水谷 夏希

Voltage-sensing phosphatase (VSP) comprises a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), achieving a unique electrochemical signal conversion. Previous studies suggest that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P ₂ ), a membrane phospholipid known to be critical for activities of diverse voltage-gated ion channels, associates with a linker connecting the VSD with the CCR of VSP and regulates VSD-CCR coupling. However, the details of PI(4,5)P ₂ interaction with the linker of VSP remain elusive. Here, we exploit advantage of sensitivity of a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), to changes in local environment to study interaction between PI(4,5)P ₂ and the linker of Ciona intestinalis VSP (Ci-VSP). We found that a conserved tyrosine residue (Y255) as well as neighboring basic residues interacts with PI(4,5)P ₂ and this interaction was maintained in G365A Ci-VSP mutant which lacks the substrate PI(4,5)P ₂ at the active site and Ci-VSP/human phosphatase and tensin homolog (PTEN) chimera which does not dephosphorylate PI(4,5)P ₂ , indicating that the linker interacts with nonsubstrate, regulatory PI(4,5)P ₂ outside the active site. Molecular dynamics simulations demonstrated that the linker formed stable interaction with PI(4,5)P ₂ in the activated state. These findings indicate that regulation of coupling to an effector region downstream of the VSD through PI(4,5)P ₂ binding to the linker is shared among voltage-dependent membrane proteins.

Abstract Background Voltage‐sensing phosphatase contains a structurally conserved S1‐S4‐based voltage‐sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N‐terminus and S1 exhibit structural change. Methods Anap, an environment‐sensitive unnatural fluorescent amino acid, was site‐specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. Results We detected robust voltage‐dependent signals from widely distributed positions including N‐terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage‐sensor domain in the down‐state. We also found that the mechanical coupling between the voltage‐sensor and phosphatase domains affects the depolarization‐induced optical signals but not the hyperpolarization‐induced signals. Conclusions These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage‐sensor domain transition as well as its coupling with the effector.

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP.