Fig. 4: ATP-binding site and inhibition mechanism.

a A cut-open view of AtCLCa-Cl⁻ protomer. TMD is shown as a cut-open view of the electrostatic potential surface. Curved arrow indicates the direction of NO₃⁻ transport in the physiological state. NTD, orange; linker, gray; CBS1, beige; CBS2, green; ATP, pink balls and sticks; Cl⁻, green sphere; Mg²⁺, yellow sphere. The dashed box is represented to show the N-terminal β-hairpin and ATP-binding site. b A zoom-in view of the interaction between the β-hairpin and TMD. Hydrogen bonds are shown as black dashed lines. The dashed box is represented to show the ATP-binding site. c A zoom-in view of ATP-binding site. d I–V curves of wild-type AtCLCa with different adenine nucleotides at a concentration of 5 mM added to the pipette solution. n = 20 (Ctrl, nucleotide free), n = 22 (ATP), n = 19 (ADP), n = 16 (AMP). P < 0.0001 (ATP vs Ctrl), P = 0.008 (ADP vs Ctrl), P = 0.1391 (AMP vs Ctrl). e I–V curves of wild-type AtCLCa with 5 mM AMP and 5 mM ATP added separately or together to the pipette solution. n = 20 (ATP), n = 16 (AMP), n = 16 (ATP + AMP). P = 0.0011 (ATP vs AMP), P = 0.011 (ATP vs ATP and AMP), P = 0.5689 (AMP vs ATP and AMP). f–i I–V curves of AtCLCa ΔN53 mutant (f), ΔN44 mutant (g), E55A/S56A mutant (h), and D753A mutant (i) with 5 mM ATP added to the pipette solution. For ΔN53 mutant (f), n = 26 (Ctrl), n = 26 (ATP), P = 0.8163. For ΔN44 mutant (g), n = 23 (Ctrl), n = 20 (ATP), P < 0.0001. For E55A/S56A mutant (h), n = 25 (Ctrl), n = 26 (ATP), P = 0.7026. For D753A mutant (i), n = 23 (Ctrl), n = 24 (ATP), P = 0.7239. For (d–i), the whole-cell currents were evoked by clamping the HEK293T cells for 2-s voltage pulses from −80 to +80 mV in 20-mV steps followed by a repolarizing step to −80 mV, and the nitrate bath solution was used. Data are mean ± s.e.m. Two-tailed t-tests were used for significance analysis of the current density at +80 mV. *P < 0.05, **P < 0.01, ***P < 0.001, ns means no significance difference. Source data are provided as a Source data file.