The ability to rationally convert a microbial opsin pump into a channel would validate a fundamental hypothesis in ion channel structure-function, which states that a key difference between a pump and a channel is the existence of a gate that prevents backflow of ions by passive conductance and enables active ion translocation against large gradients. This biophysical feat of rational conversion by protein engineering would be the first of its kind. Furthermore, while the activity-dependent safety of neural silencing by proton and chloride pumps has been evaluated in the literature, the creation of optogenetic reagents specifically for the purpose of activity-dependent safety is novel. Moreover, the translationally focused goal of engineering a calcium-free channelrhodopsin to reduce risk of excito-toxicity is in fact opposite to ongoing efforts in the field, which seek to augment calciumconductance to increase sensitivity. The creation of reagents, whose molecular properties are tailored for translational purposes, would represent a new direction in optogenetic reagent development, which to date have focused on pushing the limits of microbial opsin photocurrent magnitude, kinetics, and spectral properties.
