Abstract
Channelrhodopsin-2 is a light-gated ion channel and a major tool of optogenetics. It is used to control neuronal activity via blue light. Here we describe the construction of color-tuned high efficiency channelrhodopsins (ChRs), based on chimeras of Chlamydomonas channelrhodopsin-1 and Volvox channelrhodopsin-1. These variants show superb expression and plasma membrane integration, resulting in 3-fold larger photocurrents in HEK cells compared with channelrhodopsin-2. Further molecular engineering gave rise to chimeric variants with absorption maxima ranging from 526 to 545 nm, dovetailing well with maxima of channelrhodopsin-2 derivatives ranging from 461 to 492 nm. Additional kinetic fine-tuning led to derivatives in which the lifetimes of the open state range from 19 ms to 5 s. Finally, combining green- with blue-absorbing variants allowed independent activation of two distinct neural cell populations at 560 and 405 nm. This novel panel of channelrhodopsin variants may serve as an important toolkit element for dual-color cell stimulation in neural circuits.
Highlights
Dual-color activation of two cell types with channelrhodopsins is a major challenge because all available well expressing variants absorb blue light
We showed that the earlier reported kinetic tuning of C2 can be transferred to the new C1V1 chimera, the effects of individual mutations differ quantitatively and substantially from related mutations in C2
It was conceivable that the 39 extra amino acids in the N-terminal region of C1 contain sequences or even single amino acids that are responsible for better membrane integration or folding, both of the
Summary
Dual-color activation of two cell types with channelrhodopsins is a major challenge because all available well expressing variants absorb blue light. Despite the wide application of ChR, the use of channelrhodopsin-2 (C2) bears several limitations that often prevent sufficient depolarization in optogenetic studies These are, for example, low expression levels, small unitary conductance, inappropriate kinetics, partial inactivation, and inappropriate ion selectivity. Advanced optogenetics will depend on actuator probes, and on the simultaneous use of reporter proteins as genetically encoded fluorescent calcium indicators or voltage sensors To meet this challenge, we implemented a systematic molecular engineering approach, integrating helix swapping as global rearrangement of structural elements with subsequent mutagenesis resulting in local conformational changes or alteration of the hydrogen bond network. The recently described threedimensional structure of a distinct C1C2 chimera allows to interpret our results on a molecular level [22]
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