a, Single-channel characteristics of MthK(Δ2–17) are similar to the wild type. Top, representative single-channel recording traces from MthK(Δ2–17) in horizontal lipid bilayers made of POPE:POPG (3:1) liposomes in decane at +100 mV without and with 5 mM Ca2+. Traces are filtered at 200 Hz for display. Single-channel current–voltage curves (bottom left) and Po as a function of voltage (bottom right) for MthK(Δ2–17) (red symbols) compared to wild type (dashed black lines). For MthK(WT), data are mean ± s.e.m. of 5 measurements for all membrane potentials except at −100, 75, and 100 mV, which contain 6 measurements. For MthK(Δ2–17), data are mean ± s.e.m. of 5 (−50 mV), 6 (−75, 50 and 125 mV), 7 (−125 mV), 8 (−25 and 75 mV) and 9 (25 and 100 mV) measurements. Each measurement is from a separate bilayer. b, Relative Tl+ flux rates as a function of incubation time of MthK WT (blue) and MthK Δ2–17 (red)-containing POPE:POPG (3:1) liposomes with 5 mM Ca2+. Symbols are the mean ± s.d. from three independent experiments. c, Fluorescence quench curves for MthK(WT)-containing DOPC:POPG (3:1) liposomes after 1 or 10 s (dark and light blue, respectively) incubation with 5 mM Ca2+. Control fluorescence is in the absence of Tl+ (black). A small leak of Tl+ into liposomes was observed in the absence of Ca2+ (light grey) and in the MthK-free liposomes (dark grey). Three experiments were performed with similar results. d, Fluorescence quench curves for MthK(Δ2–17)-containing DOPC:POPG (3:1) liposomes after 1 or 10 s (brown and pink, respectively) incubation with 5 mM Ca2+. The control was performed similarly as that for the experiment with the MthK(WT) liposomes. Experiments were performed three times with similar results. e, f, Simulation snapshots illustrating salt bridges between the basic residues on the N terminus (blue) and the ring of glutamates at the intracellular pore entrance (e), and hydrophobic interactions between the N terminus (blue) and hydrophobic residues lining the pore cavity (f). Only three subunits of the MthK transmembrane domain are shown for clarity. The residues in stick representation are coloured the same as the individual subunits. The calibration bar indicates the position of the COM of the peptide parallel to the channel pore axis (see Methods). g, Convergence of the free energy profile from Umbrella Sampling simulations for the N-terminal peptide plugging the MthK pore (Fig. 4f). Convergence to within 1 kcal mol−1 was achieved in 23 ns.