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Aspartate key to proton selectivity Voltage-gated proton channels are considered completely selective for protons -- no evidence exists for the permeation of ions other than H.sup.+. A study of the underlying mechanism of the high selectivity of the human voltage-gated proton channel reveals an Asp112 residue as the selectivity filter. When this residue was changed to a neutral amino acid, the mutant channel lost proton selectivity and either became anion-selective or did not conduct any ions. The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel.sup.1, H.sub.V1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes.sup.2,3,4, histamine secretion by basophils.sup.5, sperm capacitation.sup.6, and airway pH.sup.7. The most selective ion channel known, H.sub.V1 shows no detectable permeability to other ions.sup.1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity.sup.1,8,9,10,11, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions.sup.12. Here we identify aspartate 112 as a crucial component of the selectivity filter of H.sub.V1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H.sub.V1 requires an acidic group at the selectivity filter., Author(s): Boris Musset [sup.1] , Susan M. E. Smith [sup.2] , Sindhu Rajan [sup.3] , Deri Morgan [sup.1] , Vladimir V. Cherny [sup.1] , Thomas E. DeCoursey [sup.1] Author Affiliations: [...]

Research has been conducted on the neuronal nitric oxide. The study of the full-length neuronal nitric oxide including ligands' vibrational bands, protein and amino acid side chains and prosthetic groups carried out via Fourier transform infrared difference spectroscopy is presented.

Bioluminescence in dinoflagellates is controlled by HV1 proton channels. Database searches of dinoflagellate transcriptomes and genomes yielded hits with sequence features diagnostic of all confirmed HV1, and show that HV1 is widely distributed in the dinoflagellate phylogeny including the basal species Oxyrrhis marina. Multiple sequence alignments followed by phylogenetic analysis revealed three major subfamilies of HV1 that do not correlate with presence of theca, autotrophy, geographic location, or bioluminescence. These data suggest that most dinoflagellates express a HV1 which has a function separate from bioluminescence. Sequence evidence also suggests that dinoflagellates can contain more than one HV1 gene. [ABSTRACT FROM AUTHOR]

We previously characterized a H(+) transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13(BN)) rats, and superoxide production was enhanced in low Na(+) media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na(+). We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H(+) currents. To test this hypothesis, we developed HV1(-/-) null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH4Cl prepulse in 0 sodium/BaCl2 containing media resulted in superoxide production in thick limb from wild-type but not HV1(-/-) rats (P<0.05) and more rapid recovery of intracellular pH in wild-type rats (ΔpHI 0.005 versus 0.002 U/s, P=0.046, respectively). Superoxide production was enhanced by low intracellular sodium (<10 mmol/L) in both thick limb and peritoneal macrophages only when HV1 was present. When fed a high-salt diet, blood pressure, outer medullary renal injury (tubular casts), and oxidative stress (4-hydroxynonenal staining) were significantly reduced in HV1(-/-) rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na(+) is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats. [ABSTRACT FROM AUTHOR]

The topological similarity of voltage-gated proton channels (Hv1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether Hv1s have a similar structure. We present the construction and validation of a homology model of the human Hv1 (hHv1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hHv1 by exploiting the homology of hHv1 with VSDs of K+ and Na+ channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hHv1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hHv1. Each Arg residue in the S4 helix of hHv1 was replaced by His to test accessibility using Zn2+ as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and exerimental data indicate that in the open state, Arg211 the third Arg residue in the S4 helix in hHv1, remains accessible to the internal solution and is located near the charge transfer center, Phe150. [ABSTRACT FROM AUTHOR]

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, HV1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, HV1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of HV1. When a neutral amino acid replaced Asp?112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp?185 did not impair proton selectivity, indicating that Asp?112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp?112, the mutant channel was still anion-permeable. Evidently, the proton specificity of HV1 requires an acidic group at the selectivity filter. [ABSTRACT FROM AUTHOR]

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a β hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity. [ABSTRACT FROM AUTHOR]

The three mammalian nitric-oxide synthases produce NO from arginine in a reaction requiring 3 electrons per NO, which are supplied to the catalytic center from NADPH through reductase domains incorporating FAD and FMN cofactors. The isoforms share a common reaction mechanism and requirements for reducing equivalents but differ in regulation; the endothelial and neuronal isoforms are controlled by calcium/calmodulin modulation of the electron transfer system, while the inducible isoform binds calmodulin at all physiological Ca2+ concentrations and is always on. The thermodynamics of electron transfer through the flavin domains in all three isoforms are basically similar. The major flavin states are FMN, FMNH, FMNH2, FAD, FADH, and FADH2. The FMN/FMNH couple is high potential (∼100 mV) in all three isoforms and is unlikely to be catalytically competent; the other three flavin couples form a nearly isopotential group clustered around −250 mV. Reduction of the flavins by the pyridine nucleotide couple at −325 mV is thus moderately thermodynamically favorable. The ferri/ferroheme couple in all three isoforms is ∼−270 mV in the presence of saturating arginine. Ca2+/calmodulin has no effect on the potentials of any of the couples in endothelial nitric-oxide synthase (eNOS) or neuronal nitric-oxide synthase (nNOS). The pH dependence of the flavin couples suggests the presence of ionizable groups coupled to the flavin redox/protonation states. [ABSTRACT FROM AUTHOR]

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores, and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp-Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O+ protonated the SF, breaking the Asp-Arg linkage and opening the conduction pathway, whereas Na+ or Cl- was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp-Lys SF behaved like the Asp-Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH0-H2O0-Arg+ interactions with hydronium but unfavorable Asp--X-/X+-Arg+ interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis, and signaling. [ABSTRACT FROM AUTHOR]

The voltage-gated proton channel (HV1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hHV1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+ in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pHo 7 suggests additional groups contribute. We hypothesized that Asp185, which faces position 116 in our closed-state model, contributes to Zn2+ chelation. Confirming this prediction, V116H/D185N abolished Zn2+ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+ concentration. We hypothesized that slow turn-on of H+ current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hHV1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg205 near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp185 opposes position 116 in closed channels and that Arg205 moves between them when the channel opens. [ABSTRACT FROM AUTHOR]

We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the voltage-gated proton channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1--changing His168 to Gln168, the corresponding residue in HtHV1--compromises the pHi dependence of gating in the human channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus. His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by <20 mV/unit. Thus, His168 is critical to pHi sensing in hHV1. His168, located at the inner end of the pore on the S3 transmembrane helix, is the first residue identified in HV1 that significantly impairs pH sensing when mutated. Because pHo dependence remains intact, the selective erosion of pHi dependence supports the idea that there are distinct internal and external pH sensors. Although His168 may itself be a pHi sensor, the converse mutation, Q229H, does not normalize the pHi sensitivity of the HtHV1 channel. We hypothesize that the imidazole group of His168 interacts with nearby Phe165 or other parts of hHV1 to transduce pHi into shifts of voltage-dependent gating. [ABSTRACT FROM AUTHOR]

Part of the "signature sequence" that defines the voltage-gated proton channel (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence HV1 genes. Replacing Trp207 in human HV1 (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30-38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation-π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1, ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating. [ABSTRACT FROM AUTHOR]

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hHv1), because the proton concentration is >106 times lower than that of other cations. Here we use "selectivity filter scanning" to elucidate the molecular requirements for proton-specific conduction in hHv1. Asp112, in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp112 by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for "second site suppressor" activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn2+. Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anionpermeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction. [ABSTRACT FROM AUTHOR]

By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1 -- 4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47phox/p67phox, suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47phox/p67phox, suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (Kd = 58 ± 12 riM). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes. [ABSTRACT FROM AUTHOR]

The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CAM) and mutant forms of CAM, including CAM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CAM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS). The present study was designed to evaluate the role of the four CAM EF hands in the activation of eNOS and iNOS. To assess the role of CAM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (.NO) generation as assessed by the oxyhemoglobin capture assay. CAM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain. Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS. Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent. We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex. In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive. Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified. Domains 2 and 3 of CAM contain important elements required for the Ca2+/CAM independence of .NO production by the iNOS enzyme. The disparity between cytochrome c reduction and .NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CAM constructs are depleted of Ca2+. In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CAM-binding site and associated with CAM control. [ABSTRACT FROM AUTHOR]

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Bioluminescence in dinoflagellates is controlled by H V 1 proton channels. Database searches of dinoflagellate transcriptomes and genomes yielded hits with sequence features diagnostic of all confirmed H V 1, and show that H V 1 is widely distributed in the dinoflagellate phylogeny including the basal species Oxyrrhis marina. Multiple sequence alignments followed by phylogenetic analysis revealed three major subfamilies of H V 1 that do not correlate with presence of theca, autotrophy, geographic location, or bioluminescence. These data suggest that most dinoflagellates express a H V 1 which has a function separate from bioluminescence. Sequence evidence also suggests that dinoflagellates can contain more than one H V 1 gene., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis , HtH V 1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtH V 1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pH i ). The H + conductance ( g H )- V relationship in the voltage-gated proton channel (H V 1) from other species shifts 40 mV when either pH i or pH o (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of H V 1 in many species and in numerous human tissues. The HtH V 1 channel exhibits normal pH o dependence but anomalously weak pH i dependence. In this study, we show that a single point mutation in human hH V 1-changing His 168 to Gln 168 , the corresponding residue in HtH V 1-compromises the pH i dependence of gating in the human channel so that it recapitulates the HtH V 1 response. This location was previously identified as a contributor to the rapid gating kinetics of H V 1 in Strongylocentrotus purpuratus His 168 mutation in human H V 1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pH o dependence, but changing pH i shifts the g H - V relationship on average by <20 mV/unit. Thus, His 168 is critical to pH i sensing in hH V 1. His 168 , located at the inner end of the pore on the S3 transmembrane helix, is the first residue identified in H V 1 that significantly impairs pH sensing when mutated. Because pH o dependence remains intact, the selective erosion of pH i dependence supports the idea that there are distinct internal and external pH sensors. Although His 168 may itself be a pH i sensor, the converse mutation, Q229H, does not normalize the pH i sensitivity of the HtH V 1 channel. We hypothesize that the imidazole group of His 168 interacts with nearby Phe 165 or other parts of hH V 1 to transduce pH i into shifts of voltage-dependent gating., (© 2018 Cherny et al.)

Voltage-gated proton channels, H V 1, were first reported in Helix aspersa snail neurons. These H + channels open very rapidly, two to three orders of magnitude faster than mammalian H V 1. Here we identify an H V 1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtH V 1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hH V 1 (human) at pH o 7, between 50 and 90 mV. In contrast to most H V 1, activation of HtH V 1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e 0 suggests that HtH V 1 functions as a dimer, evidently with highly cooperative gating. HtH V 1 opening is exquisitely sensitive to pH o , whereas closing is nearly independent of pH o Zn 2+ and Cd 2+ inhibit HtH V 1 currents in the micromolar range, slowing activation, shifting the proton conductance-voltage ( g H - V ) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtH V 1 possessing three of the four amino acids that coordinate Zn 2+ in mammalian H V 1. All known H V 1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the g H - V relationship for a unit change in either pH o or pH i This property is crucial for all the functions of H V 1 in many species and numerous human cells. The HtH V 1 channel exhibits normal or supernormal pH o dependence, but weak pH i dependence. Under favorable conditions, this might result in the HtH V 1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtH V 1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968)., (© 2018 Thomas et al.)

NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47 phox and p67 phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the V max of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b 558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398-719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1-550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310-529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Part of the "signature sequence" that defines the voltage-gated proton channel (H(V1)) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H(V1) genes. Replacing Trp207 in human HV1 (hH(V1)) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30-38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hH(V1). Cation-π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H(V1) that is essential to its biological functions, was compromised. In the WT hH(V1), ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H(V1) from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in H(V1) of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating., (© 2015 Cherny et al.)

Significance: NOX2 is important for host defense, and yet is implicated in a large number of diseases in which inflammation plays a role in pathogenesis. These include acute and chronic lung inflammatory diseases, stroke, traumatic brain injury, and neurodegenerative diseases, including Alzheimer's and Parkinson's Diseases., Recent Advances: Recent drug development programs have targeted several NOX isoforms that are implicated in a variety of diseases. The focus has been primarily on NOX4 and NOX1 rather than on NOX2, due, in part, to concerns about possible immunosuppressive side effects. Nevertheless, NOX2 clearly contributes to the pathogenesis of many inflammatory diseases, and its inhibition is predicted to provide a novel therapeutic approach., Critical Issues: Possible side effects that might arise from targeting NOX2 are discussed, including the possibility that such inhibition will contribute to increased infections and/or autoimmune disorders. The state of the field with regard to existing NOX2 inhibitors and targeted development of novel inhibitors is also summarized., Future Directions: NOX2 inhibitors show particular promise for the treatment of inflammatory diseases, both acute and chronic. Theoretical side effects include pro-inflammatory and autoimmune complications and should be considered in any therapeutic program, but in our opinion, available data do not indicate that they are sufficiently likely to eliminate NOX2 as a drug target, particularly when weighed against the seriousness of many NOX2-related indications. Model studies demonstrating efficacy with minimal side effects are needed to encourage future development of NOX2 inhibitors as therapeutic agents.

Precise spatiotemporal regulation of O2(-)-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67(phox) and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O2(-)-generating system consisting of isolated membranes and recombinant p47(phox) and p67(phox) proteins. Addition of soluble RacGAPs decreased O2(-) production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O2(-) production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O2(-) production by Nox2, allowing selective regulation via different signaling pathways., (© 2013 Elsevier Inc. All rights reserved.)

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hHV1), because the proton concentration is >10(6) times lower than that of other cations. Here we use "selectivity filter scanning" to elucidate the molecular requirements for proton-specific conduction in hHV1. Asp(112), in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp(112) by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for "second site suppressor" activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn(2+). Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.

The topological similarity of voltage-gated proton channels (H(V)1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H(V)1s have a similar structure. We present the construction and validation of a homology model of the human H(V)1 (hH(V)1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH(V)1 by exploiting the homology of hH(V)1 with VSDs of K(+) and Na(+) channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH(V)1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp(112). Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH(V)1. Each Arg residue in the S4 helix of hH(V)1 was replaced by His to test accessibility using Zn(2+) as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg(211), the third Arg residue in the S4 helix in hH(V)1, remains accessible to the internal solution and is located near the charge transfer center, Phe(150).

The human voltage-gated proton channel, hHV1, appears to exist mainly as a dimer. Teleologically, this is puzzling because each protomer retains the main properties that characterize this protein: proton conduction that is regulated by conformational (channel opening and closing) changes that occur in response to both voltage and pH. The HV1 dimer is mainly linked by C-terminal coiled-coil interactions. Several types of mutations produce monomeric constructs that open approximately five times faster than the wild-type dimeric channel but with weaker voltage dependence. Intriguingly, the quintessential function of the HV1 dimer, opening to allow H(+) conduction, occurs cooperatively. Both protomers undergo a conformational change, but both must undergo this transition before either can conduct. The teleological purpose of dimerization may be to steepen the voltage dependence of channel opening, at least in phagocytes. In other cells, the purpose is not understood. Finally, several single-celled species have HV that are likely monomeric., (Copyright © 2013 Elsevier Inc. All rights reserved.)

NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The voltage-gated proton channel exists as a dimer, although each protomer has a separate conduction pathway, and when forced to exist as a monomer, most major functions are retained. However, the proton channel protomers appear to interact during gating. Proton channel dimerization is thought to result mainly from coiled-coil interaction of the intracellular C-termini. Several types of evidence are discussed that suggest that the dimer conformation may not be static, but is dynamic and can sample different orientations. Zn(2+) appears to link the protomers in an orientation from which the channel(s) cannot open. A tandem WT-WT dimer exhibits signs of cooperative gating, indicating that despite the abnormal linkage, the correct orientation for opening can occur. We propose that C-terminal interaction functions mainly to tether the protomers together. Comparison of the properties of monomeric and dimeric proton channels speaks against the hypothesis that enhanced gating reflects monomer-dimer interconversion.