Figure 3.

Electrophysiological properties of the mutant channel ALMT9_E196A. A, Fluorescence and transmission images of vacuoles released from lysed tobacco protoplasts that transiently overexpress ALMT9-GFP (left) and ALMT9_E196A-GFP (right). Auto-fluorescence of chloroplasts is shown in magenta. Scale bars = 20 µm. B, Patch-clamp experimental procedure. Vacuoles were patched in excised cytosolic-side-out configuration under symmetric ionic conditions (100 mm Cl⁻_vac/ 100 mm Cl⁻_cyt). The cytosolic buffer was sequentially exchanged (100 mm Cl⁻; 100 mm Cl⁻ + 1 mm MA²⁻; 100 mm MA²⁻) on the same membrane patch. C, Representative currents of ALMT9 (left) and ALMT9_E196A (right) in presence of 100 mm Cl⁻ (Cl⁻) and 100 mm Cl⁻ + 1 mm MA²⁻ (Cl⁻ + MA²⁻) in the cytosolic buffers. Currents were evoked by a 2.5-s voltage ramp ranging from +40 mV to −100 mV. D, Relative Cl⁻ and Cl⁻ + MA²⁻ currents mediated by ALMT9 and ALMT9_E196A. Currents were normalized to the current amplitude measured at −100 mV in presence of 100 mm MA²⁻ in the cytosolic solution. E, Level of MA²⁻-activation (I_Cl⁻ _{+ MA}²⁻/ I_Cl⁻) of ALMT9- and ALMT9_E196A-mediated Cl⁻ currents at −100 mV. Data are means ± sd. Asterisks indicate statistically significant differences between ALMT9 (n = 5) and ALMT9_E196A (n = 6) currents (*P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t test).