Allosteric inhibition of muscle-type nicotinic acetylcholine receptors by a neuromuscular blocking agent pancuronium.

Muscle relaxants are indispensable for surgical anesthesia. Early studies suggested that a classical non-depolarizing muscle relaxant pancuronium competitively binds to the ligand binding site to block nicotinic acetylcholine receptors (nAChR). Our group recently showed that nAChR which has two distinct subunit combinations are expressed in zebrafish muscles, αßδε and αßδ, for which potencies of pancuronium are different. Taking advantage of the distinct potencies, we generated chimeras between two types of nAChRs and found that the extracellular ACh binding site is not associated with the pancuronium sensitivity. Furthermore, application of either 2 µM or 100 µM ACh in native αßδε or αßδ subunits yielded similar IC50 of pancuronium. These data suggest that pancuronium allosterically inhibits the activity of zebrafish nAChRs.

Receptor subunit compositions underly distinct potencies of a muscle relaxant in fast and slow muscle fibers.

A line of studies in the 1960s-1980s suggested that muscle relaxants do not work uniformly on all skeletal muscles, though its mechanism has not been clarified. We showed here that a classical non-depolarizing muscle relaxant pancuronium inhibits fast muscle fibers at lower concentration compared to slow muscle fibers in zebrafish. The difference of effective concentration was observed in locomotion caused by tactile stimulation as well as in synaptic currents of the neuromuscular junction induced by motor neuron excitation. We further showed that this difference arises from the different composition of acetylcholine receptors between slow and fast muscle fibers in the neuromuscular junction of zebrafish. It will be interesting to examine the difference of subunit composition and sensitivity to muscle relaxants in other species.

Efficient visible/NIR light-driven uncaging of hydroxylated thiazole orange-based caged compounds in aqueous media.

In photoactivation strategies with bioactive molecules, one-photon visible or two-photon near-infrared light-sensitive caged compounds are desirable tools for biological applications because they offer reduced phototoxicity and deep tissue penetration. However, visible-light-sensitive photoremovable protecting groups (PPGs) reported so far have displayed high hydrophobicity and low uncaging cross sections (ÎµΦ < 50) in aqueous media, which can obstruct the control of bioactivity with high spatial and temporal precision. In this study, we developed hydroxylated thiazole orange (HTO) derivatives as visible-light-sensitive PPGs with high uncaging cross sections (ÎµΦ ≈ 370) in aqueous solution. In addition, 2PE photolysis reactions of HTO-caged glutamate were achieved using a NIR laser (940 nm). Moreover, HTO-caged glutamate can activate N-methyl-d-aspartic acid receptors in Xenopus oocytes and mammalian cells with green-light illumination, thus allowing optical control of biological functions.

Polarized PtdIns(4,5)P2 distribution mediated by a voltage-sensing phosphatase (VSP) regulates sperm motility.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.

Dynamic structural rearrangements and functional regulation of voltage-sensing phosphatase.

The voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region. The latter contains a phosphatase domain and a C2 domain, showing remarkable similarity to the tumour suppressor enzyme PTEN. In VSP, membrane depolarization induces a conformational change in the VSD, which activates the phosphoinositide phosphatase. The final outcome in VSP is enzymatic activity in the cytoplasmic region, unlike in voltage-gated ion channels where conformational change of the transmembrane pore is induced by the VSD. Therefore, it is crucial to detect structural change in the cytoplasmic catalytic region to gain insights into the operating mechanisms of VSP. This review summarizes a recent study in which a method of genetic incorporation of a non-canonical amino acid, Anap, was used to detect dynamic membrane voltage-controlled rearrangements of the structure of the catalytic region of sea squirt VSP (Ci-VSP). Upon membrane depolarization, both the phosphatase domain and the C2 domain move in a similar time frame, suggesting that the two regions are coupled to each other. Measurement of Förster resonance energy transfer (FRET) between Anap introduced into the C2 domain of Ci-VSP and dipicrylamine in the cell membrane suggested no large movement of the enzyme towards the membrane. Fluorescence changes in Anap induced by different membrane potentials indicate the presence of multiple conformations of the active enzyme.

The hydrophobic nature of a novel membrane interface regulates the enzyme activity of a voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) contain a voltage sensor domain (VSD) similar to that of voltage-gated ion channels but lack a pore-gate domain. A VSD in a VSP regulates the cytoplasmic catalytic region (CCR). However, the mechanisms by which the VSD couples to the CCR remain elusive. Here we report a membrane interface (named 'the hydrophobic spine'), which is essential for the coupling of the VSD and CCR. Our molecular dynamics simulations suggest that the hydrophobic spine of Ciona intestinalis VSP (Ci-VSP) provides a hinge-like motion for the CCR through the loose membrane association of the phosphatase domain. Electrophysiological experiments indicate that the voltage-dependent phosphatase activity of Ci-VSP depends on the hydrophobicity and presence of an aromatic ring in the hydrophobic spine. Analysis of conformational changes in the VSD and CCR suggests that the VSP has two states with distinct enzyme activities and that the second transition depends on the hydrophobic spine.

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.

Comparison between mouse and sea urchin orthologs of voltage-gated proton channel suggests role of S3 segment in activation gating.

The voltage-gated proton channel, Hv1, is expressed in blood cells, airway epithelium, sperm and microglia, playing important roles in diverse biological contexts including phagocytosis or sperm maturation through its regulation of membrane potential and pH. The gene encoding Hv1, HVCN1, is widely found across many species and is also conserved in unicellular organisms such as algae or dinoflagellates where Hv1 plays role in calcification or bioluminescence. Voltage-gated proton channels exhibit a large variation of activation rate among different species. Here we identify an Hv1 ortholog from sea urchin, Strongylocentrotus purpuratus, SpHv1. SpHv1 retains most of key properties of Hv1 but exhibits 20-60 times more rapid activation kinetics than mammalian orthologs upon heterologous expression in HEK293T cells. Comparison between SpHv1 and mHv1 highlights novel roles of the third transmembrane segment S3 in activation gating of Hv1.

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.

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.

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.

X-ray crystal structure of voltage-gated proton channel.

The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

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.

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 a voltage sensor and a cytoplasmic phosphatase region, and the movement of the voltage sensor is coupled to the phosphatase activity. However, its coupling mechanisms still remain unclear. One possible scenario is that the phosphatase is activated only when the voltage sensor is in a fully activated state. Alternatively, the enzymatic activity of single VSP proteins could be graded in distinct activated states of the voltage sensor, and partial activation of the voltage sensor could lead to partial activation of the phosphatase. To distinguish between these two possibilities, we studied a voltage sensor mutant of zebrafish VSP, where the voltage sensor moves in two steps as evidenced by analyses of charge movements of the voltage sensor and voltage clamp fluorometry. Measurements of the phosphatase activity toward phosphatidylinositol 4,5-bisphosphate revealed that both steps of voltage sensor activation are coupled to the tuning of phosphatase activities, consistent with the idea that the phosphatase activity is graded by the magnitude of the movement 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 VSP's functions could be to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile.

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)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] 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)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-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)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) 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.

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

The voltage sensing phosphatase Ci-VSP is composed of a voltage sensor domain (VSD) and a cytoplasmic phosphatase domain. Upon membrane depolarization, movement of the VSD triggers the enzyme's phosphatase activity. To gain further insight into its operating mechanism, we studied the PI(4,5)P2 phosphatase activity of Ci-VSP expressed in Xenopus oocytes over the entire range of VSD motion by assessing the activity of coexpressed Kir2.1 channels or the fluorescence signal from a pleckstrin homology domain fused with green fluorescent protein (GFP) (PHPLC-GFP). Both assays showed greater phosphatase activity at 125 mV than at 75 mV, which corresponds to 'sensing' charges that were 90% and 75% of maximum, respectively. On the other hand, the activity at 160 mV (corresponding to 98% of the maximum 'sensing' charge) was indistinguishable from that at 125 mV. Modelling the kinetics of the PHPLC-GFP fluorescence revealed that its time course was dependent on both the level of Ci-VSP expression and the diffusion of PHPLC-GFP beneath the plasma membrane. Enzyme activity was calculated by fitting the time course of PHPLC-GFP fluorescence into the model. The voltage dependence of the enzyme activity was superimposable on the Q-V curve, which is consistent with the idea that the enzyme activity is tightly coupled to VSD movement over the entire range of membrane potentials that elicit VSD movement.

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.

Characterization of native myosin VI isolated from sea urchin eggs.

Myosin VI is a molecular motor that is ubiquitously expressed among eukaryotic cells, and thought to be involved in membrane trafficking and anchoring the organelle to actin cytoskeleton. Studies on myosin VI have been carried out using recombinant proteins, but native myosin VI has not been purified yet. Here we purified native myosin VI from sea urchin eggs and characterized its properties. We found that the native myosin VI was a monomeric and non-processive motor protein, and also showed that it moved toward the pointed end of F-actin. Ca2+ stimulated actin-activated MgATPase activity of the native myosin VI, while it lowered its motility on F-actin. Immunofluorescence microscopy showed that the myosin VI was translocated from the inner cytoplasm to the cortex after fertilization. Myosin VI may be involved in endocytic activities in fertilized eggs.