The Energy Landscape of Voltage Sensing in Ci-VSP

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The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane associated phosphatidylinositol lipid phosphatase. The enzymatic activity of the cytoplasmic phosphatase domain is directly regulated by changes in the transmembrane electric field through conformational rearrangements at the voltage-sensing domain (VSD). Ci-VSD shares high sequence and structural similarity with the VSD of voltage-gated cation and proton channels, and is the only membrane-spanning domain of Ci-VSP. Previously, we have determined the structure of Ci-VSD in its resting (Down) and activated (Up) conformations. Thus, this system provides us with a great opportunity to study the energetics of the gating mechanism of this isolated VSD. Using mutagenesis, electrophysiology, modeling and molecular dynamics simulations, we systematically investigated residue-residue interactions between the segments of the VSD and the interactions between the VSD and lipids during a complete functional gating cycle. We found that Ci-VSD can adopt mainly four conformational states: the Up and Down states determined by crystallography, in addition to a further down (‘Down-minus’) and a further up (‘Up-plus’) states. One helix turn translation of the S4 segment drives the transition between two adjacent states. Upon depolarization, the VSD rearranges from the ‘Down-minus’ state to the ‘Up-plus’ state in a sequential stepwise way, translating one positively charged residue across the membrane electric field at each transition, resulting in a gating charge of ∼3 e0. Furthermore, cysteine mutations on the extracellular side of S4 can form metal-ion bridges with lipid phosphate group and decrease the total gating charge displacement with intensity depending on the external Cd2+ concentration, while cysteine mutations on the S1−S3 segments have little effect, representing minor conformational changes of them during gating and do not contribute to the gating charge.