Figure 1
pH dependence and ion selectivity of Chrimson photocurrents. (a) Chrimson photocurrent traces measured in HEK293 cells during illumination at 600 nm (2.37 mW × mm−2) at different voltages and different extracellular pHe with symmetric intra- and extracellular 110 mM NaCl and intracellular pHi 7.2 (b) Current-voltage dependence of normalized peak photocurrents at different extracellular pHe as represented in (a) (mean ± SD; pHe 5: purple, n = 8 cells; pHe 7.2: black, n = 37 cells; pHe 9.0: red, n = 16 cells) (c) Normalized photocurrent amplitudes at −60 mV and different extracellular ionic conditions with two different intracellular solutions of 110 mM NaCl pHi 7.2 (left) and 110 mM NaCl pHi 9.0 (right) (mean ± SD; LJP corrected; normalized to extracellular 110 mM NaCl, pHe 7.2 (dashed line) t-test for comparison to symmetric ionic conditions for both intracellular conditions respectively; 110 mM KCl pHe 7.2, n = 8, p = 0.7; 110 mM GdmCl pHe 7.2, n = 8, p < 0.0001; 55 mM CaCl2 pHe 7.2, n = 12, p < 0.0001; 55 mM MgCl2 pHe 7.2, n = 12, p < 0.0001; 110 mM NMDGCl pHe 7.2, n = 14, p = 0.004; 110 mM NaCl pHe 9.0/pHi 7.2, n = 16, p < 0.0001; 110 mM NMDGCl pHe 9.0/pHi 7.2, n = 6, p < 0.0001/0.002; 110 mM NaCl pHe 9.0/pHi 9.0, n = 17; 110 mM KCl pHe 9.0, n = 5, p = 0.3; 110 mM GdmCl pHe 9.0, n = 5, p < 0.0001; 55 mM CaCl2 pHe 9.0, n = 7, p < 0.0001; 110 mM NMDGCl pHe 9.0, n = 6, p < 0.0001) (d) Reversal potential shifts with two different intracellular solutions of 110 mM NaCl pHi 7.2 (left) and 110 mM NaCl pHi 9.0 (right) compared to symmetric conditions of either extracellular 110 mM NaCl and pHe 7.2 (left) or extracellular 110 mM NaCl and pHe 9.0 (right) (Mean ± SD, LJP corrected, t-test for comparison to symmetric ionic conditions for both intracellular conditions respectively; 110 mM KCl pHe 7.2, n = 8, p = 0.05; 110 mM GdmCl pHe 7.2, n = 8, p = 0.6; 55 mM CaCl2 pHe 7.2, n = 12, p = 0.06; 55 mM MgCl2 pHe 7.2, n = 12, p = 0.005; 110 mM NMDGCl pHe 7.2, n = 14, p < 0.0001; 110 mM NaCl pHe 9.0/pHi 7.2, n = 16, p < 0.0001; 110 mM NMDGCl pHe 9.0/pHi 7.2, n = 6, p < 0.0001/0.002; 110 mM NaCl pHe 7.2/pHi 9.0, n = ; 110 mM KCl pHe 9.0, n = 5, p = 0.005; 110 mM GdmCl pHe 9.0, n = 5, p = 0.001; 55 mM CaCl2 pHe 9.0, n = 7, p = 0.0004; 110 mM NMDGCl pHe 9.0, n = 6, p < 0.0001)) (e) Fluorescence ratio F 340/F 380 (left: mean ± SE) and corresponding changes Δ(F 340/F 380) (right: mean ± SD, 0.02 s after activation, two-sample t-test p < 0.0001) of Fura-2 loaded HEK293 cells after excitation at 340 nm and 380 nm following the activation of CrChR2 T159C at 450 nm (n = 17 cells) or Chrimson at 560 nm (n = 30 cells) for 10 s in an extracellular solution of 70 mM CaCl2. Inlet: Photocurrents of both ChRs measured under the same conditions. (f) Chrimson action spectra after 10 ms excitation at different wavelength of equal photon count (Mean ± SD, pHe 5.0: purple, −60 mV, n = 6 cells; pHe 7.2: black, −60 mV, n = 9 cells; pHe 9.0: red, +30 mV, n = 4 cells).
