FIGURE 3.

Photocurrents of ChR2(H134R)-mCherry and variants in Na⁺- and Ca²⁺-based extracellular solutions. A, typical photocurrent of ChR2(H134R)-mCherry in HeLa cells measured at −120 mV upon excitation with a 500-ms light pulse (480 nm, black bar) in extracellular solution 1 (in mm, 145 NaCl, 3 KCl, 5 N-methyl-d-glucamine, 10 Hepes, 20 glucose; pH 7.35). On the right, the IV relationship from −120 mV to 20 mV in 20 mV steps is shown (n = 9). B, expression of ChR2(H134R)-mCherry (WT) and variants in HeLa cells as assessed by confocal microscopy. C and D, inward photocurrents at −120 mV in extracellular solution 1 (C) and solution 2 (D) (in mm, 10 CaCl₂, 3 KCl, 135 N-methyl-d-glucamine, 10 Hepes, 20 glucose; pH 7.35). *, p < 0.05, unpaired two-tailed t test. pF, picofarads. E, ratio between photocurrent peaks in solution 1 and 2. *, p < 0.002, unpaired two-tailed t test. F, Fluo-4 measurement of intracellular Ca²⁺ in HeLa cells transfected with the ChR2 WT and S63D mutant upon 100-ms pulses of 490-nm light. A significant increase in intracellular Ca²⁺ in ChR2-S63D as compared with WT-ChR2 expressing cells was detected. Error bars in panels A and C–F indicate S.E.