Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

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.

(R)-ketamine restores anterior insular cortex activity and cognitive deficits in social isolation-reared mice.

Chronic social isolation increases the risk of mental health problems, including cognitive impairments and depression. While subanesthetic ketamine is considered effective for cognitive impairments in patients with depression, the neural mechanisms underlying its effects are not well understood. Here we identified unique activation of the anterior insular cortex (aIC) as a characteristic feature in brain-wide regions of mice reared in social isolation and treated with (R)-ketamine, a ketamine enantiomer. Using fiber photometry recording on freely moving mice, we found that social isolation attenuates aIC neuronal activation upon social contact and that (R)-ketamine, but not (S)-ketamine, is able to counteracts this reduction. (R)-ketamine facilitated social cognition in social isolation-reared mice during the social memory test. aIC inactivation offset the effect of (R)-ketamine on social memory. Our results suggest that (R)-ketamine has promising potential as an effective intervention for social cognitive deficits by restoring aIC function.

What voltage-sensing phosphatases can reveal about the mechanisms of ion channel regulation by phosphoinositides.

Many membrane proteins including ion channels and ion transporters are regulated by membrane phospholipids such as phosphoinositides in cell membranes and organelles. Voltage-sensing phosphatase, VSP, is a voltage-sensitive phosphoinositide phosphatase which dephosphorylates PI(4,5)P2 into PI(4)P. VSP rapidly reduces the level of PI(4,5)P2 upon membrane depolarization, thus serving as a useful tool to quantitatively study phosphoinositide-regulation of ion channels and ion transporters using a cellular electrophysiology system. In this review, we focus on the application of VSPs to Kv7 family potassium channels, which have been important research targets in biophysics, pharmacology and medicine.

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.

Obsessive-Compulsive Disorder with Psychotic Features: Is It a Clinical Entity?

(1) Background: Even though the comorbidity of obsessive-compulsive disorder (OCD) and a psychotic disorder (PD), such as schizophrenia, is being increasingly recognized, the impact of this comorbidity on the clinical presentation, including insight into obsessive-compulsive symptoms and the functioning of OCD, remains unclear. (2) Methods: To investigate clinical differences between OCD patients with and without PD, 86 Japanese outpatients who met the DSM-IV-TR criteria for OCD were recruited and divided into two groups: 28 OCD patients with PD, and 58 OCD patients without PD. The two groups were cross-sectionally compared in terms of their sociodemographic profiles and clinical characteristics, including the DSM-IV-TR insight specifier and the Global Assessment of Functioning (GAF). (3) Results: The results showed that OCD patients with PD scored lower on both the insight and GAF assessments. (4) Conclusions: The present study suggests that comorbid PD in OCD is a clinical entity.

Voltage-sensing phosphatase (Vsp) regulates endocytosis-dependent nutrient absorption in chordate enterocytes.

Voltage-sensing phosphatase (Vsp) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. Vsp orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of Vsp in native tissues remains largely unknown. Here we report that zebrafish Vsp (Dr-Vsp), encoded by tpte gene, is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-Vsp-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-Vsp-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis Vsp (Ci-Vsp) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of Vsp in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species.

Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP2 regulation.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel's pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein-ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.

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.

Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum.

Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

Spotlight on the Binding Affinity of Ion Channels for Phosphoinositides: From the Study of Sperm Flagellum.

The previous studies revealed that many types of ion channels have sensitivity to PtdIns(4,5)P2, which has been mainly shown using heterologous expression system. On the other hand, there remains few evidence showing that PtdIns(4,5)P2 natively regulate the ion channel activities in physiological context. Our group recently discovered that a sperm specific K+ channel, Slo3, is natively regulated by PtdIns(4,5)P2 in sperm flagellum. Very interestingly, a principal piece, to which Slo3 specifically localized, had extremely low density of PtdIns(4,5)P2 compared to the regular cell plasma membrane. Furthermore, our studies and the previous ones also revealed that Slo3 had much stronger PtdIns(4,5)P2 affinity than KCNQ2/3 channels, which are widely regulated by endogenous PtdIns(4,5)P2 in neurons. Thus, the high-PtdIns(4,5)P2 affinity of Slo3 is well-adapted to the specialized PtdIns(4,5)P2 environment in the principal piece. This study sheds light on the relationship between PtdIns(4,5)P2-affinity of ion channels and their PtdIns(4,5)P2 environment in native cells. We discuss the current understanding about PtdIns(4,5)P2 affinity of diverse ion channels and their possible regulatory mechanism in native cellular environment.

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.

Heterologous functional expression of ascidian Nav1 channels and close relationship with the evolutionary ancestor of vertebrate Nav channels.

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary ß subunits. Detailed information on the structure-function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate ß subunits, the human ß1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II-III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.

Regulation of Neutrophil Functions by Hv1/VSOP Voltage-Gated Proton Channels.

The voltage-gated proton channel, Hv1, also termed VSOP, was discovered in 2006. It has long been suggested that proton transport through voltage-gated proton channels regulate reactive oxygen species (ROS) production in phagocytes by counteracting the charge imbalance caused by the activation of NADPH oxidase. Discovery of Hv1/VSOP not only confirmed this process in phagocytes, but also led to the elucidation of novel functions in phagocytes. The compensation of charge by Hv1/VSOP sustains ROS production and is also crucial for promoting Ca2+ influx at the plasma membrane. In addition, proton extrusion into neutrophil phagosomes by Hv1/VSOP is necessary to maintain neutral phagosomal pH for the effective killing of bacteria. Contrary to the function of Hv1/VSOP as a positive regulator for ROS generation, it has been revealed that Hv1/VSOP also acts to inhibit ROS production in neutrophils. Hv1/VSOP inhibits hypochlorous acid production by regulating degranulation, leading to reduced inflammation upon fungal infection, and suppresses the activation of extracellular signal-regulated kinase (ERK) signaling by inhibiting ROS production. Thus, Hv1/VSOP is a two-way player regulating ROS production. Here, we review the functions of Hv1/VSOP in neutrophils and discuss future perspectives.

Heterogeneity of microglial proton channel in different brain regions and its relationship with aging.

The properties of microglia largely differ depending on aging as well as on brain regions. However, there are few studies that investigated the functional importance of such heterogeneous properties of microglia at the molecular level. Voltage-gated proton channel, Hv1/VSOP, could be one of the candidates which confers functional heterogeneity among microglia since it regulates brain oxidative stress in age-dependent manner. In this study, we found that Hv1/VSOP shows brain region-dependent heterogeneity of gene expression with the highest level in the striatum. We studied the importance of Hv1/VSOP in two different brain regions, the cerebral cortex and striatum, and examined their relationship with aging (using mice of different ages). In the cortex, we observed the age-dependent impact of Hv1/VSOP on oxidative stress, microglial morphology, and gene expression profile. On the other hand, we found that the age-dependent significance of Hv1/VSOP was less obvious in the striatum than the cortex. Finally, we performed a battery of behavioral experiments on Hv1/VSOP-deficient mice both at young and aged stages to examine the effect of aging on Hv1/VSOP function. Hv1/VSOP-deficient mice specifically showed a marked difference in behavior in light/dark transition test only at aged stages, indicating that anxiety state is altered in aged Hv1/VSOP mice. This study suggests that a combination of brain region heterogeneity and animal aging underscores the functional importance of Hv1/VSOP in microglia.

Deep brain stimulation for refractory obsessive-compulsive disorder (OCD): emerging or established therapy?

A consensus has yet to emerge whether deep brain stimulation (DBS) for treatment-refractory obsessive-compulsive disorder (OCD) can be considered an established therapy. In 2014, the World Society for Stereotactic and Functional Neurosurgery (WSSFN) published consensus guidelines stating that a therapy becomes established when "at least two blinded randomized controlled clinical trials from two different groups of researchers are published, both reporting an acceptable risk-benefit ratio, at least comparable with other existing therapies. The clinical trials should be on the same brain area for the same psychiatric indication." The authors have now compiled the available evidence to make a clear statement on whether DBS for OCD is established therapy. Two blinded randomized controlled trials have been published, one with level I evidence (Yale-Brown Obsessive Compulsive Scale (Y-BOCS) score improved 37% during stimulation on), the other with level II evidence (25% improvement). A clinical cohort study (N = 70) showed 40% Y-BOCS score improvement during DBS, and a prospective international multi-center study 42% improvement (N = 30). The WSSFN states that electrical stimulation for otherwise treatment refractory OCD using a multipolar electrode implanted in the ventral anterior capsule region (including bed nucleus of stria terminalis and nucleus accumbens) remains investigational. It represents an emerging, but not yet established therapy. A multidisciplinary team involving psychiatrists and neurosurgeons is a prerequisite for such therapy, and the future of surgical treatment of psychiatric patients remains in the realm of the psychiatrist.

Regulation of hepatic oxidative stress by voltage-gated proton channels (Hv1/VSOP) in Kupffer cells and its potential relationship with glucose metabolism.

Voltage-gated proton channels (Hv1/VSOP), encoded by Hvcn1, are important regulator of reactive oxygen species (ROS) production in many types of immune cells. While in vitro studies indicate that Hv1/VSOP regulates ROS production by maintaining pH homeostasis, there are few studies investigating the functional importance of Hv1/VSOP in vivo. In the present study, we first show that Hv1/VSOP is functionally expressed in liver resident macrophage, Kupffer cells, regulating the hepatic oxidative stress in vivo. Our immunocytochemistry and electrophysiology data showed that Hvcn1 is specifically expressed in Kupffer cells, but not in hepatocytes. Furthermore, Hvcn1-deficiency drastically altered the hepatic oxidative stress. The Hvcn1-deficient mice showed high blood glucose and serum insulin but normal insulin sensitivity, indicating that these phenotypes were not linked to insulin resistance. Transcriptome analysis indicated that the gene expression of glycogen phosphorylase (Pygl) and Glucose-6-phosphatase, catalytic subunit (G6pc) were upregulated in Hvcn1-deficient liver tissues, and quantitative PCR confirmed the result for Pygl. Furthermore, we observed higher amount of glucose-6-phosphate, a key sugar intermediate for glucose in Hvcn1-deficient liver than WT, suggesting that glucose production in liver is accelerated in Hvcn1-deficient mice. The present study sheds light on the functional importance of Kupffer cells in hepatic oxidative stress and its potential relationship with glucose metabolism.

TRPM5 Negatively Regulates Calcium-Dependent Responses in Lipopolysaccharide-Stimulated B Lymphocytes.

B cells produce high amounts of cytokines and immunoglobulins in response to lipopolysaccharide (LPS) stimulation. Calcium signaling cascades are critically involved in cytokine production of T cells, and the cytosolic calcium concentration is regulated by calcium-activated monovalent cation channels (CAMs). Calcium signaling is also implicated in B cell activation; however, its involvement in the cytokine production of LPS-stimulated B cells remains less well characterized. Here, we show that the transient receptor potential melastatin 5 channel (TRPM5), which is one of the CAMs, negatively modulates calcium signaling, thereby regulating LPS-induced proliferative and inflammatory responses by B cells. LPS-stimulated B cells of Trpm5-deficient mice exhibit an increased cytosolic calcium concentration, leading to enhanced proliferation and the production of the inflammatory cytokines interleukin-6 and CXCL10. Furthermore, Trpm5-deficient mice show an exacerbation of endotoxic shock with high mortality. Our findings demonstrate the importance of TRPM5-dependent regulatory mechanisms in LPS-induced calcium signaling of splenic B cells.

The Slo3/Lrrc52 complex is sensitive to phosphoinositides.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphatase activity toward phosphoinositides. Recently, we reported that VSP is activated and generates polarized PtdIns(4,5)P2 distribution in sperm flagellum. Interestingly, such specialized PtdIns(4,5)P2 distribution appears to contribute to the activity of Slo3, a sperm-specific K+ channels. It has been already reported that Slo3 activity is upregulated by PtdIns(4,5)P2 using a heterologous expression system. However, PtdIns(4,5)P2-dependence of Slo3 activity has not been studied in heterologous expression system in the presence of auxiliary subunit of Slo3, Lrrc52, which drastically changes the electrophysiological property of Slo3. In the present study, we analyzed the regulation of Slo3 activity with Lrrc52 by VSP in Xenopus oocytes. Slo3 with Lrrc52 still exhibited similar sensitivity to VSP activity as Slo3 alone. This finding supports our previous report that VSP regulates Slo3 activity in native sperm flagellum.

Hv1/VSOP regulates neutrophil directional migration and ERK activity by tuning ROS production.

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1 µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.