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

• Published in: The Journal of biological chemistry 2013-10, Vol.288 (41), p.29911-29922
• Main Authors: Govorunova, Elena G., Sineshchekov, Oleg A., Li, Hai, Janz, Roger, Spudich, John L.
• Format: Article
• Language: English
• Subjects: Algae; Algal Proteins - genetics; Algal Proteins - metabolism; Algal Proteins - physiology; Animals; Cells, Cultured; Channelrhodopsin; Chlamydomonas reinhardtii - genetics; Chlamydomonas reinhardtii - metabolism; Chlorophyta - genetics; Chlorophyta - metabolism; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Ion Channels; Ion Channels - genetics; Ion Channels - metabolism; Ion Channels - physiology; Ion Transport - physiology; Light; Marine Biology; Membrane Biology; Membrane Potentials - physiology; Membrane Potentials - radiation effects; Neurons - metabolism; Neurons - physiology; Optogenetics; Photobiology; Photoreceptors; Phototaxis; Rats; Rats, Sprague-Dawley; Rhodopsin - genetics; Rhodopsin - metabolism; Rhodopsin - physiology; Sodium - metabolism; Spectrometry, Fluorescence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.505495

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 PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR 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 (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λmax of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pKa 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 CrChR2. PsChR 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 PsChR than with CrChR2 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 PsChR 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 (PsChR) are characterized. Conclusion: Blue-shifted PsChR has ∼3-fold greater unitary conductance, faster recovery from excitation, and higher sodium selectivity than channelrhodopsin 2 from Chlamydomonas. Significance: These properties of PsChR facilitate further analysis of light-gated channel function and are potentially useful for optogenetics.