Application of novel channelrhodopsins: molecular engineering, site-specific opsin targeting, discovery of new variants and the advancement in optogenetic devise development

Scientists from the Max-Planck-Institute of Biophysics in Frankfurt developed a mutant of ChR2, L132C that upon high-frequency blue light stimulation display excellent spike reliability and accelerated repolarisation in a Ca²⁺ dependent manner. This mutant, termed CatCh i.e. Calcium transporting Channelrhodopsin, shows a four-fold Ca²⁺ permeability compared to wildtype ChR2 and a 70-fold light sensitivity of fast spiking hippocampal neurons. The superior properties of CatCh result from the enhanced Ca²⁺ permeability. Increased [Ca²⁺]i elevate the internal surface potential, promoting voltage-gated Na+ channels activation and as a consequence increase light sensitivity. Repolarization following light-stimulation is significantly accelerated by Ca²⁺-dependent big potassium channel activation. Therefore, CatCh induces an increased light sensitivity in neurons with much accelerated response kinetics as a result of increased Ca²⁺ permeability and subsequent consequences on neuronal excitation. Due to its minimal light requirements CatCh spikes can be generated far from its spectral maximum of 474 nm e.g. with green light facilitating tissue penetration. The CatCh expression level is excellent in many different cell types and has already been applied in mice and macaque in the context of vision restoration.

CatCh exemplifies a new principle by which blue light-gated channels can be engineered to increase the light sensitivity of neuronal stimulation. The crucial characteristics of triggering precise and fast action potentials while requiring low light intensities for activation pave the way for gene-therapeutic visual restoration effort and other biological applications such as Ca²⁺ dependencies in cell organelles.

The priority application was filed September 2010 (EP11760432). The patent has been granted in EP, US, AU, CA, CN, IL, JP, MX, RU, SG and KR.

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