Characterization of engineered channelrhodopsin variants with improved properties and kinetics.

By John Y. Lin, Michael Z. Lin, Paul A. Steinbach, Roger Tsien. Biophysical Journal, 2009

709 citations recorded by the source corpus.

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.

Subfields: Biomedical Engineering, Cellular and Molecular Neuroscience

View Paper: https://www.semanticscholar.org/paper/e62af4f545a220c215c58cbd27c5f58603669a23

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In Biomedical Engineering

In Cellular and Molecular Neuroscience

Citations in the corpus

Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins (2011, neutral): “and chimeras constructed by combining channelrhodopsin”
Ultrafast optogenetic control (2010, background): “However, spike failures are also a major issue; previously, we and others have reported that optical stimulation with ChR2 in excitatory neurons does not reliably evoke spikes at frequencies above the gamma (40 Hz) rang”
Molecular and cellular approaches for diversifying and extending optogenetics. (2010, background): “…strategies for eliciting from microbial opsins increased light sensitivity (Berndt et al., 2009), increased photocurrent magnitude (Gradinaru et al., 2007, 2008; Zhao et al., 2008), faster kinetics (Gunaydin et al., 2010; Lin et al., 2009), and bistable switching behavior (Berndt et al., 2009).”
The Development and Application of Optogenetics (2011, methodology): “Next, chimera strategies (using hybrids of ChR1 and ChR2) were found to be helpful in giving rise to improved expression and spiking properties for channelrhodopsins (Lin et al. 2009, Wang et al. 2009).”
All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins (2014, methodology): “E) Under continuous illumination CheRiff partially desensitized with a time constant of 400 ms. ChR2 H134R and ChIEF desensitized significantly faster (39 ± 4 ms, n = 8 cells, P 0.001, and 49 ± 8 ms, n = 5 cells, P 0.001, respectively).”