Light-activated chimeric opsins and methods of using the same

Abstract

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 .- 51 . (canceled) 
     
     
         52 . A polynucleotide comprising a nucleotide sequence encoding a light-responsive chimeric polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:7. 
     
     
         53 . The polynucleotide of  claim 52 , wherein the light-responsive chimeric polypeptide comprises a C-terminal trafficking signal. 
     
     
         54 . The polynucleotide of  claim 53 , wherein the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:15). 
     
     
         55 . The polynucleotide of  claim 52 , wherein the nucleotide sequence is operably linked to a promoter. 
     
     
         56 . The polynucleotide of  claim 52 , wherein the polynucleotide further comprises a nucleotide sequence encoding a second light-activated polypeptide. 
     
     
         57 . The polynucleotide of  claim 56 , 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. 
     
     
         58 . 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 an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:7.   
     
     
         59 . The method of  claim 58 , 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. 
     
     
         60 . The method of  claim 58 , wherein the excitatory neuron comprises a pyramidal neuron and the inhibitory neuron comprises a parvalbumin neuron. 
     
     
         61 . The method of  claim 58 , wherein the inhibitory neuron or the excitatory neuron is present in the prefrontal cortex. 
     
     
         62 . A mammalian cell comprising a polynucleotide of  claim 52 . 
     
     
         63 . The mammalian cell of  claim 62 , wherein the cell further comprises a second light-activated polypeptide present in the cell membrane, 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. 
     
     
         64 . The mammalian cell of  claim 62 , wherein the cell is a muscle cell. 
     
     
         65 . The mammalian cell of  claim 62 , wherein the cell is a stem cell. 
     
     
         66 . The mammalian cell of  claim 62 , wherein the cell is an excitatory neuronal cell. 
     
     
         67 . The mammalian cell of  claim 62 , wherein the cell is an inhibitory neuronal cell. 
     
     
         68 . A polynucleotide comprising a nucleotide sequence encoding a light-responsive chimeric polypeptide comprising 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. 
     
     
         69 . The polynucleotide of  claim 68 , wherein the light-responsive chimeric polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs:11, 12, 13, or 14. 
     
     
         70 . The polynucleotide of  claim 68 , wherein the light-responsive chimeric polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs:11, 12, 13, or 14. 
     
     
         71 . The polynucleotide of  claim 68 , wherein the light-responsive chimeric polypeptide has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:11 and is activated at a wavelength of 405 nm, 461 nm, or 470 nm.