Engineered light-activated anion channel proteins and methods of use thereof
Aspects of the disclosure include compositions, devices, systems and methods for optogenetic modulation of action potentials in target cells. The subject devices include light-generating devices, control devices, and delivery devices for delivering light-responsive polypeptides, or nucleic acids encoding same, to target cells. The subject compositions and systems include light-activated polypeptides, nucleic acids comprising nucleotide sequences encoding these polypeptides, as well as expression systems that facilitate expression of these polypeptides in target cells. Also provided are methods of using the subject devices and systems to optogenetically manipulate action potentials in target cells, e.g., to treat a neurological or psychiatric condition in a human or animal subject.
1. A light-activated polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:1, wherein the polypeptide functions as a light-activated anion channel, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from the group consisting of T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and N297Q, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
2. The polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1.
3. The polypeptide of claim 1 , comprising an endoplasmic reticulum (ER) export polypeptide.
4. The polypeptide of claim 3 , wherein the ER export polypeptide comprises the amino acid sequence FXYENE (SEQ ID NO:92), where X is any amino acid.
5. The polypeptide of claim 1 , comprising a membrane trafficking polypeptide.
6. The polypeptide of claim 5 , wherein the membrane trafficking polypeptide comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:83).
7. The light-activated polypeptide of claim 1 , wherein the polypeptide further comprises a C167T, C167A, or C167S substitution, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
8. The light-activated polypeptide of claim 1 , wherein the polypeptide further comprises a D195A or a D195N substitution, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
9. The light-activated polypeptide of claim 1 , wherein the polypeptide comprises T98S, E129S, E140S, E162S, and T285N substitutions, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
10. The light-activated polypeptide of claim 1 , wherein the polypeptide comprises V156K, H173R, V281K and N297Q substitutions, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
11. The light-activated polypeptide of claim 1 , wherein the polypeptide comprises T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and N297Q substitutions, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
12. The light-activated polypeptide of claim 11 , wherein the polypeptide further comprises a C167T substitution, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
13. The light-activated polypeptide of claim 11 , wherein the polypeptide further comprises a C167A substitution, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
14. The light-activated polypeptide of claim 1 , wherein the first 50 N-terminal amino acid residues are replaced by a peptide having the amino acid sequence MDYGGALSAVG (SEQ ID NO:82), relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
15. A nucleic acid comprising a nucleotide sequence encoding a light-activated polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:1, wherein the polypeptide functions as a light-activated anion channel, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from the group consisting of T98S, E129S, E140S, E162S, V156K, H173R, T285N, V281K and N297Q, relative to the amino acid sequence of C1C2 (SEQ ID NO:78).
16. A recombinant expression vector comprising the nucleic acid of claim 15 .
17. A pharmaceutical composition comprising:
a) the recombinant expression vector of claim 16 ; and
b) a pharmaceutically acceptable carrier.
18. A cell comprising the nucleic acid of claim 15 .
19. A cell comprising the recombinant expression vector of claim 16 .
20. A system for modulating the membrane potential of a cell, the system comprising:
a) the recombinant vector of claim 16 ; and
b) a device configured to illuminate a target location with light.
21. The system of claim 20 , wherein the device is configured to illuminate the target location with light having a wavelength ranging from about 350 nm to about 750 nm, or from about 450 nm to about 500 nm.
22. The system of claim 20 , wherein the device is configured to:
a) constantly illuminate the target location with light;
b) illuminate the target location with pulses of light;
c) modulate the wavelength and/or the intensity of the light;
d) modulate the frequency and/or the duration of the pulses of light; or
e) illuminate the target location in response to a user input.
23. The system of claim 20 , wherein the device is configured to illuminate the target location in response to a user input, and wherein the user input comprises: the wavelength of light, the intensity of light, the duration of a light pulse, the frequency of a light pulse, and/or the target location.
24. The system of claim 20 , wherein the device is adapted to be implanted in a subject.
25. The system of claim 20 , wherein the target location is: a cell, a portion of a cell, a plurality of cells, a bundle of nerve fibers, a neuromuscular junction, a central nervous system (CNS) tissue, a peripheral nervous system (PNS) tissue, or an anatomical region.
26. A method for modulating the membrane potential of a cell in response to light, the method comprising exposing a cell to light of an activating wavelength, wherein the cell is genetically modified with the recombinant vector of claim 16 .
27. The method of claim 26 , wherein the cell is a neuron.