Mutant Chrimson Ion Channel for Faster Optogenetic Off-Kinetics
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Solution Overview
Problem
Existing light-inducible ion channels, such as ChR2, have limitations in kinetics and light sensitivity, which hinder their application in high-frequency neuronal activities, particularly in ganglion cells and interneurons, necessitating the development of mutants with faster response kinetics and improved light-sensitivity.
Innovation Solution
Mutations at specific positions (Y261, Y268, and S267) in the helix 6 of Chrimson accelerate the off-kinetics of the channel, enhancing its response speed to match the high-frequency firing rates of neurons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If channel open time is increased to improve light sensitivity, then light sensitivity is improved, but response speed decreases
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at positions 261, 267, and 268 in helix 6 of Chrimson. These point mutations alter the channel's kinetic parameters, specifically reducing the open time constant from 24.6 ms (wild type) to 3.5-12.1 ms (mutants), thereby achieving faster response speeds while maintaining functional light sensitivity through optimized molecular structure
2Speed
If channel open time is decreased to improve response speed, then response speed is improved, but light sensitivity decreases
Solution Approach 1:
The patent resolves this contradiction through precise parameter changes in the amino acid sequence. By substituting specific residues (Y261F, Y268F, S267M) in helix 6, the channel achieves accelerated off-kinetics with τoff values of 3.5-12.1 ms while preserving adequate light sensitivity, enabling the channel to respond to high-frequency neuronal activity up to 800-1000 Hz
3Speed
If mutations are introduced to accelerate off-kinetics, then response kinetics are improved, but channel structure complexity increases
Solution Approach 1:
The patent applies local quality by introducing mutations only at three specific positions (261, 267, 268) within helix 6 of the Chrimson channel. This localized modification approach achieves accelerated off-kinetics (τoff = 3.5-12.1 ms) while maintaining the overall structural integrity and simplicity of the channel protein, avoiding genome-wide or extensive structural modifications
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The mutant light-inducible ion channels achieve response speeds up to 800 to 1000 Hz, suitable for stimulating neurons at high frequencies, demonstrated in NG108-15, HEK293, and hippocampal cells.
Implementation Method 1
The mutant light-inducible ion channel comprises an amino acid sequence which has at least 90% similarity/homology and/or at least 72% identity to the full length sequence of SEQ ID NO: 1 (Chrimson), and the mutant light-inducible ion channel only differs from its parent light-inducible ion channel by a substitution at one or more position(s) selected from the positions corresponding to Y261, Y268, and S267 in SEQ ID NO: 1, which substitution(s) accelerate(s) the off-kinetics of the mutant channel
Data Source
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AI summary
The invention relates to mutant light-inducible ion channels having improved properties as compared to the parent channel, nucleic acid constructs encoding same, expression vectors carrying the nucleic acid construct, cells comprising said nucleic acid construct or expression vector, and their respective uses, as well as non- human animals comprising the mutant light-inducible ion channel, the nucleic acid construct or the expression vector as disclosed herein.