Ciona Intestinalis Voltage-sensing Phosphatase Research Articles

Dynamics of activation in the voltage-sensing domain of Ciona intestinalis phosphatase Ci-VSP

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltage-sensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Previously published crystal structures of Ci-VSD in putative resting and active conformations suggested a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners, but the microscopic details of the transition between the resting and active conformations remained unknown. Here, by combining extensive molecular dynamics simulations with a recently developed computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates that are hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism.

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

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.

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.

Coupling Mechanisms of VSD Mutants of Ci-VSP

Voltage-sensing phosphatase (VSP) shows phosphatase activity toward phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) upon membrane depolarization. Voltage sensor deficient mutants lack phosphatase activity and mutations in a short linker between voltage sensor domain (VSD) and the PTEN-like phosphatase domain reduce phosphatase activity, suggesting that activation of VSD “couples” with phosphatase activity via the linker. However, it is unclear how VSD regulates phosphatase activity. We have recently found that amino acid mutation at F234 (two amino acids downstream of the 4th arginine (R4) of S4) in Ciona intestinalis VSP (Ci-VSP) causes a remarkable decrease in the phosphatase activity whereas the gating current is normal. To understand what structural detail of VSD is optimal for inducting enzyme activity in Ci-VSP, we analyzed both VSD motions and enzyme activities of F234 mutants and other VSD mutants. Mutants of Ci-VSP were expressed in Xenopus oocytes and analyzed by the two-electrode voltage clamp. We studied the voltage-driven motion of VSD or cytoplasmic catalytic region with thiol-reactive fluorescent dye or with unnatural fluorescent amino acid, Anap, respectively. Surprisingly, in multiple mutants including F234 mutant, phosphatase activity remarkably decreased whereas the gating current remained intact. We interpret these findings based on the hypotheses: (1) VSD cannot be fully activated, leading only partial phosphatase activity, (2) the structure of the fully-activated VSD differs from that of the normal full activation state, failing to induce the full phosphatase activity, (3) local membrane structure or environment facing the cytoplasmic side of the VSD may be altered by the mutations, possibly preventing the interaction between enzyme region and the plasma membrane which may be critical for phosphatase activity (the second and the third idea are not exclusive to each other).

The Energy Landscape of Voltage Sensing in Ci-VSP

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.

Structural Determinants of Kv7.5 Potassium Channels That Confer Changes in Phosphatidylinositol 4,5-Bisphosphate (PIP2) Affinity and Signaling Sensitivities in Smooth Muscle Cells.

Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5 α-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)-induced PIP2 depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP2, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP2 affinity; these changes in PIP2 affinity have corresponding effects on channel activity. Resting affinities for PIP2 differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.

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.

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P3 or PI(3,4)P2 Our results indicate that dimerization plays a significant role in Ci-VSP function.

Domain-to-domain coupling in voltage-sensing phosphatase.

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

Allosteric substrate switching in a voltage-sensing lipid phosphatase.

Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis.

Computational Characterization of Conformational Transitions in the Voltage-Sensing Domain of Ci-VSP

The canonical voltage-sensing domain (VSD) is a conserved structural module that transduces the electric field across cellular membranes into defined protein motions of its charge-rich helix, the S4 segment. Up on depolarization, the VSD rearranges from the ‘resting’ (or ‘down’) state conformation into the ‘active’ (or ‘up’) state conformation, which in turn regulates the motion of downstream modules of the protein, such as opening of the pore domain of a voltage-gated ion channel or the activity of an enzyme. The voltage-sensing process has long attracted much interest and fruitful results have been reported using a variety of functional, spectroscopic and structural approaches. Recently, we solved crystal structures of the VSD of the Ciona intestinalis voltage-sensing phosphatase Ci-VSP in both it resting (Down) and activated (Up) conformations. Comparison of the two structures reveals an ∼ 5-A three-residue displacement of the S4 segment along, together with an ∼ 60° rotation around, its axis (called the one-click gating motion), while the S1-S3 segments remain fairly stable. Using 2-D self-learning umbrella sampling molecular dynamics simulations, combined with homology modeling and the string method, we have now calculated the free energy landscape governing the conformational changes of Ci-VSD. These calculations have allowed us to explore the energetic viability of further up (one-click up) and further down (two-clicks down) conformations, as potential mechanisms for additional charge translocation. These conformations should advance our understanding of the detailed mechanisms of voltage-gating and allow for the design of new experiments aimed to verify the present model of S4 movements and gating charge translocation.

Editorial: Phosphoinositides and their phosphatases: Linking electrical and chemical signals in biological processes.

Editorial: Phosphoinositides and their phosphatases: Linking electrical and chemical signals in biological processes.

V220 Mutants of Ciona Voltage-Sensing Domain Based Genetically Encoded Fluorescent Voltage Sensors

Genetically-encoded fluorescent voltage sensors enable the optical reporting of changes in membrane potentials. These sensors consist of a voltage-sensing domain of voltage-sensing phosphatase (usually from Ciona intestinalis Voltage-Sensing phosphatase) fused to a fluorescent protein (Super Ecliptic pHlorin A227D). To optically differentiate action potentials from sub-threshold synaptic activity, residues in the voltage-sensing domain were mutated. Surprisingly one mutation, V220T in the S4 domain shifted the voltage response to more negative potentials, since this residue does not interact with the positively charged residues in S4 responsible for gating currents. To fine tune the voltage sensitivity of this probe, we mutated the V220 position extensively testing the remaining 19 amino acids. We found that tyrosine, phenylalanine, tryptophan, aspartate, threonine, and proline shift the voltage response substantially to more negative potentials enabling optical signals from hyper-polarizing voltages. Polar amino acids (glutamine, asparagine, alanine, and serine) vastly reduce the optical signal. Hydrophobic residues leucine, isoleucine, methionine, cysteine do not shift the voltage sensitivity to more negative potentials. We combined another mutant D164N (S2 domain) capable of shifting the voltage sensitivity to left with V220F and P. This combination further shifts the voltage response to more negative potentials and shallows the curve. These results suggest that the V220 residue interacts with the plasma membrane and could be mutated in conjunction with other mutations to generate probes capable of differentiating action potentials from sub-threshold synaptic activity, and hyper-polarizing inhibitory activity.

Let’s Twist (the S4) Again

Let’s Twist (the S4) Again

A glutamate switch controls voltage-sensitive phosphatase function

Ciona intestinalis voltage sensing phosphatase Ci-VSP couples a voltage-sensing domain (VSD) to a lipid phosphatase similar to the tumor suppressor PTEN. How the VSD controls enzyme function has been unclear. Here, we present high-resolution crystal structures of the Ci-VSP enzymatic domain that reveal conformational changes in a key loop, termed the “gating loop”, that controls access to the active site by a mechanism in which residue Glu411 directly competes with substrate. Structure-based mutations that restrict gating loop conformation impair catalytic function and demonstrate that Glu411 also contributes to substrate selectivity. Structure-guided mutations further define an interaction between the gating loop and linker that connects the phosphatase to the VSD for voltage control of enzyme activity. Together, the data suggest that functional coupling between the gating loop and the linker forms the heart of the regulatory mechanism that controls voltage-dependent enzyme activation.

The Use of Engineered Voltage-Sensing Phosphatases to Study the Determinants of Substrate Specificity of Phosphoinositide Phosphatases

Ciona intestinalis voltage sensing phosphatase (Ci-VSP) is the founding member of a new class of voltage-sensing proteins, and exhibits PI(3,4,5)P3 and PI(4,5)P2 5’phosphatase activity upon membrane depolarisation (Murata and Okamura, 2007; Iwasaki et al., 2008; Halaszovich et al., 2009). Ci-VSP consists of a N-terminal voltage sensing domain (VSD) and a C-terminal phosphatase domain (PD) that is highly homologous to the phosphoinositide (PI) phosphatase PTEN (Murata et al., 2005). Recently, we reported on a voltage-sensitive chimera of the Ci-VSP voltage-sensor and the tumour suppressor PTEN (Ci-VSPTEN) in agreement with PTEN's 3’phosphatase activity (Lacroix et al., 2011). Here we extend this knowledge to a novel chimera of the VSD of Ci-VSP and the PTEN homologue TPIPα (also known as TPTE2), termed Ci-VSP/TPIP (Walker et al., 2001).Using whole cell voltage-clamp and total internal reflection microscopy with genetically-encoded phosphoinositide sensors, we show that Ci-VSP/TPIP is a voltage-sensitive PI(3,4,5)P3 and PI(4,5)P2 5’phosphatase in contrast to previous reports of TPIPα as a 3’phosphatase (Walker et al., 2001). The voltage dependence of Ci-VSP/TPIP was shifted to hyperpolarized potentials compared to Ci-VSP. Functional comparisons of Ci-VSP, Ci-VSPTEN and Ci-VSP/TPIP identified alanin/glycine 365 as the critical determinant of PTEN's substrate specificity, since Ci-VSPTEN(A365G) produced robust voltage-dependent PI(3,4,5)P3 5’ phosphatase activity, in contrast to the 3’phosphatase activity of wild-type PTEN.In conclusions, our data characterise the 5’phosphatase Ci-VSP/TPIP and reveal that engineered voltage-sensing phosphatases can be used to study the activity and substrate specificity of the important tumour suppressor PTEN in vivo.This work was supported by a Research Grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to C.R.H and by Deutsche Forschungsgemeinschaft through SFB 593 TP A12 to D.O.

Internal Switch Gates Phosphatase Activity of VSP

Voltage regulation is responsible for a host of processes in the cell from muscle contraction to neuronal action potentials. In the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)1, voltage is responsible for controlling lipid phosphatase activity. How this happens is still not known. To address this, we have solved seven crystal structures of the cytosolic fragment of Ci-VSP. The structures reveal three active site conformations, one where the active site is blocked, one where the active site is open and one where the active site is bound with substrate. These three conformations show that the active site is gated. Activity assays on structure-based mutants show that the gate is involved in catalytic turnover as well as substrate specificity. A residue from the C2 domain that lines the active site in Ci-VSP, but not in the homologous phosphatase PTEN, also contributes to selectivity. A potential interaction between the segment of the protein that gates the active site and the inter-domain linker between the voltage sensing domain and the phosphatase suggests a model for coupling the voltage sensor to activity. The results indicate that voltage regulates enzyme activity by gating the active site.1. Murata, Y., et al, (2005) Nature 435, 1239-1243.

Crystal Structure of the Cytoplasmic Phosphatase and Tensin Homolog (PTEN)-like Region of Ciona intestinalis Voltage-sensing Phosphatase Provides Insight into Substrate Specificity and Redox Regulation of the Phosphoinositide Phosphatase Activity

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

Another Look at Ci-VSP

Voltage changes are involved in a range of biological functions including neuronal signal propagation, muscle contraction and T-cell activation. Voltage sensitive proteins respond to the changes in membrane potential via a voltage-sensing domain (VSD). Voltage-gated ion channels use the VSD to open and close a pore domain, which allows for the passive movement of ions down their concentration gradients. The discovery of the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)(1) opened up a whole new way of looking at voltage sensing. This is the first and only currently known class of protein that has a VSD but no pore. Instead Ci-VSP, and its VSP homologs including fish and frogs, contains a cytoplasmic lipid phosphatase whose activity is regulated by voltage. It is also the only known VSD to work as a monomer instead of in concert with other VSD modules. It still displays similar complex motions as the VSDs from channels which is intriguing. We and others have also shown that lipids play a role in regulating the protein. Even so, many questions remain unanswered regarding how this voltage activated enzyme works. We have taken an interdisciplinary approach including biochemical and electrophysiological approaches to probe the inner workings of VSPs.1. Murata, Y., et al, (2005) Nature 435, 1239-1243.