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Nitric oxide (NO) is produced from endothelial cells and cardiomyocytes composing the myocardium and benefits cardiac function through both vascular-dependent and—independent effects. This study was purposed to investigate the possible adverse effect of NO focusing on the voltage-gated Na+ channel in cardiomyocytes. We carried out patch-clamp experiments on rat neonatal cardiomyocytes demonstrating that NOC-18, an NO donor, significantly reduced Na+ channel current in a dose-dependent manner by a long-term application for 24 h, accompanied by a reduction of Nav1.5-mRNA and the protein, and an increase of a transcription factor forkhead box protein O1 (FOXO1) in the nucleus. The effect of NOC-18 on the Na+ channel was blocked by an inhibitor of thiol oxidation N-ethylmaleimide, a disulfide reducing agent disulfide 1,4-Dithioerythritol, or a FOXO1 activator paclitaxel, suggesting that NO is a negative regulator of the voltage-gated Na+ channel through thiols in regulatory protein(s) for the channel transcription.

TRPV1, a member of the transient receptor potential (TRP) channels family, has been found to be involved in redox sensing. The crystal structure of the human TRPV1 ankyrin-repeat domain (TRPV1-ARD) was determined at 4.5 Å resolution under nonreducing conditions. This is the first report of the crystal structure of a ligand-free form of TRPV1-ARD and in particular of the human homologue. The structure showed a unique conformation in finger loop 3 near Cys258, which is most likely to be involved in inter-subunit disulfide-bond formation. Also, in human TRPV1-ARD it was possible for solvent to access Cys258. This structural feature might be related to the high sensitivity of human TRPV1 to oxidants. ESI-MS revealed that Cys258 did not form an S–OH functionality even under nonreducing conditions.

Gemcitabine is an antineoplastic drug commonly used in the treatment of several types of cancers including pancreatic cancer and non–small cell lung cancer. Although gemcitabine-induced cardiotoxicity is widely recognized, the exact mechanism of cardiac dysfunction causing arrhythmias remains unclear. The objective of this study was to electrophysiologically evaluate the proarrhythmic cardiotoxicity of gemcitabine focusing on the human rapid delayed rectifier potassium channel, hERG channel. In heterologous hERG expressing HEK293 cells (hERG-HEK cells), hERG channel current (IhERG) was reduced by gemcitabine when applied for 24 h but not immediately after the application. Gemcitabine modified the activation gating properties of the hERG channel toward the hyperpolarization direction, while inactivation, deactivation or reactivation gating properties were unaffected by gemcitabine. When gemcitabine was applied to hERG-HEK cells in combined with tunicamycin, an inhibitor of N-acetylglucosamine phosphotransferase, gemcitabine was unable to reduce IhERG or shift the activation properties toward the hyperpolarization direction. While a mannosidase I inhibitor kifunensine alone reduced IhERG and the reduction was even larger in combined with gemcitabine, kifunensine was without effect on IhERG when hERG-HEK cells were pretreated with gemcitabine for 24 h. In addition, gemcitabine down-regulated fluorescence intensity for hERG potassium channel protein in rat neonatal cardiomyocyte, although hERG mRNA was unchanged. Our results suggest the possible mechanism of arrhythmias caused by gemcitabine revealing a down-regulation of IhERG through the post-translational glycosylation disruption possibly at the early phase of hERG channel glycosylation in the endoplasmic reticulum that alters the electrical excitability of cells.

Nitric oxide (NO) is produced from endothelial cells and cardiomyocytes composing the myocardium and benefits cardiac function through both vascular-dependent and -independent effects. This study was purposed to investigate the possible adverse effect of NO focusing on the voltage-gated Na+ channel in cardiomyocytes. We carried out patch-clamp experiments on rat neonatal cardiomyocytes demonstrating that NOC-18, an NO donor, significantly reduced Na+ channel current in a dose-dependent manner by a long-term application for 24 h, accompanied by a reduction of Nav1.5-mRNA and the protein, and an increase of a transcription factor forkhead box protein O1 (FOXO1) in the nucleus. The effect of NOC-18 on the Na+ channel was blocked by an inhibitor of thiol oxidation N-ethylmaleimide or a disulfide reducing agent disulfide 1,4-Dithioerythritol, suggesting that NO is a negative regulator of the voltage-gated Na+ channel through thiols in regulatory protein(s) for the channel transcription.

Activation of the atrial natriuretic signaling pathway is intrinsic to the pathological responses associated with a range of cardiovascular diseases that stress the heart, especially those involved in sustained cardiac pressure overload which induces hypertrophy and the pathological remodeling that frequently leads to heart failure. We identify transient receptor potential cation channel, subfamily V, member 1, as a regulated molecular component, and therapeutic target of this signaling system. Data show that TRPV1 is a physical component of the natriuretic peptide A, cGMP, PKG signaling complex, interacting with the Natriuretic Peptide Receptor 1 (NPR1), and upon binding its ligand, Natriuretic Peptide A (NPPA, ANP) TRPV1 activation is subsequently suppressed through production of cGMP and PKG mediated phosphorylation of the TRPV1 channel. Further, inhibition of TRPV1, with orally delivered drugs, suppresses chamber and myocyte hypertrophy, and can longitudinally improve in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction, reversing pre-established hypertrophy induced by pressure load while restoring chamber function. TRPV1 is a physical and regulated component of the natriuretic peptide signaling system, and TRPV1 inhibition may provide a new treatment strategy for treating, and reversing the loss of function associated with cardiac hypertrophy and heart failure.

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40–47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.

Transient Receptor Potential (TRP) proteins form cation channels characterized by a wide variety of activation triggers. Here, we overview a group of TRP channels that respond to reactive redox species to transduce physiological signals, with a focus on TRPA1 and its role in oxygen physiology. Our systematic evaluation of oxidation sensitivity using cysteine-selective reactive disulphides with different redox potentials reveals that TRPA1 has the highest sensitivity to oxidants/electrophiles among the TRP channels, which enables it to sense O2. Proline hydroxylation by O2-dependent hydroxylases also regulates the O2-sensing function by inhibiting TRPA1 in normoxia; TRPA1 is activated by hypoxia through relief from the inhibition and by hyperoxia through cysteine oxidation that overrides the inhibition. TRPA1 enhances neuronal discharges induced by hyperoxia and hypoxia in the vagus to underlie respiratory adaptation to changes in O2 availability. This importance of TRPA1 in non-carotid body O2 sensors can be extended to the universal significance of redox-sensitive TRP channels in O2 adaptation.

Hypoxia sensors are essential for regulating local oxygen (O

Summary Hypoxia sensors are essential for regulating local oxygen (O2) homeostasis within the body. This is especially pertinent within the CNS, which is particularly vulnerable to O2 deprivation due to high energetic demand. Here, we reveal hypoxia-monitoring function exerted by astrocytes through an O2-regulated protein trafficking mechanism within the CNS. Strikingly, cultured mouse astrocytes isolated from the parafacial respiratory group (pFRG) and retrotrapezoid nucleus (RTN) region are capable of rapidly responding to moderate hypoxia via the sensor cation channel transient receptor potential (TRP) A1 but, unlike multimodal sensory neurons, are inert to hyperoxia and other TRPA1 activators (carbon dioxide, electrophiles, and oxidants) in normoxia. Mechanistically, O2 suppresses TRPA1 channel activity by protein internalization via O2-dependent proline hydroxylation and subsequent ubiquitination by an E3 ubiquitin ligase, NEDD4-1 (neural precursor cell-expressed developmentally down-regulated protein 4). Hypoxia inhibits this process and instantly accumulates TRPA1 proteins at the plasma membrane, inducing TRPA1-mediated Ca2+ influx that triggers ATP release from pFRG/RTN astrocytes, potentiating respiratory center activity. Furthermore, astrocyte-specific Trpa1 disruption in a mouse brainstem-spinal cord preparation impedes the amplitude augmentation of the central autonomic respiratory output during hypoxia. Thus, reversible coupling of the TRPA1 channels with O2-dependent protein translocation allows astrocytes to act as acute hypoxia sensors in the medullary respiratory center.

T-type channels are low-voltage-activated channels that play a role in the cardiovascular system particularly for pacemaker activity. Glycosylation is one of the most prevalent post-translational modifications in protein. Among various glycosylation types, the most common one is asparagine-linked (N-linked) glycosylation. The aim of this study was to elucidate the roles of N-linked glycosylation for the gating properties of the Ca

Regulation of ion channels is central to the mechanisms that underlie immediate acute physiological responses to changes in the availability of molecular oxygen (O2). A group of cation-permeable channels that are formed by transient receptor potential (TRP) proteins have been characterized as exquisite sensors of redox reactive species and as efficient actuators of electric/ionic signals in vivo. In this review, we first discuss how redox-sensitive TRP channels such as TRPA1 have recently emerged as sensors of the relatively inert oxidant O2. With regard to the physiological significance of O2 sensor TRP channels, vagal TRPA1 channels are mainly discussed with respect to their role in respiratory regulation in comparison with canonical pathways in glomus cells of the carotid body, which is a well-established O2-sensing organ. TRPM7 channels are discussed regarding hypoxia-sensing function in ischemic cell death. Also, ubiquitous expression of TRPA1 and TRPM7 together with their physiological relevance in the body is examined. Finally, based upon these studies on TRP channels, we propose a hypothesis of “O2 remodeling.” The hypothesis is that cells detect deviation of O2 availability from appropriate levels via sensors and adjust local O2 environments in vivo by controlling supply and consumption of O2 via pathways comprising cellular signals and transcription factors downstream of sensors, which consequently optimize physiological functions. This new insight into O2 adaptation through ion channels, particularly TRPs, may foster a paradigm shift in our understanding in the biological significance of O2.

Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (Popen)-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, Gmax. Subsequent single-channel conductance (γ) measurements revealed that voltage-dependence of Gmax in WT can be explained by the inward-rectifying property of γ, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and NaVAb structures suggest that such voltage dependence of Popen and γ in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40–47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.

Continuous helices extending from the transmembrane region to the cytoplasmic region form a dimeric interface to regulate activation of the voltage-gated H+ channel., The voltage-gated H+ channel (Hv) is a H+-permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1–S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively, which is mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. However, the structural basis of the interdomain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, suggesting that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. The interaction interface between the two S4 and the assembly interface of the coiled-coil domain were aligned in the same direction based on the phase angle calculation along α helices. Collectively, we propose that continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer.

Hv1 (also named, voltage-sensor only protein, VSOP) lacks an authentic pore domain, and its voltage sensor domain plays both roles in voltage sensing and proton permeation. The activities of a proton channel are intrinsic to protomers of Hv1, while Hv1 is dimeric in biological membranes; cooperative gating is exerted by interaction between two protomers. As the signature pattern conserved among voltage-gated channels and voltage-sensing phosphatase, Hv1 has multiple arginines intervened by two hydrophobic residues on the fourth transmembrane segment, S4. S4 moves upward relative to other helices upon depolarization, causing conformational change possibly leading to the formation of a proton-selective conduction pathway. However, detailed mechanisms of proton-selectivity and gating of Hv1 are unknown. Here we took an approach of PEGylation protection assay to define residues facing the aqueous environment of mouse Hv1 (mHv1). Accessibilities of two maleimide molecules, N-ethylmaleimide (NEM) and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS), were examined on cysteine introduced into individual sites. Only the first arginine on S4 (R1: R201) was inaccessible by NEM and AMS in mHv1. This is consistent with previous results of electrophysiology on the resting state channel, suggesting that the accessibility profile represents the resting state of mHv1. D108, critical for proton selectivity, was accessible by AMS and NEM, suggesting that D108 faces the vestibule. F146, a site critical for blocking by a guanidinium-reagent, was accessible by NEM, suggesting that F146 also faces the inner vestibule. These findings suggest an inner vestibule lined by several residues on S2 including F146, D108 on S1, and the C-terminal half of S4.

Key points • The voltage-gated H+ channel assembles as a dimer by the cytoplasmic coiled-coil domain. • This study focuses on understanding the structural characteristics and functional significance of dimerization. • Monomeric, trimeric and tetrameric channels can be engineered by changing the assembly state of the coiled coil by mutation, and interestingly, they show functional currents. • However, only the native dimeric form shows successful cooperative gating, which is of physiological importance in the phagosomal production of reactive oxygen species. • These results help us to understand better why the native form of the channel is a dimer from a standpoint of molecular structure and physiological function. Abstract The voltage-gated H+ channel functions as a dimer, a configuration that is different from standard tetrameric voltage-gated channels. Each channel protomer has its own permeation pathway. The C-terminal coiled-coil domain has been shown to be necessary for both dimerization and cooperative gating in the two channel protomers. Here we report the gating cooperativity in trimeric and tetrameric Hv channels engineered by altering the hydrophobic core sequence of the coiled-coil assembly domain. Trimeric and tetrameric channels exhibited more rapid and less sigmoidal kinetics of activation of H+ permeation than dimeric channels, suggesting that some channel protomers in trimers and tetramers failed to produce gating cooperativity observed in wild-type dimers. Multimerization of trimer and tetramer channels were confirmed by the biochemical analysis of proteins, including crystallography. These findings indicate that the voltage-gated H+ channel is optimally designed as a dimeric channel on a solid foundation of the sequence pattern of the coiled-coil core, with efficient cooperative gating that ensures sustained and steep voltage-dependent H+ conductance in blood cells.

TRPA1 is a member of the transient receptor potential (TRP) cation channel family that is expressed primarily on sensory neurons. This chemosensor is activated through covalent modification of multiple cysteine residues with a wide range of reactive compounds including allyl isothiocyanate (AITC), a spicy component of wasabi. The present study reports on potent and selective agonists of TRPA1, discovered through screening 1657 electrophilic molecules. In an effort to validate the mode of action of hit molecules, we noted a new TRPA1-selective agonist, JT010 (molecule 1), which opens the TRPA1 channel by covalently and site-selectively binding to Cys621 (EC50 = 0.65 nM). The results suggest that a single modification of Cys621 is sufficient to open the TRPA1 channel. The TRPA1-selective probe described herein might be useful for further mechanistic studies of TRPA1 activation.

This research was originally published in Journal of Biological Chemistry. Makoto Matsuda, Kohei Takeshita, Tatsuki Kurokawa, Souhei Sakata, Mamoru Suzuki, Eiki Yamashita, Yasushi Okamura, and Atsushi Nakagawa. 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. Journal of Biological Chemistry. 2011; 286, 23368-23377. © the American Society for Biochemistry and Molecular Biology., 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, wildtype (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.

Transient receptor potential vanilloid 1 (TRPV1) channel is a tetrameric protein that acts as a sensor for noxious stimuli such as heat and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In TRPV1 oxidation sensing, cysteine (Cys) oxidation has been considered as the principle mechanism; however, its biochemical basis remains elusive. Here, we characterize the oxidative status of Cys residues in differential redox environments and propose a model of TRPV1 activation by oxidation. Through employing a combination of non-reducing SDS-PAGE, electrophysiology, and mass spectrometry we have identified the formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys-258 and Cys-742 of human TRPV1 (hTRPV1). C258S and C742S hTRPV1 mutants have a decreased protein half-life, reflecting the role of the intersubunit disulfide bond in supporting channel stability. Interestingly, the C258S hTRPV1 mutant shows an abolished response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with hydrogen peroxide shows that Cys-258 is highly sensitive to oxidation. Our results suggest that Cys-258 residues are heterogeneously modified in the hTRPV1 tetrameric complex and comprise Cys-258 with free thiol for oxidation sensing and Cys-258, which is involved in the disulfide bond for assisting subunit dimerization. Thus, the hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4, 5-bisphosphate [PI(4, 5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4, 5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4, 5)P2 and the possible role of PI(4, 5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4, 5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4, 5)P2 using a model simulation that could explain the complicated effects of PI(4, 5)P2 on different ROCs.

The voltage-gated H+ channel (Hv) is a H+ permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1-S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively; which is mediated by the coiled-coil assembly domain in the cytoplasmic C-terminus. However, the structural basis of the inter-domain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, demonstrating that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. Thus, continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer.

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-A 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.

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.

Hv1/VSOP is a dimeric voltage-gated H(+) channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the S4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H(+) current through Hv1/VSOP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between S4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. Our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with S4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VSOP operates.

VSOP/Hv1 is a dimeric voltage-gated H+ channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for the dimer formation and intersubunit coupling remains unknown, however. Here we analyzed the high-resolution (1.45 A) crystal structure of VSOP and showed that the C-terminal cytoplasmic coiled-coil architecture mediates dimer assembly and modulates channel gating via linkage to the transmembrane S4 domain. Electrophysiological analysis and circular dichroism spectroscopy revealed that the temperature-dependent activation of VSOP is regulated by the thermostability of the coiled-coil domain. Modulation of the activation kinetics by the cytoplasmic coiled-coil domain was also altered by changing the flexibility/stability of the linker between S4 and the coiled-coil. Collectively, our results indicate that VSOP activation is modified by the coiled-coil assembly through force transmission along a continuous α-helix consisting of the S4 voltage sensor helix and the coiled-coil strands.

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.

Rhodococcus rhodochrous is an active soil bacterium belonging to the Nocardia group of high GC Gram-positive bacteria. It is rich in various enzymes and thus important in the industrial production of chemicals and bioremediation. In this work, the respiratory chain of this aerobic organism was investigated and characterized. Grown under highly aerobic conditions, the membrane fraction of R. rhodochrous cells only contained a-, b- and c-type cytochromes, suggesting that it is the cytochrome bcc-aa3-type pathway that mainly operates under these conditions. In contrast, the d-type cytochrome was also present under microaerobic conditions, indicating that the alternative pathway of the bd-type oxidase works in these circumstances. In addition, the results of H+/O ratio measurements indicate that these two pathways have different energy efficiencies.

Corynebacterium glutamicum contains at least two terminal oxidases in the respiratory chain; cytochrome aa(3)-type cytochrome c oxidase and bd-type menaquinol oxidase. Thus, the chain has two branches of electron flow. The bcc-aa(3) branch translocates three protons per electron transferred, while the bd branch translocates only one. In this study, we constructed two mutant strains, lacking of either subunit I of the cytochrome c oxidase (DeltactaD) or subunits I and II of the quinol oxidase (DeltacydAB), and also plasmids to complement the deficient genes to investigate their effects on energy conservation and cell growth. We measured H(+)/O ratios of C. glutamicum wild-type and mutant cells grown aerobically. The H(+)/O ratio of the wild-type cells grown in the semi-synthetic medium was 3.94 +/- 0.30, while the value was 2.76 +/- 0.25 for the DeltactaD mutant. In contrast, the value was 5.23 +/- 0.36 for the DeltacydAB mutant. The cells grown in the LB medium showed higher value compared to that of cells grown in the semi-synthetic medium. The DeltactaD mutant grew less than the wild-type in LB medium, while they grew about equally in semi-synthetic medium. Correlation between bioenergetics and growth of C. glutamicum was significantly affected by the growth nutrients.

VSOP/Hv1 is a protein that contains the voltage sensor domain but not pore domain [1, 2]. It exhibits properties of native voltage-gated proton channels reported in phagocytes and microglia. Addressing how proton permeates and how voltage-dependent gating is achieved in VSOP/Hv1 will lead to critical clues to understand mechanisms of voltage sensor operation and ion permeation. The putative fourth transmembrane segment (S4) of mouse VSOP (mVSOP) has three positively charged residues in a pattern similar to those conserved in other voltage-gated channels. We have previously shown that VSOP/Hv1 forms dimer and a version lacking the cytoplasmic region (V216X) expressing mainly as monomer exhibits robust voltage-dependent proton currents, suggesting that monomer constitutes proton permeation pathway [3]. However, V216X still contains some cytoplasmic stretch and it remained unknown whether a remaining stretch downstream of S4 segment is essential for proton channel activities. To address this, a series of deletion constructs of mVSOP were expressed in tsA201 cells and whole cell patch recording and western blot were performed. Surprisingly, voltage-gated outward currents were elicited in constructs with stop codon at sites upstream to the third arginine. Proton permeation was verified by measuring intracellular pH using the pH-sensitive fluorescent dye, simultaneously with whole-cell patch clamping. Therefore, mVSOP retains functions of voltage-gated proton channel only with a truncated S4 segment, neglecting some possible mechanisms of proton permeation. To gain more insights, we are currently trying to biochemically map the topology of S4 using the cysteine-targeting reagent.[References][1] Sasaki M. et al., (2006) Science 312:589-592.[2] Ramsey IS. et al., (2006) Nature 440:1213-1216.[3] Koch HP. et al., (2008) Proc. Natl. Acad. Sci. USA 105:9111-9116.

Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as H V or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein–protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.

Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the PTEN-like region with phosphoinositide-phosphatase activities. VSP is conserved from sea squirt to human, but little is known about its physiological roles. Here we characterize the molecular properties of chick ortholog of VSP (designated as Gg-VSP). Gg-VSP shares basic properties with other VSP orthologs, including voltage sensing by the transmembrane segments and phosphoinositide phsophatase activities of the cytoplasmic region. Imaging of PI(3,4)P2-sensitive fluorescent protein in Xenopus oocyte showed that membrane depolarization triggers dephosphorylation of PI(3,4,5)P3 into PI(3,4)P2. Notably, about 10% of cell populations of DF-1 cells transiently expressing Gg-VSP exhibited morphological feature of cell process outgrowths. This was accompanied by the decrease of actin filaments as detected by phalloidin-staining. Transfection of the voltage sensor mutant, Gg-VSPR153Q, showing shift of voltage dependence to a lower voltage, facilitated cell process outgrowths. Coexpression of chick PTEN cDNA suppressed this effect of Gg-VSP. Visualization of PI(3,4)P2 with the Ds-Red fused with the FAPP1-derived pleckstrin homology (PH) domain showed more fluorescent clusters than luciferase-transfected control cells, suggesting that cell process outgrowths paralleled with the increase of PI(3,4)P2. Whole mount in situ hybridization and immunostaining of chicken brain showed that Gg-VSP is expressed in neurons. These findings suggest that one of the VSP’s functions is to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile.

VSOP/Hv1 is a voltage-gated proton channel that contains the voltage sensor domain (VSD) but not pore domain. VSD of VSOP/Hv1 allows protons to permeate as well as sensing voltage. It has been reported that basic amino acids in the fourth transmembrane segment (S4) of voltage-gated ion channels play critical roles in voltage-sensing. Mouse VSOP (mVSOP) has three arginine residues (R1, R2, R3) in a pattern similar to those conserved in other voltage-gated channels. To address the role of S4 in mVSOP, we have reported that the truncated construct (A206sop) just downstream of R2 in the S4 is still ion-conductive (Biophysical Society 53th Annual Meeting, 2009). In this study, we further analysed properties of A206stop. The outward current of A206stop was almost completely blocked by zinc. Visualization of intracellular pH using BCECF (pH-sensitive ratiometric dye) showed that cytoplasm of tsA201 cell was alkalinized under the depolarization condition. Na and K ion do not permeate through A206stop. Gating properties of the proton currents through A206stop were sensitive to either intracellular pH or extracellular pH. However, voltage dependency of A206stop was weaker than that of full-length mVSOP, and the I-V relationship of A206stop was shifted rightward. These results indicate that A206stop retains the basic properties of the voltage-gated proton channel even if it lacks a half of S4. We also carried out two biochemical assays: site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by western blotting (pegylation protection) and in vitro glycosylation assays. Both showed that S4 of A206stop inserts into the membrane and the position of A206 faces intracellular aqueous environments. These findings suggest that the region downstream of the R2 position of S4 of VSOP/Hv1 is not essential for proton selectivity.

VSOP/Hv1 is a dimeric voltage-gated H+ channel unlike other tetrameric voltage-gated channels. Each subunit has its own permeation pathway, and the gating of one subunit is coupled to that of the other subunit within the dimer. We previously reported that the cytoplasmic dimer coiled-coil mediated the dimeric assembly and the gating coupling, based on the crystal structure analysis of the coiled-coil domain. The crystal structure of the VSOP coiled-coil shows an I/L core packing pattern, in which well-packed Ile/Leu residues are situated at positions ‘a’/‘d’ in the heptad repeat and are periodically observed along the entire length of the coiled-coil. However, the functional significance of the patterned sequence for the channel function remains unknown. To address this issue, we changed the packing pattern to I/I, L/L and L/I-types, and analyzed the stoichiometry of the mutant coiled-coils’ assembly. Sedimentation and crystal structure analyses showed that coiled-coils with I/I and L/L-type cores formed trimers, and the version harboring the L/I core formed a tetramer. These were consistent with the results of cross-linking analysis followed by Western blotting of the full-length proteins, indicating that the assembly stoichiometry was determined by the core type. Electrophysiological analysis revealed that only the dimer types showed slow and sigmoidal activation kinetics suggestive of cooperative gating. Thus, the I/L sequence pattern of the coiled-coil core in the natural channel is optimally designed to form a dimeric channel with slow and cooperative gating.