Souhei Sakata

Functionality of the voltage-gated proton channel truncated in S4

The voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels and voltage-sensing phosphatases. Structurally, both the VSD and the recently discovered voltage-gated proton channels (Hv channels) voltage sensor only protein (VSOP) and Hv1 contain four transmembrane segments. The fourth transmembrane segment (S4) of Hv channels contains three periodically aligned arginines (R1, R2, R3). It remains unknown where protons permeate or how voltage sensing is coupled to ion permeation in Hv channels. Here we report that Hv channels truncated just downstream of R2 in the S4 segment retain most channel properties. Two assays, site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by Western blotting and insertion into dog pancreas microsomes, both showed that S4 inserts into the membrane, even if it is truncated between the R2 and R3 positions. These findings provide important clues to the molecular mechanism underlying voltage sensing and proton permeation in Hv channels.

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P3. TLC assay showed that the 3′ phosphate of PI(3,4,5)P3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was removed by VSPs. Monitoring of PI(3,4)P2 levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PHTAPP1-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P3. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P3 is dephosphorylated at the 5′ position, PI(3,4)P2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.

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.

A Kir3.4 mutation causes Andersen–Tawil syndrome by an inhibitory effect on Kir2.1

Objective: To identify other causative genes for Andersen–Tawil syndrome, which is characterized by a triad of periodic paralysis, cardiac arrhythmia, and dysmorphic features. Andersen–Tawil syndrome is caused in a majority of cases by mutations in KCNJ2, which encodes the Kir2.1 subunit of the inwardly rectifying potassium channel. Methods: The proband exhibited episodic flaccid weakness and a characteristic TU-wave pattern, both suggestive of Andersen–Tawil syndrome, but did not harbor KCNJ2 mutations. We performed exome capture resequencing by restricting the analysis to genes that encode ion channels/associated proteins. The expression of gene products in heart and skeletal muscle tissues was examined by immunoblotting. The functional consequences of the mutation were investigated using a heterologous expression system in Xenopus oocytes, focusing on the interaction with the Kir2.1 subunit. Results: We identified a mutation in the KCNJ5 gene, which encodes the G-protein–activated inwardly rectifying potassium channel 4 (Kir3.4). Immunoblotting demonstrated significant expression of the Kir3.4 protein in human heart and skeletal muscles. The coexpression of Kir2.1 and mutant Kir3.4 in Xenopus oocytes reduced the inwardly rectifying current significantly compared with that observed in the presence of wild-type Kir3.4. Conclusions: We propose that KCNJ5 is a second gene causing Andersen–Tawil syndrome. The inhibitory effects of mutant Kir3.4 on inwardly rectifying potassium channels may account for the clinical presentation in both skeletal and heart muscles.

Coupling of the phosphatase activity of Ci-VSP to its voltage sensor activity over the entire range of voltage sensitivity

Non‐technical summary  Ci‐VSP is a protein that consists of a voltage sensor domain (VSD) and a cytoplasmic phosphatase region. The phosphatase activity is regulated by the VSD. Detailed mechanisms of how the VSD regulates the phosphatase activity are elusive. The voltage range where the phosphatase activity is coupled with the VSD provides important clues to the coupling mechanisms. This paper examined the voltage sensitivity of the phosphatase activity over a wide range of voltage by electrophysiological methods, imaging analysis and mathematical modelling. The results demonstrate that the voltage dependency of the phosphatase activity correlates with that of the VSD. Thus, the phosphatase activity of Ci‐VSP is coupled to the VSD over the entire range of voltages that elicit movement of the VSD.

Gating Mechanisms of Voltage-Gated Proton Channels

Hv1 is a voltage-gated proton-selective channel that plays critical parts in host defense, sperm motility, and cancer progression. Hv1 contains a conserved voltage-sensor domain (VSD) that is shared by a large family of voltage-gated ion channels, but it lacks a pore domain. Voltage sensitivity and proton conductivity are conferred by a unitary VSD that consists of four transmembrane helices. The architecture of Hv1 differs from that of cation channels that form a pore in the center among multiple subunits (as in most cation channels) or homologous repeats (as in voltage-gated sodium and calcium channels). Hv1 forms a dimer in which a cytoplasmic coiled coil underpins the two protomers and forms a single, long helix that is contiguous with S4, the transmembrane voltage-sensing segment. The closed-state structure of Hv1 was recently solved using X-ray crystallography. In this article, we discuss the gating mechanism of Hv1 and focus on cooperativity within dimers and their sensitivity to metal ions.

Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor.

The voltage‐sensing phosphatase (VSP) consists of the voltage sensor and the phosphatase domain. The voltage sensor movement is coupled to the phosphatase activity. To uncover the coupling mechanisms between the two domains, we made a voltage sensor mutant of VSP. Our analyses showed that this voltage sensor moves in two steps. Measurements revealed that the phosphatase activity of this mutant is associated with both the first and second step of the voltage sensor movements. Results suggest that the phosphatase activity of VSP shows graded dependence on the extent of activation of the voltage sensor.

Potential Role of Voltage‐Sensing Phosphatases in Regulation of Cell Structure Through the Production of PI(3,4)P2

Voltage‐sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the cytoplasmic domain with phosphoinositide‐phosphatase activities. The voltage sensor tightly couples with the cytoplasmic phosphatase and membrane depolarization induces dephosphorylation of several species of phosphoinositides. VSP gene is conserved from urochordate to human. There are some diversities among VSP ortholog proteins; range of voltage of voltage sensor motions as well as substrate selectivity. In contrast with recent understandings of biophysical mechanisms of VSPs, little is known about its physiological roles. Here we report that chick ortholog of VSP (designated as Gg‐VSP) induces morphological feature of cell process outgrowths with round cell body in DF‐1 fibroblasts upon its forced expression. Expression of the voltage sensor mutant, Gg‐VSPR153Q with shifted voltage dependence to a lower voltage led to more frequent changes of cell morphology than the wild‐type protein. Coexpression of PTEN that dephosphorylates PI(3,4)P2 suppressed this effect by Gg‐VSP, indicating that the increase of PI(3,4)P2 leads to changes of cell shape. In addition, visualization of PI(3,4)P2 with the fluorescent protein fused with the TAPP1‐derived pleckstrin homology (PH) domain suggested that Gg‐VSP influenced the distribution of PI(3,4)P2. These findings raise a possibility that one of the VSPs functions could be to regulate cell morphology through voltage‐sensitive tuning of phosphoinositide profile. J. Cell. Physiol. 229: 422–433, 2014.

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

Significance Voltage-sensing phosphatase (VSP) dephosphorylates phosphoinositides in a voltage-dependent manner. The molecular mechanisms by which the voltage-sensor domain of VSP activates the catalytic activity of the cytoplasmic region still remain unknown. Using a method of incorporation of a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), in the catalytic region, we revealed that some loops in the catalytic region move on membrane depolarization and that the catalytic region is located beneath the plasma membrane irrespective of the membrane potential. Furthermore, fluorescence change of Anap in the C2 domain showed multiple voltage-dependent activated states and protein conformation, which is sensitive to substrate availability in the active center. These findings provide novel insights into the mechanisms of voltage-dependent catalytic activity of VSP. 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.

Functional diversity of voltage-sensing phosphatases in two urodele amphibians.

Voltage‐sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage‐gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage‐dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn‐VSP) and Cynops VSP (Cp‐VSP), including the positively‐charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C‐terminal C2 domain of Hn‐VSP is significantly shorter than that of Cp‐VSP and other VSP orthologs. RT‐PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage‐dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed “sensing” currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp‐VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn‐VSP lacked such phosphatase activity due to the truncation of its C2 domain.