Engineered red-shifted channelrhodopsin variants
View Patent ↗The invention provides engineered red-shifted channelrhodopsin variants. In some embodiments, the channelrhodopsin variants are characterized by improved membrane trafficking, expression, and/or unique spectral and kinetic properties.
1. A polypeptide comprising a channelrhodopsin-1 (ChR1) domain, a Volvox carteri channelrhodopsin-1 (VChR1) domain and a Volvox carteri channelrhodopsin-2 (VChR2) domain, wherein said polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
2. The polypeptide of claim 1 , wherein the polypeptide has the structure:
X 1 -X 2 -X 3 -X 4
wherein X 1 is a ChR1 domain,
X 2 is a first VChR1 domain,
X 3 is a VChR2 domain and
X 4 is a second VChR1 domain.
3. The polypeptide of claim 2 , wherein one or more of the domains X 1 , X 2 , X 3 , and X 4 comprises 1, 2, 3, 4, or 5 amino acid substitutions relative to a corresponding wild-type domain.
4. The polypeptide of claim 3 , wherein the amino acid substition is at one or more positions selected from 163, 171, 174 and 266.
5. The polypeptide of claim 3 , comprising one or more amino acid substitutions selected from Glu163Thr, Leu171Ile, Leu171Val, His174Arg, and Phe266Tyr.
6. The polypeptide of claim 1 further comprising a fluorescent polypeptide.
7. The polypeptide according to claim 1 , comprising an L171I amino acid substitution.
8. The polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO:1.
9. The polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO:4.
10. The polypeptide of claim 1 , wherein the polypeptide provides a photocurrent of at least 10 pA/pF upon photostimulation with light having a wavelength of at least 600 nm in HEK293 cells.
11. The polypeptide of claim 1 , wherein the ratio of membrane to cytosol localization of the polypeptide is at least 1.0, when expressed in an HEK293 cell.
12. The polypeptide of claim 1 , further comprising a fluorescent protein fused to the C-tertninus.