Z position of gating charges relative to the HCS under varying TM potentials (−150 and −500 mV). At −150 mV (top), no structural transition in the position of the gating charges is observed staying in the up state after 5 µs. Applying −500 mV (bottom) results in further structural transitions, with the gating charges reaching the down state, indicating that higher potentials promote more extensive conformational changes.