Characterization of a Highly Efficient Blue-shifted Channelrhodopsin from the Marine Alga Platymonas subcordiformis

Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of Ps ChR, one of the phototaxis receptors in the alga Platymonas ( Tetraselmis ) subcordiformis . Ps ChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na + ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii ( Cr ChR2). Photocurrents generated by Ps ChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by Cr ChR2. Their maximal spectral sensitivity was at 445 nm, making Ps ChR the most blue-shifted channelrhodopsin so far identified. The λ max of detergent-purified Ps ChR was 437 nm at neutral pH and exhibited red shifts (p K a values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in Cr ChR2. Ps ChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with Ps ChR than with Cr ChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes Ps ChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required. Background: Channelrhodopsins are algal phototaxis receptors used in optogenetics. Results: Channel activity and photochemistry of a new channelrhodopsin ( Ps ChR) are characterized. Conclusion: Blue-shifted Ps ChR has ∼3-fold greater unitary conductance, faster recovery from excitation, and higher sodium selectivity than channelrhodopsin 2 from Chlamydomonas . Significance: These properties of Ps ChR facilitate further analysis of light-gated channel function and are potentially useful for optogenetics.