Is voltage-sensing phosphatase (VSP) regulated by phosphoinositide, Pi(4,5)P2? Question revisited with Anap.

Abstract

Among the voltage-dependent membrane protein superfamily, voltage-sensing phosphatase (VSP) is the only protein in which a voltage sensor domain (VSD) regulates an enzyme activity. The cytoplasmic catalytic region (CCR) with remarkable similarity to phosphatase and tensin homolog (PTEN), which consists of a phosphatase domain (PD) and a C2 domain, is connected to the fourth transmembrane helix (S4) of the VSD through the VSD-PD linker. Through tight coupling between the VSD and the CCR, VSP exhibits the voltage-dependent phosphoinositide phosphatase activity against mainly PI(4,5)P2. We have recently reported that interaction between hydrophobic residues at the C-terminal end of S4 and the CCR mediates coupling in Ciona intestinalis VSP (Ci-VSP) (Mizutani et al., PNAS, 2022). To gain further insights into molecular mechanism of VSP, we then focused on the VSD-PD linker. PI(4,5)P2 is thought to bind to the N-terminal PI(4,5)P2-binding motif (PBM) of PTEN which is also conserved in the C-terminal half of the VSD-PD linker among VSP orthologs. Previous studies suggest that PI(4,5)P2 modulates VSD motion and coupling through the PBM-like region of VSP. However, how PI(4,5)P2 interacts with the linker has not been fully elucidated. In this study, we expressed Ci-VSP with a fluorescent unnatural amino acid (Anap) incorporated in the PBM-like region or its vicinities in Xenopus oocytes, and analyzed PI(4,5)P2-dependent structural rearrangements of these residues by measuring fluorescence upon membrane depolarization by voltage clamp fluorometry. We compared fluorescence signals between in the presence or absence of pre-depolarization. We found activity-dependent changes in the kinetics of the signals at several residues, consistent with the idea that PI(4,5)P2 binds to the PBM-like region or its vicinities. Our finding indicates that PI(4,5)P2 is not only a substrate but also a regulating factor of VSP.