Mechanism of Voltage Gating in the Voltage-Sensing Phosphatase Ci-VSP
The conformational changes in voltage-sensing domain (VSD) are driven by the transmembrane electric field acting on charges and countercharges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes, as well as major conformations corresponding to a complete functional gating cycle in the isolated voltage-sensing domain of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1-S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated Up and resting Down states, the simulations predict two novel Ci-VSD conformations: a deeper resting state ( Down-minus) and an extended activated ( Up-plus) state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. These results show that voltage activation involves sequentially populating these four states in a stepwise way, translating one arginine across the membrane electric field per step, transferring ~3 e0 charges.