Figure 3.

Effect of cysteine substitution and thiol-reactive reagents treatment on functional activity of the protein. (A) Mean stationary photocurrents of WT ChR2, ChR2_C34A/C36A, and cysteine mutants of the channel recorded at a V_m of −120 mV in Ringer’s buffer (5 mM MOPS/NaOH, 110 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH = 7.6), and measured as in μA. The currents shown are the average of stationary currents from 7 to 61 cells. The error bars represent SE. ^∗, p ≤ 0.05. (B) Analysis of cysteine-substituted mutants after MTSEA and MTSES treatment via two-electrode voltage clamp. Each oocyte was incubated in Ringer’s buffer in presence of 1 mM sulfhydryl-specific reagents MTSEA (in black) or MTSES (in gray) at room temperature for 10 min in the measuring chamber. Currents were recorded at −120 mV before and after incubation with MTS reagents and are expressed as the percent of the stationary currents at −120 mV before treatment. The data are means ± SE from 5 to 35 oocytes. ^∗p ≤ 0.002.