FIGURE 1.

Cytosolic ATP reversibly blocks AtALMT9-mediated currents in a voltage-dependent fashion. A–C, representative traces and corresponding I-V curves of excised cytosolic-side out patches from N. benthamiana vacuoles overexpressing AtALMT9 in 30 mm malate_cyt, 100 mm malate_vac (A), 30 mm malate_cyt + 1 mm ATP_free, 100 mm malate_vac (B), and recovery (C). Currents were evoked in response to 3-s voltage pulses ranging from +60 to −160 mV in −20 mV steps, followed by a tail pulse at +60 mV. The holding potential was set at +60 mV. Red highlights the currents at the membrane potentials where the ATP inhibition was effective. D, ratio between currents measured in the absence (I_ctrl, 30 mm malate_cyt, 100 mm malate_vac) and presence of the indicated concentrations of ATP_cyt (I_ATP, 30 mm malate_cyt + X mm ATP_free, 100 mm malate_vac) at different applied membrane potentials. Data were fitted with Equation 3 (see “Experimental Procedures”; dashed line) (n = 4–7). E, dose-response curves for ATP_free at different membrane potentials. Data were fitted with Equation 1 (see “Experimental Procedures”; solid lines) to derive the dissociation constant (K_d^ATP). F, voltage dependence of the dissociation constant for ATP (K_d^ATP) in 30 mm malate_cyt, 100 mm malate_vac. Solid line, data were fitted with Equation 2. G and H, inhibitory effect of Mg-ATP. G, normalized current-voltage curves were obtained with a voltage ramp (from +60 to −160 mV in 1.5 s) with 30 mm malate_cyt, 100 mm malate_vac and in 30 mm malate_cyt, 100 mm malate_vac in the presence of 1 mm ATP_free (+1 mm ATP) or Mg-ATP (1 mm Mg-ATP). H, residual currents (I_ATP/I_ctrl; mean ± S.E., n = 3–7) plotted against the applied voltage in the presence of 1 mm ATP_free or Mg-ATP. Data were fitted with Equation 3 (dashed line). Error bars denote S.E.