Modified channelrhodopsin
View Patent ↗An object of the present invention is to provide a modified channelrhodopsin having high ion permeability (photoreactivity). The solution is to substitute the third extracellular domain counted from the N-terminal side of three extracellular domains included in a Volvox carteri-derived channelrhodopsin by a corresponding extracellular domain of a Chlamydomonas reinhardtii -derived channelrhodopsin-2.
1. A modified channelrhodopsin, which is a polypeptide obtained by substituting the third extracellular domain counted from the N-terminal side of three extracellular domains included in a Volvox carteri-derived channelrhodopsin by a corresponding extracellular domain of a Chlamydomonas reinhardtii -derived channelrhodopsin-2.
2. The modified channelrhodopsin according to claim 1 , wherein the Volvox carteri-derived channelrhodopsin contains at least amino acids at positions 67 to 322 in the amino acid sequence represented by SEQ ID NO: 1.
3. The modified channelrhodopsin according to claim 2 , which is obtained by substituting amino acids at positions 142 to 169 in the amino acid sequence represented by SEQ ID NO: 1 by amino acids at positions 143 to 170 in the amino acid sequence of a Chlamydomonas reinhardtii -derived channelrhodopsin-1 represented by SEQ ID NO: 2.
4. The modified channelrhodopsin according to claim 3 , which is any of the following (a) to (c):
(a) a polypeptide composed of the amino acid sequence represented by SEQ ID NO: 4;
(b) a polypeptide that is composed of an amino acid sequence including deletion, substitution, addition, or insertion of one or a plurality of amino acids in the amino acid sequence represented by SEQ ID NO: 4, and that has a channelrhodopsin function; and
(c) a polypeptide that is composed of an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 4, and that has a channelrhodopsin function.
5. The modified channelrhodopsin according to claim 4 , which is a polypeptide composed of the amino acid sequence represented by SEQ ID NO: 8.
6. The modified channelrhodopsin according to claim 1 , which is obtained by substituting the sixth transmembrane domain counted from the N-terminal side of seven transmembrane domains included in the Volvox carteri-derived channelrhodopsin by a corresponding transmembrane domain of a Chloromonas oogama-derived channelrhodopsin.
7. The modified channelrhodopsin according to claim 2 , which is obtained by substituting amino acids at position 323 and downstream thereof in the amino acid sequence represented by SEQ ID NO: 1 by amino acids at position 265 and downstream thereof in the amino acid sequence of a Chloromonas oogama-derived channelrhodopsin represented by SEQ ID NO: 5.
8. The modified channelrhodopsin according to claim 6 , which is any of the following (a) to (c):
(a) a polypeptide composed of the amino acid sequence represented by SEQ ID NO: 6;
(b) a polypeptide that is composed of an amino acid sequence including deletion, substitution, addition, or insertion of one or a plurality of amino acids in the amino acid sequence represented by SEQ ID NO: 6, and that has a channelrhodopsin function; and
(c) a polypeptide that is composed of an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 6, and that has a channelrhodopsin function.
9. The modified channelrhodopsin according to claim 8 , which is a polypeptide obtained by substituting His at position 172 in the polypeptide composed of the amino acid sequence represented by SEQ ID NO: 6 by another amino acid.
10. The modified channelrhodopsin according to claim 9 , which is a polypeptide composed of the amino acid sequence represented by any of SEQ ID NOS: 9 to 12.
11. The modified channelrhodopsin according to claim 6 , which is any of the following (a) to (c):
(a) a polypeptide composed of the amino acid sequence represented by SEQ ID NO: 7;
(b) a polypeptide that is composed of an amino acid sequence including deletion, substitution, addition, or insertion of one or a plurality of amino acids in the amino acid sequence represented by SEQ ID NO: 7, and that has a channelrhodopsin function; and
(c) a polypeptide that is composed of an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 7, and that has a channelrhodopsin function.
12. A polynucleotide encoding the polypeptide according to claim 1 .
13. An expression vector comprising the polynucleotide according to claim 12 functionally linked to a promoter.
14. A cell expressing the polypeptide according to claim 1 .
15. The cell according to claim 14 , wherein the cell is a neuron.
16. A method of production of a pharmaceutical for treating a subject suffering from damage to the outer retinal layers, comprising the step of formulating the polypeptide according to claim 1 with one or more pharmaceutically acceptable carriers.
17. The method according to claim 16 , wherein the damage to the outer retinal layers is retinitis pigmentosa, age-related macular degeneration, or retinal detachment.
18. A pharmaceutical composition for treating damage to the outer retinal layers, comprising the polypeptide according to claim 1 as an active ingredient.
19. A method of production of a pharmaceutical for treating a subject suffering from damage to the outer retinal layers, comprising the step of formulating the polynucleotide according to claim 12 with one or more pharmaceutically acceptable carriers.
20. A method of production of a pharmaceutical for treating a subject suffering from damage to the outer retinal layers, comprising the step of formulating the expression vector according to claim 13 with one or more pharmaceutically acceptable carriers.
21. A pharmaceutical composition for treating damage to the outer retinal layers, comprising the expression vector according to claim 13 as an active ingredient.