Fig. 2: S4 charge neutralization stabilizes distinct VSD conformations independent of Calcium.

a The VSD S4 of aBK mutants R1Q (red), R2Q (green), and R3Q (blue) are shown as a cartoon superimposed for Ca²⁺-bound (opaque) and apo (light). To ensure clarity, we depict only S4 as a cartoon, while TMD S1, S2, and S3 remain transparent. Stick side chains are shown for the charged residues 196,199, and 202. The structures were aligned based on the residues of their selectivity filter 275–282 (S5–S6 loop, the least mobile part of all). The speculatively hydrophobic gasket is focused on the electric field marked by the transparent khaki slab. b Representative traces of gating current and steady-state Q-V curves for mutants R1Q (red) and WT (black). Q-V curves were obtained by 300 µS integration of the Qoff response. The half-activation voltage was determined by fitting the data to a Boltzmann distribution, yielding V₀  = 179.82 ± 8.6 mV and z = 0.6500 ± 0.130 for WT (n = 4), and V₀ = −231.33 ± 1.78 mV and z = 0.5761 ± 0.0216 for R1Q (n = 5). Data are shown as mean ± SEM. The apo condition was achieved with 5 mM EGTA. c Schematic representation showing the resting and active VSD conformational states. In the resting VSD scheme, green dotted lines indicate hydrogen bonds with a distance of <3.5 Å. In the active VSD scheme, these interactions are represented by violet dotted lines. The yellow dotted lines in both schemes indicate possible π-cation interactions with distance <4 Å.