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. 2019 Jan 4;294(11):3806–3821. doi: 10.1074/jbc.RA118.006996

Figure 4.

Figure 4.

Physiological screening for calcium and proton selectivity. a, change in Fura-2 340/380 nm emission ratio (panel i) and SNARF-5F 610/640 nm emission ratio (panel ii) mediated by previously described channelrhodopsins. Cells expressing fluorophores only were used as controls (mCherry in panel i and EGFP in panel ii) (n = 20–35 cells each). WT, WT ChR2. Black bar, illumination period, 470 nm for 10 s; irradiance, 4.6 milliwatts/mm2 (panel i); illumination period, 500 nm, 1.5 s; irradiance, 4 milliwatts/mm2 (panel ii). b and c, outcome of the calcium and proton selectivity of the screen for channelrhodopsins emerging from the screen of Fig. 1, showing the Fura-2 340/380 ratio (b, panel i) and calcium concentration (c, panel i) after blue light illumination (delivered as in a) in HEK cells expressing the indicated ChR2 mutants (n = 12–69 cells) and the SNARF-5F 610/640 ratio (b, panel ii) and pH (c, panel ii) after green light illumination (delivered as in a but with 1-s pulses) in HEK cells expressing the indicated ChR2 mutants (n = 8–35 cells). Gray dots indicate data from individual cells. d and e, population data for photocurrent density ratios measured using whole-cell patch clamp in HEK cells in ion-specific extracellular solutions (see “Experimental procedures” for details), calcium photocurrent (Icalcium) measured in 90 mm CaCl2, pH 7.4, divided by sodium photocurrent (Isodium) measured in 145 mm NaCl, pH 7.4 (d), and proton photocurrent (Iproton) measured in 135 mm NMDG, pH 6.4, divided by sodium photocurrent (Isodium) (e) of WT ChR2 and mutants with improved ion selectivity identified from b and c using illumination conditions 470 nm, 1 s, and 10 milliwatts/mm2 (n = 5–12 HEK293FT cells each). In d and e, peak and steady-state photocurrent ratios for WT ChR2 are indicated by dashed lines and dotted lines, respectively. Plotted are means and error bars representing S.E. Statistics for b, panels i and ii, d, and e, *, p < 0.05; **, p < 0.01; ***, p < 0.001, non-Bonferroni–corrected t test comparing mutant versus WT.