Optogenetic approaches enable remote control of excitable cell activity with unique spatiotemporal resolution. They are of key importance for a deeper understanding of neural networks and bear potential for the development of innovative medical treatments. The Institute for Auditory Neurociences develops the optical cochlear implant (oCI), which promises improved hearing restoration for the deaf. Hearing restoration by the future oCI is based on optical activation of spiral ganglion neurons via light-gated ion channels, so-called Channelrhodopins (ChRs).
We optimize ChRs by structure-guided mutagenesis to provide suitable action spectra, kinetics, and ion selectivity. We take advantage of results from time-resolved spectroscopy and high-resolution structure determination and conduct in-depth electrophysiological characterizations of promising variants. Genomic sequencing of the aquatic microbiome yields an increasing number of genes known to encode for ChRs. In order to identify advanced optogenes, we conduct biophysical characterizations of ChR variants with alterations at putative key residues.
High-frequency photostimulation of neurons at light intensities which are safely applicable in vivo remains challenging, due to the trade-off between fast channel closing kinetics and light sensitivity and the low single-channel conductance of ChRs. Robust ChR expression increases the photocurrent by increasing the number of channels but could possibly result in cytotoxicity. ChRs with enhanced plasma membrane targeting have shown to improve the success rate of fast neuronal photostimulation. Consequently, we aim for a further improvement of ChRs' plasma membrane targeting. We also plan to generate a synthetic ChR that combines high single-channel conductance and fast gating. We will employ PCR-based random mutagenesis techniques to introduce locally restricted variations in key domains for channel function. Subsequent library testing with an automated patch-clamp system in combination with optogenetic stimulation will likely fuel the development of a light-gated, large-conductance depolarizing ion channel that provides fast photoswitching at low light intensities.