Fig. 1: I384 residue controls electromechanical coupling in the Shaker potassium channel.

A Structure of the Shaker channel (PDB:7SIP). Voltage-sensing domains (VSD) and pore domains (PD) and S4–S5 helixes are labeled. B Location of I384 at the VSD–PD interface and its contacting residues. C, D The I384C mutant shows markedly slower deactivation compared to WT, with GV curves (N = 4) sharper and more closely aligned with QV (N = 5). Inset shows the tail current of I384C in black and WT in red returning to −120 mV. E, F WT displays typical ionic and gating currents, with GV and QV (n = 3 for both) separated. G–H I384L requires stronger depolarization for activation, with a shallower GV slope (N = 4) and altered gating current (N = 5) The inset highlights the coexistence of both gating and ionic current at the beginning of the pulse. I, J Fluorescence recordings from a TMR labeled I384L (A359C) suggest normal VSD movements (N = 3 for GV/QV/FV curves). Note there is no slow component in the fluorescence traces. K Single-channel recordings from I384L reveal unchanged conductance but increased flickering. L, M I384R abolishes ionic conduction in 12 external K⁺, leaving only gating currents, with the QV shifted strongly to negative voltages (N = 4 for I384R). N Gating current kinetics of I384R are faster than WT in the W434F background (N = 3 for both). All the data are shown as Mean ± SEM and N is the number biological replicates. For C, E, G, prepulse and returning pulse are both -120 mV and ∆V = 10 mV. For D, F, H, the data were fitted with a two-state model (details in method section) for easy visualization and calculation of change in ∆V_1/2. Fitting results could be found in Supplementary Tables 1-2.