CAG-ChIEF-tdTOMATO AAV (Serotype 6)

Channelrhodopsins 1 and 2 (ChR1 and ChR2) of Chlamydomonas reinhardtii are small membrane channels that are directly gated by light. Of the two ChR proteins of Chlamydomonas, ChR2 has received the most attention as a neuroscience tool because heterologously expressed Chop2 naturally binds endogenous all-trans-retinal to form functional ChR2 in the mammalian nervous system, allowing experimenters to selectively stimulate genetically targeted neurons with blue light without the need for exogenous cofactors. Multiple studies have demonstrated the utility of ChR2 in mapping neural circuits, inducing synaptic plasticity, restoring vision in rhodopsin-deficient animals, and studying behavior in freely moving animals. ChR2 is generally unable to induce high-fidelity action potentials above 30 Hz because the response to subsequent light exposure is significantly reduced after the initial response due to inactivation. Reduced inactivation of ChR1 is a more desirable property of ChR because lower levels of inactivation result in more consistent responses to repeated stimulation. However, ChR1 is not sufficient to control neuronal excitability because the number of protons permeating the channel is insufficient to depolarize neurons above threshold at physiological pH. By creating chimeras of the ChR1 and ChR2 transmembrane domains, combined with site-directed mutagenesis, a ChR variant was developed, named ChEF, that exhibits significantly less deactivation during sustained light stimulation. ChEF undergoes only 33% deactivation, compared to 77% for ChR2. A point mutation of Ile170 to Val in ChEF (generating “ChIEF”) accelerates the rate of channel closure while maintaining a low rate of deactivation, resulting in more consistent responses when stimulated at frequencies above 25 Hz in HEK293 cells and cultured hippocampal neurons. In addition, these variants alter spectral response, light sensitivity, and channel selectivity. ChEF and ChIEF allow for more precise control of the timing of depolarization and can induce action potential trains that more closely resemble natural pulse patterns.