Fig 1.
Expression screening of 12 different NaRs in mammalian cultured cells.
A, Confocal images of 3 strong expressed NaRs in ND7/23 cells. NaRs were tagged with eGFP at the C-terminus. Scale bar = 25 μm. B, Na+ pump currents amplitudes in ND7/23 cells at 0 mV holding potential in a whole-cell patch configuration (N = 4–10). Photocurrents were evoked with 0.8 mW/mm2 irradiance at 520 nm except for SrNaR (550 nm) and AR3 (560 nm). Data represent the mean ± SEM (standard error of the mean). ND: Not detected.
Fig 2.
Basic characteristics of pumping functions.
(A)-(C), representative photocurrent of 3 NaRs (GFP versions). Membrane voltage was held at 0 mV. 520 nm light (A, B) or 550 nm (C) light (0.8 mW/mm2) was illuminated during the time indicated by a green bar. (D) Current-voltage relation (I/V plot) of AR3 (black), FdNaR (blue), NyNaR (red) and SrNaR (purple). (E) Action spectrum, wavelength dependency of photocurrents. green: KR2, blue: FdNaR, red: NyNaR, purple: SrNaR, black: AR3. The light intensities at all wavelengths were 0.2 mW/mm2. (F) Light sensitivity of photocurrents from AR3 (black), FdNaR (blue) and NyNaR (red). Data represent the mean ± SE. G-J, H+ and Na+ transport activity of each NaR measured by a pH electrode after expression in E.coli. The cells were illuminated (0.14 mW/mm2) between 0 and 150 sec. The measurement was performed in the absence (blue) and presence (green) of CCCP.
Fig 3.
Spectroscopic characteristics of FdNaR, SrNaR and KR2.
Transient absorption spectra (left), time evolutions of transient absorption changes (middle) and photocycle schema of FdNaR (A), SrNaR (B) and KR2 (C) reconstituted in DOPC in 100 mM NaCl (pH 8.0). The results of KR2 were reported from [13]. Although the K was not observed in the results for SrNaR, it is expected to exist immediately after absorption of light as FdNaR, KR2 and all other microbial rhodopsins.
Fig 4.
Expression and functional characterization of NaR rhodopsins as optogenetic silencing tools in mammalian neurons.
(A) Confocal images of AAV-transduced neurons with different NaR pumps in comparison with the targeting-enhanced proton pump Arch-3.0 The scale bar is 10 μm. Schematic representation of fusion constructs with fluorophore and trafficking sequence (ts, and endoplasmatic export sequence (er,). (B) Typical photocurrent traces in voltage-clamp shown in green under continuous light stimulation (300 ms, 532 nm, 15–20 mW/mm2). Corresponding current-clamp recording at 20 Hz light stimulation (3 ms pulse width, 300 ms, 15–20 mW/mm2) are shown in red. (C) Upper bar diagram depicts population data of average peak photocurrent of NaR compared to AR3. Lower panel represents the amplitude of hyperpolarization relative to resting membrane potential. (D) Adaptation of peak photocurrent upon repetitive light stimulation (3 ms light pulses for 300 ms at 15–20 mW/mm2 at 2 Hz, 20 Hz and 40 Hz at different shade of red). Example traces are shown in the upper panel for FdNaR. Population data for the different constructs are shown in the lower panel (n > 5 cells). (E) Current ramps are used to evaluate optical silencing capability of neuronal activity for NyNaR. During current ramps (2 pA ms-1 for 500 ms) either continuous light (green), 40 Hz stimulation (red) or no light (blue) stimulation was applied. Efficiency of optical silencing was measured both as a shift in time of first action potential (T) and as the change in number of evoked action potentials. (F) Summary of optical silencing efficacy using the two light stimulation protocols (two-tailed Student t-test). All error bars are given as SEM (standard error of mean).