Figure EV2. Inhibition of CLC‐a‐mediated nitrate/H⁺ antiport activity by PI(3,5)P₂ .

1. Current recordings from a vacuole successively exposed to low‐chloride bath solution (black traces; left: control; right: wash‐out) and to bath solution containing PI(3,5)P₂ (red traces), at concentrations of 10 and 300 nM, as indicated. Pipette solution contained 200 mM nitrate. Currents were elicited by 3‐s voltage steps ranging from −97 to +63 mV in 20‐mV increments. The zero current level is indicated by dashed lines.
2. Dose–response analysis of nitrate/H⁺ current inhibition. Normalized residual time‐dependent currents (I_norm), determined at +63 mV, were plotted against the applied PI(3,5)P₂ concentration. Data points represent mean ± SEM of n = 5 vacuoles. Data fitting with the Hill equation (continuous line) yielded a half‐inhibition concentration of 8.5 nM, a Hill coefficient of 0.71 and a residual current at saturating [PI(3,5)P₂] of 0.14.