(A) Left, representative current-voltage relationship for GluD2-A654T Lc, with (blue) and without (black) pentamidine (100 μM). The subtracted current (gray) shows clear inward rectification and block at positive voltages. Right, currents recorded at −60 mV were larger for GluD2-A654T than for WT (n = 6 cells, p=0.0033, two-sample t-test), and were strongly reduced with 100 μM pentamidine for A654T (n = 6 cells, p=0.0045, paired t-test) but not for the WT GluD2 (n = 6 cells, p=0.34, paired t-test). (B) Left, ratio of the currents recorded at −60 mV under 380 and 535 nm light, for A654T and the fifteen cysteine mutants engineered on the A654T background (n = 3–8 cells, one-sample t-test, or Wilcoxon when normality is not verified, compared with a theoretical mean value of 1). Right, representative change in holding current when switching between dark, 380 and 535 nm light, for A654T and for five mutants that show modulation of the holding current when switching between 380 and 535 nm light (p<0.05 except for Q669C where p=0.09). (C) Left, representative current traces (Vm = −60 mV) recorded for A654T and A654T-I677C in darkness, then under 380 nm light (violet), upon application of pentamidine (blue, 100 μM), and then under 535 nm after pentamidine washout (green). Right, percent photoswitching (PS = Blockphoto/Blockpenta) for A654T and the cysteine mutants that show both clear leak current and pentamidine block. Photoswitching reached 15.5 ± 3.1% for Q666C (p=0.016, n = 4),–1.9 ± 0.4% for Q669C (p=0.008, n = 6), 14.6 ± 1.7% for D670C (p=0.003, n = 4), 20.5 ± 4.2% for Q674C (p=0.008, n = 5) and 46.3 ± 4.3% for I677C (p=1.34E-05, n = 8, one-sample t-test, or Wilcoxon when normality is not verified, compared with a theoretical mean value of 0). Photoswitching was absent in the control A654T mutant (0.37 ± 0.9%, p=0.72, n = 5) and in the other two cysteine mutants K673C (−0.9 ± 1.06%, p=0.43) and D676C (0.39 ± 0.57%, p=0.53). (D) Model of GluD2 showing the location of A654 (red), Q666, D670, Q674 and I677C (violet), Q669 (green), and all the non-photocontrollable cysteine mutants (yellow). Data are presented as mean value ± sem. Source files of individual data points used for the quantitative analysis are available in the Figure 2—source data 1.