Differential interaction of primary and quaternary ammonium ions with the rectification controller carboxylate. (A) Chemical structures of DA10 and DQA10. (B–E) Steady-state spermine block was characterized as described in Fig. 2, in either 10 or 100 µM spermine for WT Kir2.1 or Kir2.1[D172N] channels, as indicated. (F) K_app, reflecting blocker binding to the deep binding site, was determined by fitting a single Boltzmann relationship (I/I_control=1/[1+K_app(0 mV)*e^(zδFV/RT)]) to the conductance–voltage relations at low concentrations of each blocker (10 µM for DA10 and DQA10 and 1 µM for spermine). (G) ΔΔG values were determined as ΔG_D172N − ΔG_{WT Kir2.1,} based on the K_app constants determined in F. ΔΔG reflects the effect of the D172N mutation on the binding energy of each blocker.