Fig. 1: Branched aliphatic side chains form a barrier to ions within the conduction pathway.

a Ribbon representation of the KirBac3.1 pore (cyan); for clarity, only two subunits are shown. The side chains of Leu124, Tyr132 and Thr96 are depicted (yellow sticks). A scale bar indicates the distance (Å) from the centre of mass of Thr96. SF indicates the location of the selectivity filter. Insert shows a longitudinal section depicting the accessible surface of the pore interior, highlighting the steric barrier provided by the Leu124 cluster. Potassium ions at binding sites are depicted in purple. b Profile of the pore radius through the transmembrane domain of KirBac3.1 calculated using HOLE⁵⁶. Distances are relative to the molecular axis. c Schematic of the ACMA assay. A lipid-soluble proton-sensitive dye (ACMA) is equilibrated with proteoliposomes (green circle) in isotonic solution (K⁺_in /Na⁺_out), prior to addition of the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Protons moving into the liposome via CCCP bind ACMA to form ACMA-H⁺, which does not fluoresce, resulting in a decrease in total fluorescence emission. Proton influx is balanced by K⁺ efflux through Kir. Protonated ACMA cannot pass out through the membrane. Limiting fluorescence is determined by addition of the specific K⁺ ionophore valinomycin. The total fluorescence change measured in the assay is summed from individual proteoliposomes. d Summary functional assay data for the Leu124M (n = 5 independent samples) and Tyr132I (n = 3) point mutants represented as mean ± SEM. Values of n for control samples are 5, 6 and 3 for liposome-only, KirBac3.1 and KirBac3.1-SCS samples respectively. Data analysis was by two-sided t-test analysis of variance and refers to pairwise comparisons to KirBac3.1-SCS as indicated (ns = non-significant; *p ≤ 0.05). Comparative p-values for KirBac3.1-SCS to Y132I and L124M are 0.265 and 0.249, respectively. Dunnett’s test was applied to adjust for multiple comparisons to each control. e PMF along the molecular axis, oriented to match the inset in (a). Distances along z are relative to the centre of mass of the four Thr96 sidechains. Source data are provided as a Source data file.