In situ introduction of positive charge to the inner cavity decreases spermine-dependent rectification. (A–D) Representative currents illustrating rectification induced by spermine in WT-N160D, and in V129C, L157C, and L164C mutant channels (mutation in second half of N160D-N160D dimer background), before and after complete modification by MTSEA⁺ (modification not shown). Two separate voltage protocols are shown for WT-N160D. One or the other is used for each cysteine mutant construct. Current scale indicates 0.5 nA in each case. (E) Steady-state current in spermine relative to control (Grel) plotted vs. voltage, fitted with Boltzmann functions (no offset) as indicated.