Light-activated chimeric opsins and methods of using the same
Provided herein are compositions comprising light-activated chimeric proteins expressed on plasma membranes and methods of using the same to selectively depolarize excitatory or inhibitory neurons.
1. A mammalian cell comprising a polynucleotide comprising a nucleotide sequence encoding a light-responsive chimeric polypeptide comprising, in order from N-terminus to C-terminus:
a) an amino acid sequence having at least 90% amino acid sequence identity to amino acids 1-163 of the ChR1 amino acid sequence set forth in SEQ ID NO:16; and
b) an amino acid sequence having at least 90% amino acid sequence identity to amino acids 120-300 of SEQ ID NO:17.
2. The mammalian cell of claim 1 , wherein the light-responsive chimeric polypeptide comprises a C-terminal trafficking signal.
3. The mammalian cell of claim 2 , wherein the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:15).
4. The mammalian cell of claim 1 , wherein the nucleotide sequence is operably linked to a promoter.
5. The mammalian cell of claim 1 , wherein the light-responsive chimeric polypeptide comprises a Glu to Thr amino acid substitution at position 122 relative to the amino acid sequence set forth in SEQ ID NO:16.
6. The mammalian cell of claim 1 , wherein the light-responsive chimeric polypeptide comprises a Glu to Thr amino acid substitution at position 162 relative to the amino acid sequence set forth in SEQ ID NO:16.
7. The mammalian cell of claim 1 , wherein the light-responsive chimeric polypeptide comprises a Glu to Thr amino acid substitution at position 122 and a Glu to Thr amino acid substitution at position 162 relative to the amino acid sequence set forth in SEQ ID NO:16.
8. The mammalian cell of claim 1 , wherein the cell is a neuronal cell.
9. The mammalian cell of claim 1 , wherein the cell is a muscle cell.
10. The mammalian cell of claim 1 , wherein the cell is a stem cell.
11. The mammalian cell of claim 1 , wherein the cell is an excitatory neuronal cell.
12. The mammalian cell of claim 1 , wherein the cell is an inhibitory neuronal cell.
13. The mammalian cell of claim 1 , wherein the cell further comprises a second light-activated polypeptide present in the cell membrane.
14. The mammalian cell of claim 13 , wherein the second light-activated polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs:11, 12, 13, or 14.
15. A method of depolarizing an excitatory or an inhibitory neuron, the method comprising exposing the neuron to light of a wavelength between about 540 nm to about 560 nm, wherein the neuron expresses in its cell membrane a light-responsive chimeric polypeptide comprising, in order from N-terminus to C-terminus:
a) an amino acid sequence having at least 90% amino acid sequence identity to amino acids 1-163 of the ChR1 amino acid sequence set forth in SEQ ID NO:16; and
b) an amino acid sequence having at least 90% amino acid sequence identity to amino acids 120-300 of SEQ ID NO:17.
16. The method of claim 15 , wherein the neuron is an excitatory neuron present in a microcircuit, and where the method further comprises expressing a second light-activated polypeptide in an inhibitory neuron, wherein the second light-activated polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs:11, 12, 13, or 14, and wherein the inhibitory neuron is exposed to light of a second wavelength.
17. The method of claim 15 , wherein the excitatory neuron is present in the prefrontal cortex.
18. The method of claim 15 , wherein the excitatory neuron comprises a pyramidal neuron.
19. The method of claim 15 , wherein the inhibitory neuron is present in the prefrontal cortex.
20. The method of claim 15 , wherein the inhibitory neuron comprises a parvalbumin neuron.