S4-based voltage sensors have three major conformations.

Voltage sensors containing the charged S4 membrane segment display a gating charge vs. voltage (Q-V) curve that depends on the initial voltage. The voltage-dependent phosphatase (Ci-VSP), which does not have a conducting pore, shows the same phenomenon and the Q-V recorded with a depolarized initial voltage is more stable by at least 3RT. The leftward shift of the Q-V curve under prolonged depolarization was studied in the Ci-VSP by using electrophysiological and site-directed fluorescence measurements. The fluorescence shows two components: one that traces the time course of the charge movement between the resting and active states and a slower component that traces the transition between the active state and a more stable state we call the relaxed state. Temperature dependence shows a large negative enthalpic change when going from the active to the relaxed state that is almost compensated by a large negative entropic change. The Q-V curve midpoint measured for pulses that move the sensor between the resting and active states, but not long enough to evolve into the relaxed states, show a periodicity of 120 degrees, indicating a 3(10) secondary structure of the S4 segment when determined under histidine scanning. We hypothesize that the S4 segment moves as a 3(10) helix between the resting and active states and that it converts to an alpha-helix when evolving into the relaxed state, which is most likely to be the state captured in the crystal structures.