Panel A: A schematic representation of the DIV_VSD movement upon depolarization, showing a graph with the x-axis labeled in milliseconds and the y-axis in microamperes, indicating the gating current slow component. Panel B: A schematic of the D 3_D 4 linker movement, with a graph showing the fluorescence signal decrease over time, labeled in percentages and milliseconds. Panel C: A schematic of the IFM movement, with a graph showing the fluorescence signal increase over time, labeled in percentages and milliseconds. Panel D: A schematic of the pore movement, with a graph showing the fluorescence signal change over time, labeled in percentages and milliseconds.
The IFM motif links DIV VSD activation to inactivation gate. (A) Upon depolarization, DIV VSD (not shown for simplicity) activates and due to its slow kinetics, manifests as the slow component in the gating current. (B) Activation of DIV VSD triggers the DIII–DIV linker (in yellow) movement that attaches via hydrogen bonds, creating a fast decrease in ANAP fluorescence signal (L1319ANAP), suggesting a transition to more hydrophilic environment. This movement is faster than fast inactivation and follows the time course of the DIV VSD movement. (C) Movement of the linker is concomitant with the fast movement of the IFM motif (in red, F1304ANAP) that produces the fast component of the increase in fluorescence (more hydrophobic) of the F1304ANAP signal. Subsequently, a second, slower increase of F1304ANAP fluorescence occurs that has the same time constant as the fast inactivation. The increase in the fluorescence signal is also consistent with the notion of IFM motif binding to a hydrophobic pocket. (D) DIII S6 closure (in green, Q1293ANAP) tracks the fast inactivation state and occurs with a delayed On, similar to what was observed in the pronase-treated giant squid axon (modified from Liu et al., 2025).