Structural dynamics in the resting and activated states of the voltage sensor of Ci-VSP from dipolar distance measurements

The mechanism of electromechanical transduction in voltage sensing domains remains controversial. Here, we have probed the conformation of the voltage sensor of Ci-VSP in different functional states by means of EPR-based distance measurements. Ci-VSP is a voltage-sensing phosphatase from Ciona intestinalis. Although it is coupled to a cytoplasmic phosphatase, its voltage-sensing domain (VSD) is homologous to voltage sensors found in voltage-gated ion channels. It therefore serves as an excellent model to study voltage sensor movement independent of the interaction with pore domain. On the basis of voltage dependence of Ci-VSP sensing currents (Q-V curves), it is agreed that, at 0 mV, the S4 of wild-type Ci-VSP is in the resting conformation (down state). The arginine at position 217, located in the extracellular end of S4, has a strong effect on the voltage dependence of Ci-VSP sensing currents. Mutations at arginine 217 with a neutral or negative residue (R217Q or R217E), lead to a large leftward shifts in the Q-V curve so that, at 0 mV, the sensor is in the activated conformation (up state). This provides a unique opportunity to monitor the conformational differences in the VSD between resting and activated states in the absence of membrane potential. We expressed and purified a series of double cysteine mutants in the isolated voltage sensor (S1 to S4) of Ci-VSP in wild-type and R217E backgrounds, and measured distances using CW-based dipolar broadenings (for short distances, 8 to 20 Å) and double electron-electron resonance (DEER) spectroscopy (for longer distances, 20 to 50 Å). Our preliminary analysis of the distance measurements suggest defined conformational differences between resting and activated states of the VSD