Structural change of the cytoplasmic N-terminus and S1 segment of voltage-sensing phosphatase reported by Anap.

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.

Role of K364 next to the active site cysteine in voltage-dependent phosphatase activity of Ci-VSP.

Voltage-sensing phosphatase (VSP) consists of the voltage sensor domain (VSD) similar to that of voltage-gated ion channels and the cytoplasmic phosphatase region with remarkable similarity to the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Membrane depolarization activates VSD, leading to dephosphorylation of three species of phosphoinositides (phosphatidylinositol phosphates (PIPs)), PI(3,4,5)P3, PI(4,5)P2, and PI(3,4)P2. VSP dephosphorylates 3- and 5-phosphate of PIPs, unlike PTEN, which shows rigid 3-phosphate specificity. In this study, a bioinformatics search showed that some mammals have VSP orthologs with amino acid diversity in the active center motif, Cx5R, which is highly conserved among protein tyrosine phosphatases and PTEN-related phosphatases; lysine next to the active site cysteine in the Cx5R motif was substituted for methionine in VSP orthologs of Tasmanian devil, koala, and prairie deer mouse, and leucine in opossum. Since lysine at the corresponding site in PTEN is known to be critical for enzyme activities, we attempted to address the significance of amino acid diversity among VSP orthologs at this site. K364 was changed to different amino acids in sea squirt VSP (Ci-VSP), and voltage-dependent phosphatase activity in Xenopus oocyte was studied using fluorescent probes for PI(4,5)P2 and PI(3,4)P2. All mutants retained both 5-phosphatase and 3-phosphatase activity, indicating that lysine at this site is dispensable for 3-phosphatase activity, unlike PTEN. Notably, K364M mutant showed increased activity both of 5-phosphatase and 3-phosphatase compared with the wild type (WT). It also showed slower kinetics of voltage sensor motion. Malachite green assay of K364M mutant did not show significant difference of phosphatase activity from WT, suggesting tighter interaction between substrate binding and voltage sensing. Mutation corresponding to K364M in the zebrafish VSP led to enhanced voltage-dependent dephosphorylation of PI(4,5)P2. Further studies will provide clues to understanding of substrate preference in PIPs phosphatases as well as to customization of a molecular tool.

Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltage-sensing phosphatase (VSP).

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.

Engineering voltage sensing phosphatase (VSP).

Voltage sensing phosphatase (VSP), consists of a voltage sensor domain (VSD) like that found in voltage-gated ion channels and a phosphoinositide (PIP) phosphatase region exhibiting remarkable structural similarity to a tumor suppressor enzyme, PTEN. Membrane depolarization activates the enzyme activity through tight coupling between the VSD and enzyme region. The VSD of VSP has a unique nature; it is a self-contained module that can be transferred to other proteins, conferring voltage sensitivity. Thanks to this nature, numerous versions of gene-encoded voltage indicators (GEVIs) have been developed through combination of a fluorescent protein with the VSD of VSP. In addition, VSP itself can also serve as a tool to alter PIP levels in cells. Cellular levels of PIPs, PI(4,5)P2 in particular, can be acutely and transiently reduced using a simple voltage protocol after heterologous expression of VSP. Recent progress in our understanding of the molecular structure and mechanisms underlying VSP facilitates optimization of its molecular properties for its use as a molecular tool.

Engineering an enhanced voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestinalis VSP. This modified Dr-VSP (CiDr-VSPmChe L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P2 sensitivity of ion channels in living cells.

Distinct functional properties of two electrogenic isoforms of the SLC34 Na-Pi cotransporter.

Inorganic phosphate (Pi ) is crucial for proper cellular function in all organisms. In mammals, type II Na-Pi cotransporters encoded by members of the Slc34 gene family play major roles in the maintenance of Pi homeostasis. However, the molecular mechanisms regulating Na-Pi cotransporter activity within the plasma membrane are largely unknown. In the present study, we used two approaches to examine the effect of changing plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) levels on the activities of two electrogenic Na-Pi cotransporters, NaPi-IIa and NaPi-IIb. To deplete plasma membrane PI(4,5)P2 in Xenopus oocytes, we utilized Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which dephosphorylates PI(4,5)P2 to phosphatidylinositol 4-phosphate (PI(4)P). Upon activation of Ci-VSP, NaPi-IIb currents were significantly decreased, whereas NaPi-IIa currents were unaffected. We also used the rapamycin-inducible Pseudojanin (PJ) system to deplete both PI(4,5)P2 and PI(4)P from the plasma membrane of cultured Neuro 2a cells. Depletion of PI(4,5)P2 and PI(4)P using PJ significantly reduced NaPi-IIb activity, but NaPi-IIa activity was unaffected, which excluded the possibility that NaPi-IIa is equally sensitive to PI(4,5)P2 and PI(4)P. These results indicate that NaPi-IIb activity is regulated by PI(4,5)P2 , whereas NaPi-IIa is not sensitive to either PI(4,5)P2 or PI(4)P. In addition, patch clamp recording of NaPi-IIa and NaPi-IIb currents in cultured mammalian cells enabled kinetic analysis with higher temporal resolution, revealing their distinct kinetic properties.