水谷 夏希

Polyphosphoinositides (PPIns) are essential components of membrane lipids and play crucial roles in cell signaling in eukaryotes. Phosphatidylinositol4,5-bisphosphate (PI(4,5)P2) is a species of PPIns enriched in the plasma membrane and regulates numerous membrane proteins, including ion channels, transporters, and receptors, primarily through direct binding to positively charged residues such as lysine and arginine. Despite recent advances in structural biology and biophysics, the specific contributions of individual amino acid residues to PI(4,5)P2 binding in membrane proteins remain unclear. These questions have been explored by functional characterization of mutant proteins with site-specific amino acid replacement and their comparison with the WT proteins. Here, we apply genetic code expansion to investigate the role of lysine residues in the PI(4,5)P2 sensitivity of ion channels. A caged lysine compound, hydroxycoumarin-lysine (HCK), was incorporated at several key lysine residues critical for PI(4,5)P2 sensitivity in the mouse inward-rectifier potassium channel Kir2.1, expressed in Xenopus oocytes. Caging of lysine by introducing HCK at K182 or K187 completely silenced Kir2.1 currents, but light-induced uncaging restored current activity. Voltage-sensing phosphatase assays revealed that this current increase was accompanied by enhanced PI(4,5)P2 sensitivity. On the other hand, introducing HCK at K219, which forms a secondary PI(4,5)P2-binding region, did not fully eliminate Kir2.1 currents, and uncaging resulted in an approximately twofold increase in current. Analysis of uncaging and PI(4,5)P2 sensitivity in Kir2.1-K219HCK revealed that the region C-terminal to residue K219 is dispensable when assembled with the full-length protein. Genetic code expansion using caged lysine provides a valuable tool for studying the mechanisms of PI(4,5)P2 regulation in ion channels, complementing existing approaches.

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.