Susan M.E. Smith

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel.

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.

Voltage-gated proton channel in a dinoflagellate

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH–V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating

Voltage‐gated proton channels are strongly inhibited by Zn2+, which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn2+. We hypothesize that high‐affinity Zn2+ binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn2+ effects were weaker in monomeric channels. Mutation of His193 and His140 in various combinations and in tandem dimers revealed that channel opening was slowed by Zn2+ only when at least one His was present in each monomer, suggesting that in wild‐type (WT) HV1, Zn2+ binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn2+ appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer–dimer interconversion occurs during activation of phagocytes appears to be ruled out.

Construction and validation of a homology model of the human voltage-gated proton channel hHV1

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 Asp112. 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 experimental 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.

Ebselen and Congeners Inhibit NADPH Oxidase 2-Dependent Superoxide Generation by Interrupting the Binding of Regulatory Subunits

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.

Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHV1.

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, 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.

Oligomerization of the voltage gated proton channel

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. Zn2+ 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.

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hHV1

A conserved tryptophan uniquely present in the voltage-sensing domain of voltage-gated proton channels is surprisingly crucial to four channel-defining properties.

HV1 Acts as a Sodium Sensor and Promotes Superoxide Production in Medullary Thick Ascending Limb of Dahl Salt-Sensitive Rats

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.13BN) 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 (&Dgr;pHI 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.

NOX2 As a Target for Drug Development: Indications, Possible Complications, and Progress.

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 Alzheimers and Parkinsons 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.