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Figure 1. Molecular design of eKR2 and its spectral characteristics. (a) KR2 variants with different modifications at the N- or/and C- terminus to improve membrane targeting. (b) Representative confocal images (0.5-µm equatorial slices) of ND7/23 cells expressing the wild type (top) and the best construct eKR2 (bottom) showing the fluorescence of the fusion protein in green (left), the membrane marker in magenta (middle), and their co-localization in white (false colors); scale bar 10 µm. (c) Representative photocurrents of wild-type KR2 (grey) and eKR2 (light blue) recorded from ND7/23 cells (525 nm, 45 mW mm−2). (d) Membrane targeting of all constructs determined from the confocal images (mean ± SEM with individual data points); *p < 0.05, **p < 0.01, ***p < 0.001 compared to wild type or the indicated variant. (e) Light-induced (525 nm) stationary currents at 0 mV recorded from ND7/23 cells (symmetric 110 mM [Na+], pH 7.2) for all variants (mean ± SEM with individual data points); *p < 0.05, **p < 0.01, ***p < 0.001 compared to wild type or the indicated variant. (f) Normalized photocurrent amplitude (0 mV) of eKR2 in ND7/23 cells at different wavelengths (equal photon flux) of activation light (mean ± SEM; n = 6); fitted with a Weibull function, symmetric 110 mM [Na+], pH 7.2. (g) Stationary photocurrent (mean ± SEM; n = 6) of eKR2 in ND7/23 cells upon increasing intensities of activation light (525 nm); normalized to maximal value, symmetric 110 mM [Na+]i/e, pH 7.2.
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Figure 2. Photocurrents of eKR2 and CsR in ND7/23 cells at 0 mV and varying intracellular ionic compositions. (a) Representative photocurrents of eKR2 after illumination with 525 nm light (0 mV) at different intracellular [Na+]i at 110 mM [Na+]e and pHe 7.2 with the off-kinetics (inset). (b) Stationary photocurrents of eKR2 normalized to respective cell capacitance extracted from measurements at different intracellular [Na+]i (pHi 7.2) and corresponding apparent time constants from the bi-exponential decline of these currents after light-off; 110 mM [Na+]e, pHe 7.2 (mean ± SD and individual data points, 0 mV). (c) Stationary photocurrents of eKR2 normalized to cell capacitance extracted from measurements at different intracellular pH values ([Na+]i 1 mM) and corresponding apparent time constants from the bi-exponential decline of the currents after light-off; 110 mM [Na+]e, pHe 7.2 (mean ± SD and individual data points, 0 mV). (d) Representative photocurrents of the H+ pump CsR after illumination (bar) with 525 nm light at 0 mV and intracellular pHi 6.0, 7.2, and 9.0; 1 mM [Na+]i (110 mM [Na+]e, pHe 7.2). (e) Stationary photocurrents of CsR normalized to cell capacitance extracted from measurements at conditions described in (d) and corresponding apparent time constants from the bi-exponential decline of the currents after light-off (mean ± SD and individual data points, 0 mV).
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Figure 3. Voltage dependence of eKR2 photocurrents at different intra- and extracellular ionic compositions. (a) Photocurrent traces of eKR2 at high H+ gradients (0 mV). Photocurrents from the same cell at symmetric pHi 9.0 (left) and after the extracellular buffer was exchanged to pHe 5.0 (right) to create a high H+ gradient; top: 110 mM symmetric [Na+]i/e; bottom: 1 mM symmetric [Na+]i/e. (b) Voltage dependence of the stationary photocurrents of eKR2 at 110 mM [Na+]i, pHi 7.2 and different extracellular pHe values and [Na+]e as indicated (normalized to the symmetric condition; LJP corrected; mean ± SEM, n = 6, 5, and 6). (c) Same experiment as in (b) but at 1 mM [Na+]i, pHi 7.2 (normalized to the symmetric condition; LJP corrected; mean ± SEM, n = 8, 11, 8, and 6). (d) Same experiment as in (b) but at 110 mM [Na+]i, pHi 9.0 (normalized to the symmetric condition; LJP corrected; mean ± SEM, n = 7, 7, and 7). (e) Same experiment as in (b) but at 1 mM [Na+]i, pHi 9.0 (normalized to the symmetric condition; LJP corrected; mean ± SEM, n = 6, 5, 6, and 6).
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Figure 4. Laser-induced eKR2 photocurrents and flash photolysis with recombinant KR2. Comparison of results from different ionic and pH conditions (a) 110 mM [Na+]i/e at pH 7.2 (mean ± SD, n = 3), (b) 1 mM [Na+]i/e at pHi/e 7.2 (mean ± SD, n = 4), and (c) 1 mM [Na+]i/e at pH 9.0 (mean ± SD, n = 5). Top: Averaged normalized single turnover eKR2 photocurrents (0 mV) induced by a 7-ns laser flash (525 nm) and corresponding components of the fit of the current decay with a tri-exponential function; inset: pie chart with integrals of the three exponential decay components. Middle: contour plots from flash photolysis measurements of recombinant KR2 under the same conditions together with time traces at the indicated wavelengths. Bottom: time constants resulting from exponential fitting (decay) of the indicated time trace; points represent SVD filtered raw data, line is only guidance to the eye.
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Figure 5. Neuronal silencing performance of eKR2. (a) Confocal image of a hippocampal neuron in culture (Z stack) with zoom-ins (0.5-µm equatorial slice) on dendrites and soma; scale bar corresponds to 10 µm. (b) Left: Representative voltage traces recorded in current-clamp configuration using a squared somatic current injection protocol to determine the rheobase (540 nm LED, 500 ms) Right: Dependence of average rheobase on illumination intensity (mean ± SD; n = 10). Dotted line represents the average rheobase without illumination. (c) Left: current-clamp traces at different light intensities using a somatic current injection ramp protocol with 0.44 pA/ms (0 to 400 pA in 900 ms). Right: Cell hyperpolarization at 10 mW/mm2 green light stimulation (mean ± SD; n = 11). Delta membrane voltage was calculated as the minimum membrane voltage value under light stimulation minus the membrane voltage at 0 somatic current injection and no light stimulus. (d) Relative number of spikes during the ramp protocol at different light intensities normalized to no light stimulation (mean ± SD; n = 11). (e) Pulse trial protocol traces of an eKR2-expressing neuron with (light blue) and without (black) light stimulation; pulse width 10 ms, LJP corrected. (f) Firing probability during pulse trial protocol pre-, during, and post- light stimulation (mean ± SD; n = 11). Left: At AP threshold; middle: injection amplitudes at AP threshold plus 300 pA. Right: At AP threshold plus 500 pA (only eKR2). The Kruskal Wallis test was used followed by Dunn’s test.
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Figure 6. Red-shift of O-state absorption by Na+. Structure of KR2 (PDB:4XTL)16 on the left with close up on the proposed transient Na+ binding site close to D25115,31,49. At high [Na+] the presence of the charge (middle) caused a red-shift of the retinal absorption in the O state compared to the ground state, whereas the absorption was similar to the ground state absorbance in absence of Na+ (right). Scheme created using the dark state structure, no implications regarding the retinal isomerization state intended; helix2 is removed for visibility.
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