Fig. 4: Ca2+-flux at physiological levels of [CaCl2]e in ND7/23 cells.
From: Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling
A–C Fura-2-AM imaging on CoChR WT (blue), C2-LC (red), CapChR1 (green) and CapChR2 (orange). A Overview of the experimental setup: Fura-2-AM loaded ND7/23 cells with a resting membrane potential were illuminated with 470 nm (~0.08 mW/mm2, saturating light intensities) light to allow an influx of calcium ions through the expressed ChR in the presence and absence of bath Mg2+. B Mean imaging response of the denoted constructs under the two measured conditions (mean as coloured line and shadows represent SEM). C Quantified peak responses after 10 s of illumination. Single dots on the right of the columns represents one cell under those conditions (Bar: mean ± SEM). Number of replicates in (B) and (C) (+Mg2+/−Mg2+): CoChR n = 30/30; C2-LC n = 32/30; CapChR1: n = 21/21; CapChR2: n = 24/21. Two-sided, unpaired Wilcoxon–Mann–Whitney-Test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. P values for comparison to C2-LC in C: CoChR P = 0.35; CapChR1 P = 0.44; CapChR2 P = 5.07 × 10−10. D–H Voltage-clamped calcium imaging on CapChR1 and 2. D Experimental design for voltage-clamped measurements on ND7/23 cells. Cells were loaded with membrane-impermeable Fura-2 via the patch pipette (internal buffer: 110 mM [NaCl]i and divalent cation free, see the “Methods” section). A baseline measurement was started for both the 380 and 340 nm channels (5 acquisitions), with subsequent 470 nm illumination (100 ms, ~0.08 mW/mm2, ~0.008 mJ/mm² per F340/380 ratio acquisition, saturating illumination for both CapChRs) to measure calcium influx through the CapChRs. A membrane voltage of −80 mV was applied at each illumination cycle. E Calcium imaging response (ratio of 340/380 nm fluorescence; R340/380) for both CapChRs at physiological pH and ion concentrations. F Fluorescence change (ΔR) after 100 ms of illumination at −80 mV holding potential. G and H Fluorescence change (ΔR) for CapChR1 (green) and CapChR2 (orange) after 100 ms of illumination at −80 mV holding potential and at different extracellular calcium and sodium concentrations. Box middle line: Mean; Box outer edges ± SEM; Box whiskers: 1.5 × SEM. Number of replicates in G and H: CapChR1/CapChR2: 144 mM [NaCl]e/0 mM [CaCl2]e: n = 7/8, 143.8 mM [NaCl]e/0.1 mM [CaCl2]e: n = 7/6, 143 mM [NaCl]e/0.5 mM [CaCl2]e: n = 7/6, 142 mM [NaCl]e/1 mM [CaCl2]e: n = 6/5, 140 mM [NaCl]e/2 mM [CaCl2]e: n = 45/29, 134 mM [NaCl]e/5 mM [CaCl2]e: n = 7/9, 124 mM [NaCl]e/10 mM [CaCl2]e: n = 6/11, 104 mM [NaCl]e/20 mM [CaCl2]e: n = 8/5. n = X biologically independent cells. AU arbitrary units.
