Fig. 3: A tripartite interaction pocket for electromechanical coupling and the structure of activated but not relaxed voltage sensors.

A Intracellular view of the uncoupled structure (green) and coupled structure (orange) with their S4-S5 linkers highlighted. In the uncoupled structure, S4-S5 linkers undergo a translational movement towards the pore, forming a much tighter collar around the S6 helices. B, C In the coupled structure (orange), Y485 at the bottom of the S6 helix interacts with E395 and R394 in the C-terminus end of the S4-S5 linker from the adjacent subunit. I384 from the same subunit is lodged in a hydrophobic pocket formed by F484 and Y485. In the uncoupled structure (green), however, R384 jumps out of the hydrophobic pocket and forms a salt bridge with E395 in the adjacent unit, which repulses R394 away from facing the S6 helix, abolishing completely the interactions with Y485. D Side view of the VSD of the uncoupled channel and the WT channel. E Coulombic density for the VSD in I384R. All the side chains of the gating charges can be clearly resolved. F Comparison of the WT and I384R voltage sensors. In both cases, all gating charges (R362, R365, R368, and R371) have moved passed the hydrophobic plug around the I287 and F290 region. Both VSDs are in a fully activated state. G, H Hysteresis of the VSDs in WT and I384R (N = 4 for both). Holding at 0 mV for a prolonged time (>15 s) leads to a ~20 mV shift of the QV curve to the left in the WT channels. However, in I384R, no such shift was observed. It seems that at 0 mV, the VSDs in I384R do not enter the relaxed state. I Helical movements in S4 due to the S4-S5 linker. The tight conformation of the S4-S5 linker shifted the C terminus of the S4-S5 linker by 4.1 Å. This shift is transduced to the N-terminus end of the linker and the S4 helix as well, shifting them 1.7 Å and 1.4 Å, respectively. Data are shown as Mean ± SEM. N is the number of biological replicates.