Fig. 5: Spermine enters wild type but not disulfide-linked pores.

a Summary of potassium flux experiments investigating spermine block in wild-type and disulfide-linked A133C-T136C channels. Data are shown as mean ± SEM (n = 4 (channel-free, wild type)) or 3 (A133C-T136C disulfide, A133C-T136C reduced experiments); details of replicates are in Supplementary Table 4. Each black or white circle represents the mean of an individual experiment. b Comparative dimensions of permeant cations (K⁺ is depicted in deep cyan, oxygen in magenta, carbon in yellow and nitrogen in blue). c The PMF represents the energetic barrier to spermine block. Positions 1 to 4 refer to the positioning of a middle carbon (C8) of spermine relative to the pore—shown in the schematic below. d The voltage-dependency of spermine block is illustrated by the trend in probability density of spermine entering the pore at field strengths of 0, 25 and 50 mV nm⁻¹. The shaded region of each panel corresponds to 80% of structures in the MD simulations, shown relative to positions 1–4. The distance of C8 relative to the center of mass of Thr96 is annotated for major peaks (red). In the region 18.0–27.5 Å, the occupancy sequentially changes from ~25 to 40–50% with increasing field (0–25 to 50 mV nm⁻¹). The field-free distribution of spermine in the cavity predicts only a small probability of spermine occupancy at position 4.