Opsin polypeptides and methods of use thereof
The present disclosure provides opsins, including variant opsins with increased activity and/or increased trafficking to the plasma membrane. The opsins are useful in therapeutic and screening applications, which are also provided.
1. An isolated polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide comprising, in order from amino terminus to carboxyl terminus:
a) an opsin 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:33;
b) a membrane trafficking signal comprising the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 10); and
c) an endoplasmic reticulum (ER) export signal comprising the amino acid sequence FXYENE (SEQ ID NO: 4), where X is any amino acid.
2. The polynucleotide of claim 1 , wherein the nucleotide sequence is operably linked to a promoter that provides for neuron-selective expression.
3. The polynucleotide of claim 1 , wherein the nucleotide sequence is codon optimized for expression in a mammalian cell.
4. The polynucleotide of claim 1 , wherein the ER export signal comprises the amino acid sequence FCYENE (SEQ ID NO:5).
5. A recombinant expression vector comprising the isolated polynucleotide of claim 1 .
6. The recombinant expression vector of claim 5 , wherein the recombinant vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
7. A mammalian cell comprising a light-activated fusion polypeptide expressed on the cell membrane, wherein the light-activated fusion polypeptide is capable of mediating a hyperpolarizing current in the cell when the cell is illuminated with light, and wherein the light-activated fusion polypeptide comprises, in order from amino terminus to carboxyl terminus:
a) an opsin 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:33;
b) a membrane trafficking signal comprising the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 10); and
c) an endoplasmic reticulum (ER) export signal comprising the amino acid sequence FXYENE (SEQ ID NO: 4), where X is any amino acid.
8. The mammalian cell of claim 7 , wherein the cell is a neuronal cell.
9. A method of modulating the voltage potential of a cell in response to a light stimulus, the method comprising exposing a cell to a light stimulus, wherein the cell that is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises, in order from amino terminus to carboxyl terminus:
i) an opsin 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:33;
ii) a membrane trafficking signal comprising the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 10); and
ii) an endoplasmic reticulum (ER) export signal comprising the amino acid sequence FXYENE (SEQ ID NO: 4), where X is any amino acid,
wherein the encoded fusion polypeptide is expressed in the cell, and wherein, in response to exposure to a light stimulus, the voltage potential of the cell is modulated.
10. The method of claim 9 , wherein the cell is a neuronal cell.
11. The method of claim 9 , wherein the cell is in vitro.
12. The method of claim 9 , wherein the cell is in vivo.
13. The method of claim 9 , wherein the light stimulus is provided by an implantable light source.
14. The method of claim 9 , wherein the light stimulus is provided by a light-emitting diode.