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.