Embodiment 15. The red-shifted variant ChRmine protein according to Embodiment 13, wherein: the substitution at the 146th isoleucine or a corresponding position is with a serine, cysteine, threonine, or methionine; the substitution at the 174th glycine or a corresponding position is with a serine, cysteine, threonine, or methionine; or the substitution at the 178th phenylalanine or a corresponding position is with phenylalanine, when the corresponding amino acid is not phenylalanine, tyrosine, or Tryptophan.
Embodiment 16. The red-shifted variant ChRmine protein according to any one of Embodiments 12 to 15, wherein the parent ChRmine protein has a sequence selected from SEQ ID NOs: 1 to 29 or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29.
Embodiment 17. The red-shifted variant ChRmine protein according to any one of Embodiments 12 to 16, wherein, compared to the parent ChRmine protein, the red-shifted variant ChRmine protein has one or both of: i) a substitution at the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and ii) a substitution at the glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.
Embodiment 18. The red-shifted variant ChRmine protein according to Embodiment 17, wherein, compared to the parent ChRmine protein, the red-shifted variant ChRmine protein has one or both of: i) a methionine substitution at the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and ii) a serine substitution at the glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.
Embodiment 19. The red-shifted variant ChRmine protein according to any one of Embodiments 12 to 18, having the sequence of SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31, wherein the variations in the sequence having at least 80% sequence identity to SEQ ID NO: 31 exclude the amino acid substitution used to produce the red-shifted variant ChRmine protein.
Embodiment 20. The red-shifted variant ChRmine protein according to any one of Embodiments 12 to 18, having the sequence of SEQ ID NO: 31.
Embodiment 21. A high-speed and red-shifted variant ChRmine protein having faster kinetics and red-shifted spectrum compared to a parent ChRmine protein, wherein the high-speed and red-shifted variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein.
Embodiment 22. The high-speed and red-shifted variant ChRmine protein according to Embodiment 21, comprising: i) one or more amino acid substitutions in Schiff base counterion of the parent ChRmine protein or one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.
Embodiment 23. The high-speed and red-shifted variant ChRmine protein according to Embodiment 21 or 22, comprising: i) one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein and ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.
Embodiment 24. The high-speed and red-shifted variant ChRmine protein according to Embodiment 23, wherein the one or more amino acid substitutions that alter the pore electrostatic potential are selected from: 33rd histidine or a corresponding position; 92nd aspartate or a corresponding position; 154th glutamate or a corresponding position; 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position.
Embodiment 25. The high-speed and red-shifted variant ChRmine protein according to Embodiment 24, wherein: 33rd histidine or a corresponding position is substituted with histidine, when the corresponding amino acid is not histidine, arginine, or lysine.
Embodiment 26. The high-speed and red-shifted variant ChRmine protein according to Embodiment 24, wherein: each of 92nd aspartate or a corresponding position, 154th glutamate or a corresponding position, 158th glutamate or a corresponding position, 242nd aspartate or a corresponding position, and 246th glutamate or a corresponding position is independently substituted with aspartate, glutamate, asparagine, or glutamine.
Embodiment 27. The high-speed and red-shifted variant ChRmine protein according to any one of Embodiments 22 to 26, wherein the one or more amino acid substitutions in the RBP of the parent ChRmine protein comprise substitutions in one or more of: 146th isoleucine or a corresponding position; 174th glycine or a corresponding position; and 178th phenylalanine or a corresponding position.
Embodiment 28. The high-speed and red-shifted variant ChRmine protein according to Embodiment 27, wherein: the substitution at the 146th isoleucine or a corresponding position is with a serine, cysteine, threonine, or methionine; the substitution at the 174th glycine or a corresponding position is with a serine, cysteine, threonine, or methionine; or the substitution at the 178th phenylalanine or a corresponding position is with phenylalanine, when the corresponding amino acid is not phenylalanine, tyrosine, or Tryptophan.
Embodiment 29. The high-speed and red-shifted variant ChRmine protein according to any one of Embodiments 21 to 28, wherein the parent ChRmine protein has a sequence selected from SEQ ID NOs: 1 to 29 or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29.
Embodiment 30. The high-speed and red-shifted variant ChRmine protein according to any one of Embodiments 21 to 29, wherein, compared to the parent ChRmine protein, the high-speed and red-shifted variant ChRmine protein has one or more of: i) a substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein; ii) a substitution at the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and iii) a substitution at the glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.
Embodiment 31. The high-speed and red-shifted variant ChRmine protein according to Embodiment 30, wherein, compared to the parent ChRmine protein, the high-speed and red-shifted variant ChRmine protein has one or more of: i) an arginine substitution at the histidine residue in the 33rd position or the corresponding residue in the first transmembrane domain of the parent ChRmine protein; ii) a methionine substitution at the isoleucine residue in the 146th position or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and iii) a serine substitution at the glycine residue in the 174th position or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.
Embodiment 32. The high-speed and red-shifted variant ChRmine protein according to any one of Embodiments 21 to 31, having the sequence if SEQ ID NO: 32 or a sequence having at least 80% sequence identity to SEQ ID NO: 32, wherein the variations in the sequence having at least 80% sequence identity to SEQ ID NO: 32 exclude the amino acid substitution used to produce the high-speed and red-shifted variant ChRmine protein.
Embodiment 33. The high-speed and red-shifted variant ChRmine protein according to any one of Embodiments 21 to 31, having the sequence of SEQ ID NO: 32.
Embodiment 34. A nucleic acid encoding for a variant ChRmine protein according to any one of the preceding Embodiments.
Embodiment 35. A genetically modified cell comprising the nucleic acid according to Embodiment 34.
Embodiment 36. An optogenetic method comprising:
Embodiment 37. The optogenetic method according to Embodiment 36, wherein the subject is a mammal.
Embodiment 38. The optogenetic method according to Embodiment 37, wherein the mammal is a rodent, a primate, a bovine, a porcine, a feline, or a canine.
Embodiment 39. A method comprising: genetically modifying a subject to express in a cell and/or organ the variant ChRmine protein according to any one of Embodiments 1 to 33.
Embodiment 40. The method of Embodiment 39, further comprising applying stimulating light to the modified cell and/or organ, and imaging the subject's cell and/or organ.
Embodiment 41. The method of Embodiment 40, wherein the cell and/or organ can belong to the cardiovascular system, the gastrointestinal system, urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic/endocrine system.
The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.
Light—a crucial energy source and environmental signal—is typically captured by motile organisms using rhodopsins, which are largely classified into two groups: microbial and animal, both consisting of a seven-transmembrane (7TM) protein (opsin) and a covalently-bound chromophore (retinal). Light absorption induces retinal isomerization followed by the photocycle, a series of photochemical reactions (Zhang et al., 2011, Ernst et al., 2014; Deisseroth and Hegemann, 2017), which in microbial rhodopsins ultimately exerts direct biochemical action (examples include pumps, channels, sensors, and enzymes (Kandori, 2020; Kato, 2021). Targeted expression of these proteins (especially of the channel- and pump-type) in specific cell types, when applied along with precise light delivery, enables causal study of cellular activity in behaving organisms (optogenetics) (Deisseroth, 2015; Kurihara and Sudo, 2015; Deisseroth, 2021).
In optogenetics, cation-conducting channelrhodopsins (cation ChRs or CCRs) are typically used for activation of target cells (Deisseroth and Hegemann, 2017). The initial description of a CCR (CrChR1 from the chlorophyte C. reinhardtii; Nagel et al., 2002) was followed by characterization of variants that were discovered or designed with new functions spanning ion selectivity, photocurrent amplitude, absorption, sensitivity, and speed (Deisseroth and Hegemann, 2017). Natural CCRs include CrChR2 (ChR2 from C. reinhardtii) (Nagel et al., 2003), VChR1 (ChR1 from V. carteri) (Zhang et al., 2008), and Chrimson (from C. noctigama) (Klapoetke et al., 2014); these were initially described chiefly from chlorophyte algae, but identification of ChRs from other species further expanded the toolkit. In 2016-17, a subfamily of microbial rhodopsins was reported from the cryptophyte G. theta (Govorunova et al., 2016; Yamauchi et al., 2017), identified as CCRs but more homologous to archaeal ion-pumps such as H. salinarum bacteriorhodopsin (HsBR). Moreover, unlike chlorophyte CCRs, cryptophyte CCRs share three amino acids on TM3 crucial for outward proton (H+) pumping [the DTD motif (Inoue et al., 2013); D85, T89, and D96 in HsBR] and have been referred to as bacteriorhodopsin-like cation ChRs or BCCRs (Sineshchekov et al., 2017) (
A high-resolution structure for this family of proteins would facilitate understanding structure-function relationships among pump- and channel-type rhodopsins and designing next-generation optogenetic tools. This previously led to creation of the initial anion-conducting ChRs (ACRs, Berndt et al., 2014, 2016; Kato et al., 2012; Wietek et al., 2014)). To that end, this disclosure provides the cryo-electron microscopy (cryo-EM) structure of ChRmine at 2.0 Å resolution. The information about the structure was also used to create variants with faster speed and greater red-shift while preserving high current and light sensitivity. These variant channelrhodopsins as disclosed herein can be used in optical neuroscience research and for targeted functional analysis in diverse systems.
Our initial efforts to crystallize ChRmine yielded low-resolution crystals; we therefore turned to single-particle cryo-EM (
Overall Structure and Comparison with HsBR and C1C2
The cryo-EM density map revealed that the quaternary structure of ChRmine is strikingly different from that of other structure-resolved ChRs (Kato et al., 2012) (
The monomer of ChRmine consists of an extracellular N-terminal domain (residues 10-26), an intracellular C-terminal domain (residues 271-279), and 7 TM domains (within residues 27-270), connected by three intracellular loops (ICL1-3) and three extracellular loops (ECL1-3) (
To explore how ChRmine can be structurally like ion-pumping rhodopsins and yet function as a channel, we compared ChRmine with an archaeal ion-pumping rhodopsin (HsBR), and a chlorophyte CCR (C1C2, the chimera derived from CrChR1 and CrChR2). Consistent with the sequence similarity (
In all microbial rhodopsins, the retinal is covalently bound to a TM7 lysine to form the protonated Schiff base; this positive charge is stabilized by 1-2 carboxylates on the extracellular side (
The Schiff base region of ChRmine is strikingly different from that of both types of rhodopsins (HsBR and C1C2;
Three water molecules (w1, w2, w3) occupy the space between the Schiff base and D115 created by the unwinding of TM3. Notably, w2 and w3 are well superposed onto the carboxyl oxygens of D85 in HsBR, suggesting that these waters structurally mimic D85 (
To explore the function of the counterion and proton acceptor candidates, D115 and D253, we measured photocurrent amplitudes of wild-type (WT), D115N, and D253N ChRmine in HEK293 cells; both D115N and D253N abolished photocurrents (
Next, to identify which carboxylate works as a primary proton acceptor in the M intermediate, we performed flash photolysis of D115N and D253N (
Ion-Conducting Pore within the Monomer
To explore the location and shape of the ion-conducting pathway, we first analyzed the configuration of cavities within the monomer. ChRmine displays markedly larger intracellular and extracellular cavities compared to C1C2 and HsBR (
Second, ChRmine exhibits two intracellular vestibules (IV) with distinct electrostatic potentials (
Third, the ICS architecture of ChRmine and C1C2 are different. In C1C2, the ICS is mainly formed by Y109, E122, and H173 (E122 and H173 are H-bonded to each other). In ChRmine, the corresponding residues are L47, A74, and D126, respectively, which participate in the formation of the ICS, but D126 forms a more extensive H-bonding network with Q71, Q130, Y260, and a water (
Fourth, the size and path of the extracellular cavities significantly differ between ChRmine and C1C2. C1C2 has two extracellular vestibules (EV1 and EV2), but ChRmine lacks the vestibule corresponding to EV1, while the volume of ChRmine's sole EV is significantly expanded (due in large part to TM3 unwinding;
While ChRmine resembles HsBR in some ways (primary sequence, overall arrangement of the secondary structural elements of the monomer, and quaternary structure of the trimer;
In a second major channel-enabling feature, the unwinding of TM3 and resulting long ECL1 contribute to creation of a large extracellular cavity in ChRmine. The helical structure of extracellular TM3 is unfolded beginning at Y116, and the C-shaped structure of ECL1 protrudes to the center of the trimer interface. This contrasts with the ECL1 of HsBR, which forms a β-sheet and is in a position that half-occludes the extracellular pore (
Notably, the ECL1 of C1C2 also forms a β-sheet structure like HsBR and moderately narrows the entrance of the pore—one of the reasons that the extracellular cavity of C1C2 is smaller than that of ChRmine (
Like HsBR, ChRmine forms a trimer; here we find that ChRmine has an unexpected additional opening at the trimer interface (
ChRmine exhibits three intermolecular H-bond interactions between adjacent protomers: S138 with E69, the main chain amide of R136 with E69, and Y156 with H96 (
To further test this hypothesis, we performed all-atom molecular dynamics simulations of ChRmine in either the dark state or the M intermediate (light state) (
We next sought to enhance the speed and spectral-response of ChRmine for all-optical experiments (
In a separate line of investigation we had found that other mutations predicted to alter pore electrostatic potential can also affect speed (Kato et al., 2018; Kim et al., 2018). However, it was unclear that these mutations along the ion-conduction pathway in dimeric ChRs would translate to the structurally-divergent ChRmine, where ion conduction has distinctive properties [for example, we discovered that ChRmine exhibits high monovalent cation selectivity (excluding Ca2+ and Mg2+, and strikingly favoring K+ over Na+;
We next sought to modify the spectral properties, specifically red light actuation to improve compatibility with blue-light-activated genetically encoded Ca2+ indicators (GECIs). Previous studies have shown that mutations in the retinal binding pocket (RBP) can change spectral properties, including peak and shape of the action spectrum (Kato et al., 2015a; Oda et al., 2018; Pan et al., 2014); however, sequence identity is low (˜20%) between ChRmine and structurally-resolved CCRs (Marshel et al., 2019), which precluded effective homology modeling of the ChRmine RBP before the structure was solved (
We designated the faster or accelerated-kinetics variant (H33R) as hsChRmine (for high-speed), and the optimal red-shifted variant (I146M/G174S) as rsChRmine (for red-shifted) (
Finally, we compared two-photon (2P) spectra of ChRmine variants with ChroME2 (Sridharan et al., 2021). We found that the spectral shift of rsChRmine was even greater with 2P; at 825 nm, little rsChRmine current was detected, whereas WT ChRmine at 825 nm exhibited 40% of the maximal photocurrent elicited at 1050 nm. Furthermore, the 2P spectra of ChroME2f/2s were found to be blue-shifted relative to ChRmine, as with 1P stim (
Minimal Cross-Talk for all-Optical Experiments with rsChRmine
A blue shoulder persists in the action spectra of all published ChRs; the distinctive spectral properties of rsChRmine thus raised the prospect of minimizing the optical cross-talk that is problematic for all-optical neuroscience. We characterized spike fidelity as a function of pulse width and irradiance for rs- and WT ChRmine in brain slices (
To test potential utility for all-optical experiments, we characterized compatibility with the green GECI XCaMP-G (Inoue et al., 2019) in cultured neurons using 1P stimulation (
These results indicated that rsChRmine could be useful for new kinds of simultaneous optical imaging and control in vivo. To explicitly test this, we first applied Frame-Projected Independent-Fiber Photometry (FIP) for simultaneous recording (Kim et al., 2016) and perturbation of activity in pyramidal (Pyr) neurons of medial prefrontal cortex (mPFC) of mice (
To quantify independence of optical information channels in a practical setting, we tested for incidental stimulation of the targeted cells by 470 nm blue light pulses that are intended for GCaMP imaging, not red-shifted opsin stimulation. rsChRmine- and ChrimsonR-expressing cells exhibited little evoked change in fluorescence even up to 20 μW of 470 nm light, while WT ChRmine exhibited significant fluorescent changes from 3 μW (
Lastly, we asked whether the shifted spectrum of rsChRmine might allow stimulation of activity in a targeted neural population during simultaneous recording of activity in both the stimulated and downstream neural populations. We therefore combined presynaptic rsChRmine stimulation with XCaMP-B recording, alongside postsynaptic GCaMP6f recording, by expressing both rsChRmine and XCaMP-B in Pyr neurons, and GCaMP6f in parvalbumin-expressing (PV) interneurons in mPFC (
Wild-type ChRmine (M1-R304, five amino acids at the C terminus truncated from the previous construct (Marshel et al., 2019)) was modified to include an N-terminal influenza hemagglutinin (HA) signal sequence and FLAG-tag epitope, and C-terminal enhanced green fluorescent protein (eGFP) and 10× histidine tag; the N-terminal and C-terminal tags are removable by human rhinovirus 3C protease cleavage. The construct was expressed in Spodoptera frugiperda (Sf9) insect cells using the pFastBac baculovirus system. Sf9 insect cells were grown in suspension to a density of 3.5×106 cells/mL, infected with ChRmine baculovirus and shaken at 27.5° C. for 24 h. Then, 10 μM all-trans-retinal (ATR) (Sigma-Aldrich) was supplemented to the culture and shaken continued for 24 more hours. The cell pellets were lysed with a hypotonic lysis buffer (20 mM HEPES-NaOH pH 7.5, 20 mM NaCl, 10 mM MgCl2, 1 mM benzamidine, 1 μg/ml leupeptin, 10 μM ATR), and cell pellets were collected by centrifugation at 10,000×g for 30 min. The above process was repeated twice; then, cell pellets were disrupted by homogenizing with a glass dounce homogenizer in a hypertonic lysis buffer (20 mM HEPES-NaOH pH 7.5, 1 M NaCl, 10 mM MgCl2, 1 mM benzamidine, 1 μg/ml leupeptin, 10 μM ATR), and crude membrane fraction was collected by ultracentrifugation (45Ti rotor, 125,000×g for 1 h). The above process was repeated twice; then, the membrane fraction was homogenized with a glass douncer in a solubilization buffer (1% n-dodecyl-β-D-maltoside (DDM) (EMD Millipore), 0.2% cholesteryl hemisuccinate (CHS) (Sigma-Aldrich), 20 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 20% glycerol, 5 mM imidazole, 1 mM benzamidine, 1 μg/ml leupeptin) and solubilized for 2 h in 4° C. The insoluble cell debris was removed by centrifugation (125,000×g, 1 h), and the supernatant was mixed with the Ni-NTA superflow resin (QIAGEN) for 1 h in 4° C. The Ni-NTA resin was collected into a glass chromatography column, washed with 2.5 CV wash 1 buffer (0.05% DDM, 0.01% CHS, 20 mM HEPES-NaOH pH7.5, 100 mM NaCl, 50 mM imidazole), 2.5 CV wash 2 buffer (0.05% DDM, 0.06% GDN (glyco-diosgenin), 0.016% CHS, 20 mM HEPES-NaOH pH7.5, 100 mM NaCl, 50 mM imidazole), and 2.5 CV wash 3 buffer (0.06% GDN, 0.006% CHS, 20 mM HEPES-NaOH pH7.5, 100 mM NaCl, 50 mM imidazole), and was eluted in a wash 3 buffer supplemented with 300 mM imidazole. After cleavage of the FLAG tag and eGFP-His10 tag His-tagged 3C protease, the sample was reloaded onto the Ni-NTA column to capture the cleaved eGFP-His10. The flow-through containing ChRmine was collected, concentrated, and purified through gel-filtration chromatography in a final buffer (20 mM HEPES-NaOH pH7.5, 100 mM NaCl, 0.03% GDN, 0.003% CHS).
Mouse monoclonal antibodies against ChRmine were raised according to previously-described methods (Jaenecke et al., 2018). Briefly, a proteoliposome antigen was prepared by reconstituting purified, functional ChRmine at high density into phospholipid vesicles consisting of a 10:1 mixture of chicken egg yolk phosphatidylcholine (egg PC; Avanti Polar Lipids) and the adjuvant lipid A (Sigma-Aldrich) to facilitate immune response. BALB/c mice were immunized with the proteoliposome antigen using three injections at two-week intervals. Antibody-producing hybridoma cell lines were generated using a conventional fusion protocol. Biotinylated proteoliposomes were prepared by reconstituting ChRmine with a mixture of egg PC and 1,2-dipal-mitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (16:0 biotinyl Cap-PE; Avanti), and used as binding targets for conformation-specific antibody selection. The targets were immobilized onto streptavidin-coated microplates (Nunc). Hybridoma clones producing antibodies recognizing conformational epitopes in ChRmine were selected by an enzyme-linked immunosorbent assay on immobilized biotinylated proteoliposomes (liposome ELISA), allowing positive selection of the antibodies that recognized the native conformation of ChRmine. Additional screening for reduced antibody binding to SDS-denatured ChRmine was used for negative selection against linear epitope-recognizing antibodies. Stable complex formation between ChRmine and each antibody clone was checked using fluorescence-detection size-exclusion chromatography. The sequence of the Fab from the antibody clone number YN7002_7 (named as Fab02) was determined via standard 5′-RACE using total RNA isolated from hybridoma cells.
Purified ChRmine was mixed with a fourfold molar excess of Fab, and the coupling reaction proceeded at 4° C. overnight. The ChRmine-Fab02 complex was purified by size exclusion chromatography on a Superdex 200 increase 10/300 GL column (Cytiva) in 20 mM HEPES-NaOH pH7.5, 100 mM NaCl, 0.03% GDN, 0.003% CHS. Peak fractions were concentrated to about 15 mg/mL for electron microscopy studies.
Cryo-EM images were acquired at 300 kV on a Krios G3i microscope (Thermo Fisher Scientific) equipped with a Gatan BioQuantum energy filter and a K3 direct detection camera in the electron counting mode. The movie dataset was collected in a correlated double sampling (CDS) mode, using a nine-hole image shift strategy in the SerialEM software (Mastronarde, 2005b), with a nominal defocus range of 0.8 to 1.6 μm. The 3,528 movies were acquired at a dose rate of 6.3 e−/pixel/s, at a pixel size of 0.83 Å and a total dose of 46 e−/Å2.
Image processing was performed in RELION-3.1 (Zivanov et al., 2018). Beam-induced motion correction and dose weighting were performed with RELION's implementation of the MotionCor2 algorithm (Zheng et al., 2017), and CTF parameters were estimated with CTFFIND-4.1.13 (Rohou and Grigorieff, 2015). Particles were first picked using the Laplacian-of-gaussian algorithm, and 2D class average images were generated as templates for reference-based auto-picking. Reference-based picked 2,958,159 particles were subjected to several rounds of 2D and 3D classifications. The selected 555,801 particles were subjected to a 3D auto-refinement, resulting in a 2.8 Å map. Subsequently, Bayesian polishing (Zivanov et al., 2019) and CTF refinement (Zivanov et al., 2020), followed by a 3D auto-refinement, resulted in a 2.6 Å map. Micelle and constant regions of Fab fragments densities were subtracted from particle images, and the subtracted particles were subjected to a masked 3D classification without alignment. After a 3D auto-refinement of selected 185,895 particles, three runs of CTF refinement were performed in order as follows: refining magnification anisotropy; refining optical aberrations; refining per-particle defocus and per-micrograph astigmatism. Another round of 3D auto-refinement yielded a 2.13 Å map. These particles were subjected to, a second round of Bayesian polishing, CTF refinement and a transmembrane region-focused 3D auto-refinement with the reconstruction algorithm SIDESPLITTER (Ramlaul et al., 2020), resulting in the final map at a global resolution of 2.02 Å.
An initial model was formed by rigid body fitting of the C1C2 (PDB: 3UG9) (Kato et al., 2012). This starting model was then subjected to iterative rounds of manual and automated refinement in Coot (Emsley and Cowtan, 2004) and Refmac5 (Murshudov et al., 2011) in Servalcat pipeline (Yamashita et al., 2021), respectively. The Refmac5 refinement was performed with the constraint of C3 symmetry. The final model was visually inspected for general fit to the map, and geometry was further evaluated using Molprobity (Chen et al., 2010). The final refinement statistics is summarized in Table 1. All molecular graphics figures were prepared with UCSF Chimera (Pettersen et al., 2004), UCSF ChimeraX (Goddard et al., 2018), and Cuemol (see world-wide-website: cuelmol.org).
The ion-conducting pores were calculated by the software HOLLOW using a grid-spacing of 1.0 Å. The electrostatic potentials of the pores are calculated by PDB2PQR server (Baker et al., 2001; Dolinsky et al., 2004). Trimer opening radii of the ChRmine was calculated with HOLE.
The retinal isomers were analyzed with an HPLC system equipped with a silica column (particle size 3 μm, 150×6.0 mm; Pack SIL, YMC, Japan), a pump (PU-4580, JASCO, Japan) and a UV-Visible detector (UV-4570, JASCO, Japan). The purified sample in a buffer containing 20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 0.035% GDN, 0.0035% CHS (GDN:CHS=10:1) were dark-adapted for two days at 4° C. A 75 μL sample and 280 μL of 90% (v/v) methanol aqueous solution were mixed on ice and then 25 μL of 2 M hydroxylamine (NH2OH) was added to convert retinal chromophore into retinal oxime, which was extracted with 800 μL of n-hexane. A 200 μL of the extract was injected into the HPLC system. The solvent containing 15% ethyl acetate and 0.15% ethanol in hexane was used as a mobile phase at a flow rate of 1.0 mL min−1. Illumination was performed on ice with green light (530±5 nm) for 20 s for samples under illumination and 60 s for light adaptation. The molar composition of the sample was calculated from the areas of the peaks and the molar extinction coefficients at 360 nm (all-trans-15-syn: 54,900 M−1 cm−1; all-trans-15-anti: 51,600 M−1 cm−1; 13-cis-15-syn, 49,000 M−1 cm−1; 13-cis-15-anti: 52,100 M−1 cm−1; 11-cis-15-syn: 35,000 M−1 cm−1; 11-cis-15-anti: 29,600 M−1 cm−1) (Trehan et al., 1990).
For HS-AFM imaging of lipid-reconstituted ChRmine, we applied membrane scaffolding proteins (MSP), which were developed for nanodisc technology (Bayburt et al., 2002; Denisov and Sligar, 2016). We followed the manufacturer's protocol for the nanodisc (Sigma-Aldrich, St. Louis, MO, USA) with minor modifications as described previously (Shibata et al., 2018). Briefly, for reconstituted lipids, we used a mixture of phospholipids, asolectin from soybean (Sigma-Aldrich, No. 11145). Asolectin (120 μg) was dissolved in chloroform and then evaporated under N2 gas to completely remove the solvent. Then, the lipids were suspended in 50 μL buffer A (20 mM HEPES-KOH pH 7.4, 100 mM NaCl, and 4% DDM) and sonicated for ˜1 min with a tip-sonicator. Next, dissolved membrane proteins (1 nmol) and MSP (50 μL, 1 mg/mL) (MSP1E3D1, Sigma-Aldrich, No. M7074) were added to the lipid suspension and mixed for ˜1 h while rotating in the dark at 4° C. Finally, we added 60 mg Bio-beads SM-2 (Bio-Rad, Hercules, CA, USA, No. 1523920) and dialyzed the samples in detergent overnight at 4° C. According to the manufacturer's protocol, nanodisc samples should be fractionated on a column to purify the nanodiscs based on size (˜10 nm in diameter). Here, we did not purify the reconstituted samples, but obtained flat membranes with limited sizes<30 nm in diameter.
A homemade HS-AFM operated in tapping mode was used (Shibata et al., 2017, 2018). An optical beam deflection detector detected the cantilever (Olympus, Tokyo, Japan: BL-AC10DS-A2) deflection using an infrared (IR) laser at 780 nm and 0.7 mW. The IR beam was focused onto the back side of the cantilever covered with a gold film through a ×60 objective lens (Nikon, Tokyo, Japan: CFI S Plan Fluor ELWD 60×). The reflected IR beam was detected by a two-segmented PIN photodiode. The free oscillation amplitude of the cantilever was ˜1 nm and set-point amplitude was approximately 90% of the free amplitude for feedback control of HS-AFM observation. An amorphous carbon tip (˜500 nm length), grown by electron beam deposition by scanning electron microscope, was used as an AFM probe. As a HS-AFM substrate, a mica surface treated with 0.01% (3-aminopropyl) triethoxysilane (Shin-Etsu Silicone, Tokyo, Japan) was used. All HS-AFM experiments were carried out in buffer solution containing 20 mM Tris-HCl pH 8.0 and 100 mM NaCl at room temperature (24-26° C.) and data analyses were conducted using laboratory-developed software based on IgorPro 8 software (WaveMetrics, USA). We usually used a scan area of 43×32 nm2 with 130×95 pixels. HS-AFM images were captured at frame rates of 2 fps. All HS-AFM images were processed by Gaussian noise-reduction filters.
For pH titration, the final purified product (20 mM HEPES-NaOH pH7.5, 100 mM NaCl, 0.03% GDN, 0.003% CHS) was diluted with 100 mM of the respective pH buffer (StockOptions pH Buffer Kit), and the UV-Vis spectra were measured.
For the laser flash photolysis spectroscopy, wildtype ChRmine was solubilized in 20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 0.035% GDN, 0.0035% CHS (GDN:CHS=10:1) or 20 mM sodium acetate pH 4.0, 100 mM NaCl, 0.03% GDN, 0.003% CHS (GDN:CHS=10:1), and ChRmine D115N and D253N mutants were solubilized in 20 mM sodium acetate pH 4.0, 100 mM NaCl, 0.03% GDN, 0.003% CHS (GDN:CHS=10:1). The optical density of the protein solution was adjusted to ˜0.4 (protein concentration ˜0.28 mg/mL) at the absorption maximum wavelengths. The laser flash photolysis measurements were conducted as previously described (Inoue et al., 2013). ChRmine wildtype at pH 7.5 was excited by the second harmonics of a nanosecond-pulsed Nd3+-YAG laser (excitation wavelength (λexc)=532 nm, 4.5 mJ/pulse, 1.4-0.5 Hz, INDI40, Spectra-Physics, CA), and nano-second pulse from an optical parametric oscillator (4.5 mJ/pulse, basiScan, Spectra-Physics, CA) pumped by the third harmonics of Nd3+-YAG laser (λ=355 nm, INDI40, Spectra-Physics, CA) was used for the excitation of ChRmine wildtype at pH 4.0 (λexc=505 nm), ChRmine D115N (λexc=488 nm) and D253N (λexc=500 nm). Transient absorption spectra were obtained by monitoring the intensity change of white-light from a Xe-arc lamp (L9289-01, Hamamatsu Photonics, Japan) passed through the sample with an ICCD linear array detector (C8808-01, Hamamatsu, Japan). To increase the signal-to-noise (S/N) ratio, 45-60 spectra were averaged, and the singular-value-decomposition (SVD) analysis was applied. To measure the time-evolution of transient absorption change at specific wavelengths, the light of Xe-arc lamp (L9289-01, Hamamatsu Photonics, Japan) was monochromated by monochromators (S-10, SOMA OPTICS, Japan) and the change in the intensity after the photo-excitation was monitored with a photomultiplier tube (R10699, Hamamatsu Photonics, Japan). To increase S/N ratio, 100-200 signals were averaged. The time-evolution of transient absorption change was analyzed by global multi-exponential fitting to determine the time constant of each reaction step and absorption spectra of the photo-intermediates. Some reaction steps were reproduced by double or triple exponentials. In this case, the averaged time constant calculated by
All ChRmine mutant plasmids were constructed in AAV-CaMKIIa or pcDNA 3.1 backbones using overlapping PCR as described previously (Fenno et al., 2020; Marshel et al., 2019). ChRmine-Oscarlet-Kv2.1, WT, rs and hs mutants were transferred to an Elav3 backbone using AgeI and MluI sites for creating transgenic fish lines. Every plasmid was sequence-verified.
For neuronal transfection, 2.0 μg plasmid DNA was mixed with 1.875 μL 2 M CaCl2) (final Ca2+ concentration 250 mM) in 15 μL H2O. To DNA-CaCl2 we added 15 μL of 2×HEPES-buffered saline pH 7.05. After 20 min at room temperature (20-22° C.), the mix was added dropwise into each well (from which the growth medium had been removed and replaced with pre-warmed minimal essential medium (MEM)) and transfection proceeded for 45-60 min at 37° C., after which each well was washed with 3×1 ml warm MEM before the original growth medium was returned. Neurons were allowed to express transfected DNA for 6-8 days prior to experiments.
For HEK cell transfection, 0.8 μg plasmid DNA was mixed with 2 μL Lipofectamine 2000 (Invitrogen) in 100 μL Opti-MEM (Invitrogen, incubated at room temperature (20-22° C.) for 20 minutes, and the mix was added dropwise into each well (from which the growth medium had been removed and replaced with 400 μL pre-warmed Opti-MEM). Transfection proceeded for two hours at 37° C., after which the transfection media was replaced by normal HEK cells growth media. Cells were allowed to express transfected DNA for 2-3 days prior to experiments.
AAV-8 (Y733F), was produced by the Stanford Neuroscience Gene Vector and Virus Core. In brief, AAV8 was produced by standard triple transfection of AAV 293 cells (Agilent). At 72 h post transfection, the cells were collected and lysed by a freeze-thaw procedure. Viral particles were then purified by an iodixanol step-gradient ultracentrifugation method. The iodixanol was diluted and the AAV was concentrated using a 100-kDa molecular mass-cutoff ultrafiltration device. Genomic titer was determined by quantitative PCR. All viruses were tested in cultured neurons for expected expression patterns prior to use in vivo.
HEK293 cells transfected with pcDNA3.1(+) plasmids were placed in an extracellular tyrode medium (150 mM NaCl, 4 mM KCl, 2 mM CaCl2), 2 mM MgCl2, 10 mM HEPES pH 7.4, and 10 mM glucose). Borosilicate patch pipettes (Harvard Apparatus) with resistance of 4-6 Mohm were filled with intracellular medium (140 mM potassium-gluconate, 10 mM EGTA, 2 mM MgCl2 and 10 mM HEPES pH 7.2). Light was delivered with the Spectra X Light engine (Lumencor) connected to the fluorescence port of a Leica DM LFSA microscope with a 580 nm filter for orange light generation.
Channel kinetics and photocurrent amplitudes were measured in voltage clamp mode at −70 mV holding potential. To determine channel kinetics and photocurrent amplitudes, traces were first smoothed using a lowpass Gaussian filter with a −3 dB cutoff for signal attenuation and noise reduction at 1,000 Hz and then analyzed in Clampfit software (Axon Instruments). Liquid junction potentials were corrected using the Clampex built-in liquid junction potential calculator as previously described. Statistical analysis was performed with t-test or one-way ANOVA, and the Kruskal-Wallis test for non-parametric data, using Prism 7 (GraphPad) software. Data collection across opsins was randomized and distributed to minimize across-group differences in expression time, room temperature, and related experimental factors.
HEK293 cells and devices for the measurement were prepared as described in the previous section. For the high sodium extracellular/high potassium intracellular condition, we used sodium bath solution containing 120 mM NaCl, 4 mM KCl, 2 mM CaCl2), 2 mM MgCl2, and 10 mM HEPES pH 7.2 (with glucose added up to osm 310 mOsm), along with potassium pipette solution containing 120 mM KCl, 10 mM EGTA, 4 mM NaCl, 2 mM CaCl2), 2 mM MgCl2, and 10 mM HEPES pH 7.2 (with glucose added up to osm ˜290). For the high potassium extracellular/high sodium intracellular condition, NaCl and KCl concentrations were reversed, and all other ionic concentrations were kept constant. For ion selectivity measurements, ions in both bath and pipette solutions were replaced with either 120 mM NaCl, 120 mM KCl, 80 mM CaCl2), 80 mM MgCl2 or 120 mM NMDG-Cl, with all other components at low concentrations (4 mM NaCl, 4 mM KCl, 2 mM CaCl2), 2 mM MgCl2, and 10 mM HEPES). Glucose was added to increase intracellular solution to 310 mOsm and extracellular solution to 290 osm. Photocurrent amplitudes were measured at −70 mV holding membrane potential. Equilibrium potentials were measured by holding membrane potentials from −75 mV to +45 mV in steps of 10 mV.
Primary rat hippocampal cultured neurons were transfected with pAAV ChRmine-bearing plasmids and were measured in the same setup as described in the HEK293 electrophysiology section. Voltage clamp recordings were performed in the presence of bath-applied tetrodotoxin (TTX, 1 μM, Tocris). For screening of action spectra, cells were held at resting potential of −70 mV, with 1.0 mW/mm2 light delivery for 1 sec at wavelengths (in nm) of 390, 438, 485, 513, 585 and 650, which were generated using filters of corresponding peak wavelengths and 15-30 nm bandwidth. Channel kinetics and photocurrent amplitudes were measured at −70 mV holding membrane potential. Liquid junction potentials were corrected using the Clampex built-in liquid junction potential calculator as previously described. Current clamp measurements were performed in the presence of glutamatergic synaptic blockers: 6-cyano-7-nitroquinoxaline-2,3,-dione (CNQX; 10 μM, Tocris) and D(−)-2-amino-5-phosphonovaleric acid (APV; 25 μM, Tocris).
For light pulse-width experiments, 585 nm light with 5 Hz frequency and 0.7 mW/mm2 intensity was used at varying pulse-width values (in ms) of 0.5, 1, 2, 5 and 10. For light sensitivity experiments, 585 nm light with 5 Hz frequency and 5 ms pulse-width was used at varying light power densities (in mW/mm2) of 0.003, 0.01, 0.03, 0.1, 0.3, 0.7, and 1.0. For spike fidelity experiments, 585 nm light with 0.7 mW/mm2 power density was used, with 1 ms pulse-width for ChRmine variants. Data collection across opsins was randomized and distributed to minimize across-group differences in expression time, room temperature, and related experimental factors. Statistical analysis was performed with t-test or one-way ANOVA, and the Kruskal-Wallis test with Dunn's test for multiple comparisons for non-parametric data, using Python and Prism 7 (GraphPad) software.
All two-photon electrophysiology experiments were performed with cultured hippocampal neurons in the same intracellular and extracellular solutions as for one-photon electrophysiology characterization. Experiments were conducted on a commercial microscope (Bruker Ultima running PrairieView v5.4) using a Nikon 16×/0.8 NA (CFI75) long-working distance objective for light delivery. For two-photon stimulation, spiral scanning was performed through a defined spiral ROI with 15 μm diameter, with 10 rotations per spiral, and 1.3 ms total exposure duration with 80 MHz laser repetition rate (Coherent Discovery). The axial point-spread-function FWHM of the two-photon stimulation beam was measured to be 6.9+/−0.2 μm at 920 nm using 1 μm diameter beads (Invitrogen Focal Check Slide #1, F36909).
For two-photon action spectra characterization, recordings were conducted in voltage clamp mode at holding voltage of −75 mV. Action spectra were measured in randomized trial order at wavelengths (in nm) of 825, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, and 1300 at a laser power of 20 mW. 10 rotations/spiral, 15 mm diameter spirals, 1.3 ms duration, and 80-MHz laser repetition rate. We measured the focal shift as we systematically varied wavelengths from 825 to 1300 nm and found that there was a ˜25 um difference in focus between 825 and 1300 nm. Therefore, the z-focus was adjusted to compensate for empirically measured focal shifts during randomized wavelength delivery.
No steps were taken to compensate for the potential effects of pulse broadening due to spectral dispersion. All measurements were normalized by the maximum value of the single recording session and then averaged across cells.
A midline incision was made to expose the skull and small craniotomies were made above the injection sites using a Meisinger Carbide Burr size 1/4. All virus dilutions were performed in ice-cold PBS and all viruses were produced at the Stanford Gene and Viral Vector Core. Virus injections were delivered with a 10 μL syringe (World Precision Instruments) and 33-gauge beveled needle (World Precision Instruments), injected at 100 nL min−1 using an injection pump (World Precision Instruments). For slice physiology experiments, mice were injected with either AAV8-CaMKIIα-ChRmine-p2A-oscarlet (2.0e13 vg/mL) or AAV8-CaMKIIα-rsChRmine-p2A-Oscarlet (7.30e12 vg/mL). One microliter of virus was stereotactically injected bilaterally into the motor cortex of 8-12 week old mice at 1.7 mm AP, 0.75 mm ML, and 1.5 mm DV from the bregma. For fiber photometry experiments, mice were injected with either AAV8-CaMKIIα-GcaMP6m-2A-opsin where “opsin” is one of the three opsins shown in
Recordings of rsChRmine and ChRmine-expressing pyramidal cells were performed in acute slices from wild-type C57BL/6 mice 4-5 weeks after virus injection. Coronal slices 300 μm in thickness were prepared after intracardial perfusion with ice-cold N-methyl-d-glutamine (NMDG) containing cutting solution: 93 mM NMDG, 2.5 mM KCl, 25 mM glucose, 1.2 mM NaH2PO4, 10 mM MgSO4, 0.5 mM CaCl2), 30 mM NaHCO3, 5 mM Na ascorbate, 3 mM Na pyruvate, 2 mM thiourea and 20 mM HEPES pH 7.3-7.4. Slices were incubated for 12 min at 34° C., and then were transported to room temperature oxygenated artificial cerebrospinal fluid (ACSF) solution: 124 mM NaCl, 2.5 mM KCl, 24 mM NaHCO3, 2 mM CaCl2), 2 mM MgSO4, 1.2 mM NaH2PO4, 12.5 mM glucose and 5 mM HEPES pH 7.3-7.4.
Current clamp measurements were performed as described in the in vitro electrophysiology section. Briefly, 585 nm light with 5 Hz frequency and 0.7 mW/mm2 intensity was used at varying pulse-width values (in ms) of 0.5, 1, 2, 5 and 10 to test pulse width, and 585 nm light with 5 Hz frequency and 5 ms pulse-width was used at varying light power densities (in mW/mm2) of 0.003, 0.01, 0.03, 0.1, 0.3, 0.7, and 1.0. For spike fidelity experiments, 585 nm light with 0.7 mW/mm2 power density was used, with 1 ms pulse-width for ChRmine variants. Data collection across opsins was randomized and distributed to minimize across-group differences in expression time, room temperature, and related experimental factors.
In Vitro Characterization Preparatory to all-Optical Set-Up
Dissociated hippocampal neurons were cultured and infected with both red-shifted opsin variants and XcaMP-G or XcaMP-B as previously described (Marshel et al., 2019). One microliter viral suspension of WT ChRmine (AAV8-CaMKIIα-ChRmine-oScarlet-Kv2.1, 1.3e13 vg/mL), rsChRmine (AAV8-CaMKIIα-rsChRmine-oScarlet-Kv2.1, 8.8e12 vg/mL), or hsChRmine (AAV8-CaMKIIα-hsChRmine-oScarlet-Kv2.1, 1.8e13 vg/ml) mixed with 1 μL XcaMP-G (AAV8-CaMKIIα-XcaMP-G, 6.9e12 vg/mL)) or 1 μL XcaMP-B (AAV8-CaMKIIα-XcaMP-B, 2.4e13 vg/mL) was added after 5 DIV. Cultured neurons were used between 12 and 14 DIV for experiments. Coverslips of cultured neurons were transferred from the culture medium to a recording bath filled with Tyrode's solution containing (129 mM NaCl, 5 mM KCl, 30 mM glucose, 25 mM HEPES-NaOH pH 7.4, 1 mM MgCl2 and 3 mM CaCl2)) supplemented with 10 μM CNQX and 25 μM APV to prevent contamination from spontaneous and recurrent synaptic activity. Optical stimulation and imaging were performed using a 40×/0.6-NA objective (Leica), sCMOS camera (Hamamatsu, ORCA-Flash4.0) and LED light source (Spectra X Light engine, Lumencor), all coupled to a Leica DMI 6000 B microscope. XcaMP-B or XcaMP-G were excited by 390 nm (Semrock, FF01-390/18) or 488 nm (Semrock, LL01-488-12.5), respectively, with the Spectra X Light engine. XcaMP-B emission was reflected off a quad wavelength dichroic mirror (Semrock, FF409/493/573/652-Di02) for various color light stimulation, and passed through a triple-band emission filter (Semrock, FF01-432/523/702-25). XcaMP-G emission was reflected off a dual wavelength dichroic mirror (Chroma, ZT488/594rpc) for orange light stimulation or another mirror (ZT488/640rpc) for red light stimulation, and passed through a 535-30-nm emission filter (Chroma, ET535/30m). Red-responsive opsins were activated with a Spectra X Light engine filtered either with 585 nm orange light (Semrock, FF01-585/29-25, 2.0 mW/mm2) or 635 nm red light (Semrock, FF01-635/18-25, 2.0 mW/mm2). For light sensitivity experiments, 434 nm blue light (Semrock, 434/17), 488 nm cyan light (Semrock, LL01-488-12.5), or 570 nm green light (Chroma, HQ570/20m) with 400 ms pulse-width was used at varying light power densities (in mW/mm2) of 0.013, 0.066, 0.30 and 1.0.
Fluorescence of XcaMP-B or XcaMP-G was imaged using low-intensity 385 nm (10 μW/mm2) or 488 nm (8 μW/mm2) laser light, respectively, without substantially activating red-responsive opsin. Images were acquired at 20 Hz using MicroManager (http://micro-manager.org). Light for stimulation was controlled by LabVIEW (National Instruments) and applied every 10 sec at an exposure time of 10, 50, 200 and 800 msec. Imaging data were analyzed in MATLAB (MathWorks). Circular regions of interest (ROIs) were drawn manually based on the averaged image. We performed background subtraction before calculating Ca2+ signals. ΔF/F responses were calculated to normalize the signal in each ROI, by dividing by its mean value of total fluorescence intensity and subtracting 1. Noise was calculated as the standard deviation of the total ΔF/F fluctuation 3 sec before the stimulation. Signal-to-noise ratio (SNR) was then computed as ΔF/F response divided by noise. Peak amplitude was calculated from the maximum value during 2 sec after stimulus cessation. To compare red-responsive opsins to triggered XcaMP-G kinetics, we calculated 200 msec exposure-triggered Ca2+ transients. Rise time (tpeak) was defined as the time-to-peak from the cessation of the light stimulus to the time point at which maximal-amplitude fluorescence was reached. The decay constants (tau) were determined by single-exponential fit from the peak of the fluorescence response for 2 sec after stimulation.
We collected bulk fluorescence from targeted brain regions using a single optical fiber while delivering excitation light for fiber photometry as described previously (Inoue et al., 2019; Kim et al., 2016). We have extended these methods to the case of dual excitation wavelengths (380 and 470 nm) with stimulation wavelength (590, 720, or 750 nm) delivered through the same fiber to allow tracking of activity in distinct cell populations (sender and receiver) during optogenetic stimulation of the sender population. A low fluorescence 400-mm-diameter 0.66-NA mono fiberoptic cannula (Doric Lenses) was implanted above mPFC for fiber photometry. Cannulas were secured to the skull using a base layer of adhesive dental cement (C&B-Metabond, Parkell), followed by a second layer of cranioplastic cement (Ortho-Jet, Lang). Experiments were conducted 4-6 weeks later for FIP recordings to allow for sufficient viral expression and postsurgery recovery. One end of the patchcord terminated in an SMA connector (Thorlabs, SMISMA) mounted at the working distance of the objective, and the other end terminated in 2.5-mm-diameter stainless steel ferrules. These ferrules were coupled via bronze sleeves (Doric, SLEEVE_BR_2.5) to ferrules implanted into a mouse. Fiber faces were imaged through a 20×/0.75-NA objective (Nikon, CFI Plan Apo Lambda 20×) through a series of reconfigurable dichroic mirrors.
In the standard configuration, the three LEDs (M385F1, M470F3, and M595F2, Thorlabs) were filtered with 380-14 nm, 473 nm, and 586-20 nm bandpass filters (FF01-380/14-25, LL01-473-25, and FF01-586/20-25, Semrock). Excitation and optogenetic stimulation light from two sources (470 and 590 nm) was passed to a 525 nm longpass dichroic mirror (T525lpxr, Chroma), and then combined with 380 nm light using a second 425 nm longpass dichroic (T425lpxr, Chroma) before finally being coupled into the optical fiber patch cord using a triple multiband dichroic (69013bs, Chroma). Fluorescence emission passed through multi-bandpass fluorescence emission filter (Semrock, FF01-425/527/685-25) for XcaMP-B and GcaMP6 recording. 575 nm shortpass filter (Edmund, 575 nm 25 mm diameter, O.D. 4.0 Shortpass filter) was directed into the tube lens to minimize direct LED emission detected by the camera. Imaging optical powers of 380 nm and 470 nm were used at the far end of the patch cord at 5 μW and 2.5 μW, respectively. The fluorescence image was focused onto the sensor of a sCMOS camera (Hamamatsu, ORCA-Flash4.0) through a tube lens (Thorlabs, AC254-035-A-ML).
A custom MATLAB (Mathworks, Natick, MA) GUI was written to control the sample illumination protocol as well as provide power modulation pulses to the LEDs (National Instruments, NI PCIe-6343-X) which temporally align the respective LED illumination with camera frame acquisition (HCImage, Hamamatsu). The generic illumination protocol would repeat a sequence of three-frame sampling periods: one isosbestic at 380 nm, one signal at 470 nm and one optogenetic at >470 nm (
To quantify spectral cross-excitation of the opsin from signal illumination, the 470 nm LED was additionally pulsed during the optogenetic sampling period. The pulse duration of this additional illumination was matched to the signal pulse width (23 ms). The minimum excitation power for the sweep was equal to that used for the signal pulse (2.5 μW). The digital camera acquired data at a total of 30 Hz. Therefore, due to the sequential 3-frame sampling protocol, the isosbestic and signal samples were each acquired at 10 Hz and all optogenetic stimulation would similarly occur at a rate of 10 Hz. The duration of this 10 Hz optogenetic stimulation was 2 seconds. To quantify the excitation efficiency of the opsin to orange light at 594 nm, the associated LED was pulsed during the optogenetic sampling period (10 ms pulse width). For light-intensity sweeps, four samples at each power were randomly interleaved with a random ITI between 20 and 30 seconds. Optogenetic excitation in the NIR window at 720 nm and 750 nm were separately characterized using this same protocol (Inoue et al., 2019; Kim et al., 2016). For 720 nm optogenetic stimulation, the 594-nm LED was replaced with a 730-nm LED (M730L5, Thorlabs). The 730-nm laser was filtered with a 716-43 nm bandpass filter (Semrock, FF01-716/43-25). For 750 nm optogenetic stimulation, the 594-nm LED was replaced with a 750-nm laser (CivilLaser). The 750-nm laser was filtered with a 750-10 nm bandpass filter (Thorlabs, FB750-10).
The Pyr-PV impulse response data were acquired using the same optical configuration. A 594 nm LED was delivered using 10 ms pulse width and 1 mW of power. The pulse frequency (1, 2, 5, 10, 20 Hz) and pulse number (10, 20, 30, 40, 60, 80, 120) were controlled by TTL signals delivered by a microcontroller (Arduino, Uno) communicating with MATLAB (MathWorks). Four samples at each frequency and number were randomly interleaved with an ITI 30 seconds.
The fluorescence signal was calculated with custom written MATLAB scripts. We fit a double exponential to a thresholded version of the fluorescence time series and subtracted the best fit from the unthresholded signal to account for slow bleaching artifacts. Fluorescence signal was normalized within each mouse by calculating the ΔF/F as (F−baseline (F))/baseline (F), where the baseline was taken from the average during 5 s before optogenetic stimulation. Peak ΔF/F amplitude was calculated from the maximum value during 2 s after the stimulus cessation. Noise was calculated as the standard deviation of the ΔF/F fluctuation during 5 s before optogenetic stimulation. Signal-to-noise ratio (SNR) response was then computed as ΔF/F response divided by noise. Every measurement point (light intensity and wavelength) represents the average of four trials at 20-30 second intervals. The optical EPD50 in
To analyze the expression pattern of opsin and GcaMP, immunohistochemistry was performed in brain tissue removed from virus-injected mice. Animals were anesthetized and transcardially perfused with ice-cold 1×PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were dissected, post-fixed in the same fixatives overnight at 4° C. Tissues were cut into 60-μm-thick slices with a vibratome (Leica, VT1000) and floated in PBS. For immunohistochemistry, brain slices were blocked with 3% normal donkey serum/0.3% Triton X-100/PBS and incubated with primary antibody diluted in the blocking buffer at 4° C. overnight on a shaker. The antibody used was mouse monoclonal anti-HA tag (1:500, Fisher Scientific A26183). After washing with 0.3% Triton X-100/PBS, tissue sections were incubated with the secondary antibody, Alexa Fluor 647-conjugated donkey anti-mouse antibody (1:500, A-31571, Thermo Fisher Scientific) and DAPI for 2 h at R.T. and mounted on slides in a tissue-mounting medium containing anti-fade, Polyvinyl alcohol mounting medium with DABCO (Millipore Sigma). Confocal imaging of GcaMP fluorescence, HA antibody staining for localization of the opsin, and DAPI for cytoarchitecture was performed using a Leica TCS SP8 or TCS SP5 confocal scanning laser microscope with a 10×/NA-0.4 or 25×/NA-0.95 water objective. Co-localization was performed using 25× images by annotating GcaMP6m expressing cell body locations and then overlaying these annotations and verifying expression in the anti-HA image. Quantitative analysis of GcaMP expression level of individual mice was performed using 10× image (5-6 z slices at 3 μm intervals through each section) by annotating GcaMP6m expression. The fluorescence intensity of GcaMP6m was quantified from the slice with the highest fluorescence intensity by setting up a 400 μm square ROI directly under the fiber tract using ImageJ (NIH).
For the electrophysiology experiments, pClamp 10.6 (Molecular Devices), Python, and Prism 7 (GraphPad) software were used to record and analyze data. Non-parametric tests (Wilcoxon rank-sum test and the signed rank test) were used for singular comparisons. For multiple comparisons, Kruskal-Wallis test was performed and was followed by Dunn's test for post-hoc comparisons. The peak photocurrent was identified as the largest difference in current in the interval from laser onset to laser offset. Tau-off was calculated by fitting a mono exponential curve to the waveform from laser offset to the baseline. Time-to-peak was calculated by measuring the time difference between laser onset and peak current.
To calculate action spectra, we first normalized photocurrents to the peak photocurrent for each cell. Then, these normalized spectra were averaged across cells to produce the action spectra for each opsin variant in both one-photon and two-photon measurements. To calculate EPD50, photocurrents were first normalized to the photocurrent elicited at the highest light power. Linear interpolation was then used to infer the light power level that produced 50% of the max photocurrent.
The unusual properties of ChRmine have opened up new avenues of investigation for optogenetics in the study of cell-specific activity within biological systems (Chen et al., 2021; Marshel et al., 2019); alongside extremely-large photocurrents, red-shifted actuation, and light sensitivity (Marshel et al., 2019), ChRmine exhibits virtually no Ca2+ conductance (
Arginine conformation in the dark state and the function of ion-transporting rhodopsins. R82 in HsBR is highly conserved among microbial rhodopsins, but two different conformations are seen in the dark state: outward-facing and parallel. In all structurally-resolved channelrhodopsins (C1C2 (PDB ID: 3UG9) (Kato et al., 2012), CrChR2 (PDB ID: 6EID) (Volkov et al., 2017), C1Chrimson (PDB ID: 5ZIH) (Oda et al., 2018), GtACR1 (PDB ID: 6CSM) (Kim et al., 2018), and ChRmine), the arginine residue faces outward. In contrast, in many ion-pumping rhodopsins, including HsBR (PDB ID: 5ZIM) (Hasegawa et al., 2018), HwBR (PDB ID: 4QID), cruxrhodopsin-3 (PDB ID: 4JR8) (Chan et al., 2014), deltarhodopsin (PDB ID: 4FBZ) (Zhang et al., 2013), GR (PDB ID: 6NWD) (Morizumi et al., 2019), Archaerhodopsin-1 (PDB ID: 1UAZ) (Enami et al., 2006), Archaerhodopsin-2 (PDB ID: 2EI4) (Yoshimura and Kouyama, 2008), PR from the Mediterranean Sea at a depth of 12 m (Med12BPR, PDB ID: 4JQ6) (Ran et al., 2013), PR from the Pacific Ocean near Hawaii at a depth of 75 m (HOT75BPR, PDB ID: 4KLY) (Ran et al., 2013), CsR (6GYH) (Fudim et al., 2019), HsHR (PDB ID: 1E12) (Kolbe et al., 2000), NpHR (PDB ID: 3A7K) (Kouyama et al., 2010), CiR (PDB ID: 5ZTK) (Yun et al., 2021), KR2 (PDB ID: 3X3C) (Kato et al., 2015), and schizorhodopsin 4 (PDB ID: 7E4G) (Higuchi et al., 2021), the tip of arginine runs parallel to the membrane and faces TM1. The arginine in the parallel conformation narrows or blocks the extracellular cavity of the ion-translocating pathway; thus, this conformation would contribute to preventing large ion flux in ion-pumping rhodopsins.
Notably, CsR (the outward proton-pumping rhodopsin from Coccomyxa subellipsoidea), also has arginine (R83) in the parallel conformation in the dark state (Fudim et al., 2019), and R83Q mutation or mutation of the adjacent tyrosine (Y57K) converts functionality from proton pump to proton channel (Vogt et al., 2015). Moreover, computational analysis of HsBR with
R82Q or Y57K mutation reveals that these mutations significantly change the conformation of R82Q or R82, respectively; most notably, R82 faces outward in the Y57K simulation (Vogt et al., 2015). These observations suggest that the conformation of the arginine in the dark state is one of the structural elements distinguishing channel- and pump-type rhodopsins. Interestingly, previous studies have reported that the arginine of some ion-pumping rhodopsins is maintained in the parallel conformation during the photocycle (Kouyama et al., 2015; Kovalev et al., 2020), but the corresponding arginine in HsBR transiently changes from parallel to outward-facing to facilitate proton release to the extracellular solvent (Kuhlbrandt, 2000; Nango et al., 2016). Since channelrhodopsins presumably evolved from ion-pumping rhodopsins (Inoue et al., 2015), these studies suggest that mutations accumulated near the arginine of ion-pumping rhodopsins gradually stabilized the outward-facing conformation; these rearrangements enlarged the extracellular cavity, enabling the large ion flux of channelrhodopsins.
The conformational change of the monomer pore during simulation: In addition to the opening of the trimer pore, we also observed that the size of the monomer pore increased during the light state simulation (
Despite similarities to HsBR, ChRmine exhibits several atypical properties in its high-resolution structure, including its long twisted ECL1. ECL1 not only significantly distorts the architecture around the Schiff base but also enlarges the extracellular cavity within the monomer. Interestingly, the length and sequence of ECL1 are not highly conserved in this subfamily, and cation selectivity has been reported to be different between pump-like ChRs that we would predict to have long vs. short ECL1 domains (Sineshchekov et al., 2020). Furthermore, we report that ChRmine (with its long ECL1) is remarkably selective for monovalent cations (especially K+;
Proton donor and acceptor: In HsBR, D85 receives a proton from the protonated Schiff base and releases it to the extracellular bulk solvent. D96 receives a proton from the intracellular bulk solvent and provides it to the deprotonated Schiff base. These proton movements generate net flow of proton from the intracellular to extracellular side; these two functionally important residues, together with T89, are called DTD motif.
In GtCCR2, a ChRmine homolog in the BCCR family, both D85 and D96 are conserved (D87 and D98, respectively) but the proposed proton translocation pathway is completely different; GtCCR2 does not show outward proton-pumping activity (Sineshchekov et al., 2017), and the proton is shuttled back and forth between the Schiff base and D85. While the deprotonation and re-protonation of D98 are assumed to occur and the deprotonation would be necessary for the channel gating, D98 never gives the proton to the deprotonated Schiff base (Sineshchekov et al., 2017). If some channelrhodopsins retain residual pumping activity (Feldbauer et al., 2009), D85 homologs presumably could release a proton to the extracellular bulk solvent after receiving a proton from the Schiff base. However, in contrast to the proton acceptor, it remains elusive which residue works as a proton donor to the deprotonated Schiff base. D96 in HsBR is also conserved in ChRmine (D126), but is exposed to intracellular bulk solvent in our structure, and the calculated pKa of D126 is as low as 6.28. D126 may be unlikely to work as the sole proton donor (
Indeed, our current structural information of ChRmine has already provided a framework for further development of ChRmine-based optogenetic tools: hs, rs, and frChRmine (
Published structures of ChRs were experimentally determined only using crystallography. However, we find that the combination of antibody and single-particle cryo-EM techniques (Wu et al., 2012) is powerful enough to determine the high-resolution structure of small proteins like ChRmine, thus representing a new and promising option for structural analysis of microbial rhodopsins alongside X-ray crystallography. The two technologies, as well as structure prediction methods, may complement each other and thus expedite structural biology of microbial rhodopsins, and the resulting information will lead to both further development of optogenetics and basic mechanistic understanding of these remarkable photoreceptor proteins.
A small population of early intermediates: initial structural changes in ChRmine: Our 2.0 Å cryo-EM map allowed accurate modeling of ATR and surrounding residues, but the C13 and C14 atoms of ATR and W223 showed weaker density in the region. Moreover, positive and negative Fo−Fc difference densities were observed around W223, suggesting that this cryo-EM density map contains information on a small population of early intermediate states (possibly the K intermediate; (
Further structural work of value would include molecular dynamics simulations and structures of the intermediate states. Investigating cooperativity/allostery properties linking different monomers within the trimer could not only help tune light-response properties of optogenetic tools, but also could illuminate the basic science of ChR origins, including the conversion from trimer to dimer assembly-logic during the evolutionary separation of non-pump-like ChRs from the presumably pump-like ancestors. Recent discoveries and applications using optogenetics may be extended with these ChRmine variants to a broad range of opportunities, capitalizing upon the remarkably high photocurrents and light-sensitivity of the parent opsin, and the improved properties of the variants (Bansal et al., 2021; Sahel et al., 2021). Structural insight and structure-guided design of microbial opsins continue to open pathways for discovery and understanding, extending now to the third of the three major known ChR types: cation-conducting (Kato et al., 2012), anion-conducting (Kim et al., 2018; Kato et al., 2018), and pump-like channelrhodopsins.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art considering the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase “means for” or the exact phrase “step for” is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. § 112(6) is not invoked.
This application claims priority to U.S. Provisional Application No. 63/302,419 (filed Jan. 24, 2022), which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/061091 | 1/23/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63302419 | Jan 2022 | US |
