Human Voltage-gated Proton Channel Research Articles

HIFs: New arginine mimic inhibitors of the Hv1 channel with improved VSD-ligand interactions.

The human voltage-gated proton channel Hv1 is a drug target for cancer, ischemic stroke, and neuroinflammation. It resides on the plasma membrane and endocytic compartments of a variety of cell types, where it mediates outward proton movement and regulates the activity of NOX enzymes. Its voltage-sensing domain (VSD) contains a gated and proton-selective conduction pathway, which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). Mutation of Hv1 residue F150 to alanine (F150A) was previously found to increase 2GBI apparent binding affinity more than two orders of magnitude. Here, we explore the contribution of aromatic interactions between the inhibitor and the channel in the presence and absence of the F150A mutation, using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated phenylalanines via nonsense suppression methodology. Our data suggest that the increase in apparent binding affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150. We used this information to design new arginine mimics with improved affinity for the nonrearranged binding site of the wild-type channel. The new compounds, named "Hv1 Inhibitor Flexibles" (HIFs), consist of two "prongs," an aminoimidazole ring, and an aromatic group connected by extended flexible linkers. Some HIF compounds display inhibitory properties that are superior to those of 2GBI, thus providing a promising scaffold for further development of high-affinity Hv1 inhibitors.

A Novel Model of the Dimeric Human Hv1 Channel Reveals a Putative ATP Binding Site

A multi-template model of the human voltage-gated proton channel (hHv1) was developed by combining the recently solved NMR structural ensemble of the hHv1 (PDBID: 5OQK) with the coiled-coil C-terminal domain (PDBID: 3A2A), bridging them through the chimeric mHv1cc structure (PDBID: 3WKV). The model was built and refined as a dimer within Rosetta's Membrane framework, using the symmetry extracted from the coiled-coil and including experimentally derived constraints during the relaxation. The dimeric model was then subjected to extensive Gaussian Accelerated Molecular Dynamics simulations in a POPC bilayer, and representative snapshots were extracted from the trajectory by clustering analysis. Blind docking simulations of an ATP molecule on this structural ensemble revealed a putative ATP binding site located on the intracellular portion of the permeation pathway, below the hydrophobic gasket. The same region was identified by two sequence-based ATP binding site predictors: ATPSite and ATPint; and a third meta-predictor which combines sequence and structure-based features: ATPBind. According to the in-silico predictions, we found that whole-cell hHv1 current measured in presence of intracellular 3 mM ATP (patch-clamp, Jurkat T cells, n=7) showed a higher amplitude (148.3±27.0 vs 35.6±4.6 pA at 20 mV p<0.05) and a hyperpolarized Vthreshold (−25.7±2.0 vs −12.0±3.7 mV, p<0.05), compared to control cells (without ATP, n=5). The combination of several in-silico methodologies allowed us to develop a novel model of dimeric hHv1 channel, and predict its direct modulation by ATP. Moreover, experimental results confirmed that ATP is able to activate the channel. In a physiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation such as pancreatic β-cells, cancer cells or immune cells.

Hydrophobic Gasket Mutation Produces Proton Selective Gating Pore Currents in Closed Human Voltage-Gated Proton Channels

A ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this apparent “hydrophobic gasket” (HG) when the channel opens. In the human voltage-gated proton channel, hHV1, not only does S4 movement occur during gating, but proton current also flows through this “gating pore” in open channels. To probe the role of the HG in the function of hHV1, we replaced putative HG residues with less hydrophobic residues or acidic Asp.

Molecular Basis of Voltage-Gated Proton Channel's Intracellular Trafficking

Voltage-gated proton channel (HV1) presents a physiological role of great importance to phagocytosis and sub sequential death of microorganisms in phagocytic cells, such as neutrophils and microglia. Recently various studies describe the contribution of this channel in a higher malignity of tumors and its proliferation. Additionally, HV1 channels have been related to augmented tissue damage in cerebral stroke. Even though these pathological processes have been associated with a higher superficial expression of HV1 channels, little is known about how their abundance in the plasma membrane is regulated. Thus, by combining molecular approaches, in this work we have studied the molecular mechanisms of HV1 channel's intracellular trafficking, associating modifications in its C-terminal domain with its intracellular localization. Therefore, full length human voltage-gated proton channel (hHV1 WT) was fused with the fluorescent protein mCherry and site-directed mutagenesis were made in order to delete the C-terminal domain completely (V220X) or partially (I242X and I256X). Electrophysiological recordings of CHO-K1 cells transfected with hHV1 WT and the C-terminal truncated mutants showed that only the V220X mutant significantly reduced the proton current density. Thus, suggesting that the surface expression of this mutant is compromised. High-resolution microscopy analysis of hHV1 WT intracellular localization through co-transfection with 8 different intracellular organelles markers fused with EGFP resulted in a marked co-localization with Rab11, which is present in recycling endossomes. Furthermore, acute treatment of hHV1 WT transfected CHO-K1 cells with microtubule and actin disruptive agents, nocodazole and cytochalasin D respectively, diminished the proton current density significantly. Overall these results indicate an association of hHV1 WT with recycling endosomes, a key role of both cytoskeletal components and of the C-terminal domain in hHV1 forward trafficking.

Nuclear Magnetic Resonance Solution Structure and Functional Behavior of the Human Proton Channel.

The human voltage-gated proton channel [Hv1(1) or VSDO(2)] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1-S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn2+-induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H+ concentration is required for the protonation of side chains in the Zn2+-induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel.

Is the Hydrophobic Gasket a Secondary Selectivity Filter in the Human Voltage Gated Proton Channel HHV1?

Is the Hydrophobic Gasket a Secondary Selectivity Filter in the Human Voltage Gated Proton Channel HHV1?

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

Histidine168 is crucial for ΔpH-dependent gating of the human voltage-gated proton channel, hHV1.

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.

A Designer Peptide Toxin Isolated by Phage Display that Inhibits the Human Voltage-Gated Proton Channel, hHv1

A Designer Peptide Toxin Isolated by Phage Display that Inhibits the Human Voltage-Gated Proton Channel, hHv1

Identification of an HV1 voltage‐gated proton channel in insects

The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722.

Tryptophan 207 is Crucial to the Unique Properties of the Human Voltage Gated Proton Channel, hHv1

Tryptophan 207 is Crucial to the Unique Properties of the Human Voltage Gated Proton Channel, hHv1

Hv1 Proton Channel Resting-State Voltage Sensor Model Structures are Refined by Experimental Mapping of Zinc-Coordinating Residues

Voltage sensor (VS) activation likely involves an outward movement of the S4 helix from its resting state conformation, but high resolution resting-state VS structures are rare and experimental data is needed to spatially constrain model structures. Zn2+ potently (EC50 ≈ 1 uM, pHO 6.5) attenuates H+ currents in the human voltage-gated proton channel (hHv1) by shifting the apparent POPEN-V relation toward more positive potentials (Cherny, et al. 1999). Zn2+ is likely to be coordinated by two extracellularly-accessible His residues, H140 (S2 helix) and H193 (S3-S4 linker) in the resting conformation of the Hv1 VS domain (Ramsey et al., 2006). The intermediate potency (EC50 20-70 uM) of Zn2+ in single H140A or H193A mutant channels suggests that additional residues may also participate resting-state Zn2+ coordination. Candidate residues include the acidic side chains of E119 (S1) and D123 (S1-S2 linker), which may be near H140 and H193 in the mouse Hv1-based voltage sensor chimeric protein mHv1cc (Takeshita et al., 2014). To identify Zn2+-coordinating residues in hHv1, we mutated candidate acidic residues to His in the background of either H140A or H193A and measured the potency of Zn2+ to attenuate voltage-gated H+ currents. We find that E119H, but not D123H or D130H, is sufficient to reconstitute potent (low micromolar EC50) block of current by Zn2+ when combined with either H193A or H140A, indicating that E119 is likely to coordinate Zn2+ in conjunction with H140 and H193 in WT hHv1. Our experimental data limit the distances of E119, H140 and H193 side chains from the coordinated Zn2+ ion and therefore strongly constrain the positions of these groups, allowing us to generate refined resting-state hHv1 VS model structures.

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

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.

Free Energy Simulations of Ion Translocation through Voltage-Gated Proton Channel Hv1

The human voltage-gated proton channel (hHV1) is a transmembrane protein that is responsible for the selective permeation of protons across cell membranes in nasal mucosa, sperm, and white blood cells. hHV1 is a four-helix bundle (S1-S4) with anionic Asp112 on S1 forming a salt bridge with cationic Arg residues on helix S4 in the narrow region of the pore [Kulleperuma et al., J. Gen. Physiol. 141, 445-465 (2013)]. Mutation of Asp112 to Val abrogates channel properties. Unexpectedly, replacing Asp112 by a smaller neutral residue such as Ser turns HV1 into an anion selective channel that conducts Cl-, as does the double mutant D112V-V116S [Musset et al., Nature 480, 273-277 (2012); Morgan et al., J. Gen. Physiol. 142, 625-640 (2013)]. Although HV1 and its mutants exhibit drastic differences in ion permeation, the molecular basis of proton selectivity in WT and anion selectivity in mutants remains unexplained.As the first step towards elucidating the charge selectivity of HV1, we perform molecular dynamics simulations with umbrella sampling to compute the free energy profile for the translocation of Na+ and Cl- ions through the pore of a homology model of HV1 and its mutants. The calculations are repeated in conformational states of the channel differing in the extent of hydration of the pore and in the relative arrangements of pore residues. Results show how ion solvation and electrostatic interactions with charged side chains in the pore lumen modulate the energetics of ion permeation in HV1 and suggest a structural basis for charge selectivity.

Detergent screening of the human voltage-gated proton channel using fluorescence-detection size-exclusion chromatography.

The human voltage-gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino- and carboxy-termini. The protein is activated by membrane depolarization, similar to other voltage-sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three-dimensional structures for homologues of this voltage-gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino-terminus of the human Hv1 proton channel (termed EGFP-Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence-detection size-exclusion chromatography. The EGFP-Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3-12 and the nonionic n-dodecyl-β-d-maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size-exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full-length human Hv1 proton channel.

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1.

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His(244) or His(266) residue. The binding of Zn(2+) to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co(2+), Ca(2+), Ni(2+), Mg(2+), and Mn(2+). The strength of Ca(2+), Co(2+), Mg(2+), Mn(2+) and Ni(2+) binding to the site close to His(244) is stronger than that of these divalent metal ions binding to the site close to His(266).

Selectivity Filter Scanning of the Human Voltage Gated Proton Channel Hhv1

Extraordinary is crucial to all proton conducting molecules, including the human voltage gated proton channel, hHV1, because [H+] is minuscule. 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 essential to the WT channel [Musset et al., 2011. Nature 480:273-277]. We neutralized Asp112 by mutating it to Ala (D112A), then introduced Asp at each position along S1 from 108 to 118, searching for second site suppressor activity. All mutants except for D112A/V109D lacked even the anion conduction exhibited by D112A. Proton specific conduction was restored with Asp or Glu at position 116. The D112V/V116D channel resembled WT in selectivity, kinetics, and ΔpH dependent gating. Both R211H and R211H/D112V/V116D were inhibited by internally applied Zn2+ when the channel was open, indicating similar S4 accessibility. At position 109 Asp allowed anion permeation in combination with D112A, but did not rescue function in the nonconducting D112V mutant, indicating that is established external to the constriction at Phe150. The three positions (109, 112, 116) that permit conduction all line the pore in our homology model, delineating the conduction pathway. Evidently, proton selective conduction requires a carboxyl group to face the pore at a constriction in the external vestibule. Molecular dynamics studies indicate reorganization of ionic networks in response to mutations and suggest that the distribution of charged groups in the external vestibule modulates charge selectivity. That the filter functions in a new location helps define local environmental features that produce proton selective conduction.

Molecular Dynamics Studies of Ion Permeation in Human Voltage-Gated Proton Channel

The human voltage-gated proton channel (hHv1) is a transmembrane protein responsible for selective proton permeation across cell membranes in nasal mucosa, sperm, and white blood cells. Its pathological states include male infertility, allergies, and diseases such as cystic fibrosis, asthma, and lupus. Its involvement in ischemic stroke and invasiveness of breast cancer cells has substantiated hHv1 as a therapeutic target for drug designs for which require the understanding of hHv1 structure and proton conduction mechanism. We recently constructed and validated a homology model (Kulleperuma et al., 2013) of hHv1 characterized by the presence of a salt bridge between anionic D112 and cationic R208 side chains in the narrow region of the hydrated pore. Thanks to the pairing of these and other charged residues in ionic networks, the distribution of charged and polar residues in the wild-type channel is a priori compatible with permeation of either cations or anions. However, a static-field electrostatic barrier opposing cation movement arises in neutral mutants of residue D112, consistent with the observation that these mutants are selective to anions (Musset et al., 2011). Our recent experiments show that proton selectivity is restored in double mutant D112V/V116D, while D112S and D112V/D116S are anion-selective and D112V does not conduct ions. Atomistic molecular dynamics simulations of these mutants in lipid bilayers show that their structures differ in the organization of ionic side chains in the external vestibule and suggest that, consistent with the above analysis, the distribution of charged groups plays a role in modulating the selectivity of the pore for either anions or protons. To elucidate the molecular mechanism of permeation and selectivity, we are conducting free energy simulations for the translocation of protons and other ions in both wild-type and mutant forms of hHv1.

The Resting State of Human Proton Channel from Functional and Structural Determinations

The human voltage gated proton channel (Hv1) plays an important role in a wide variety of physiological functions, including male fertility, immune response and metastatic proliferation. Upon transmembrane voltage stimulus, the rearrangement of positively charged S4 leads to highly selective and directional conduction of proton. Hv1 carries out this function on the structural blueprint of the S4-containing voltage sensing domains (VSD). As a way to understand Hv1 gating, we have developed a simple biochemical preparation of functional Hv1 as shown by established fluorescence based proton flux assay. We have probed Hv1 in the functional proteoliposome by site-directed spin labeling EPR spectroscopic (SDSL-EPR) methods. Mobility and accessibility information show that the secondary structure of transmembrane segments is generally consistent with the expected VSD topology. Solvent accessibility measurements revealed that the gating Arginines (205, 208 and 211) are located on the lower part of S4, the internal leaflet of the bilayer. The presence of a narrow solvent occluded region strongly suggests a short proton conduction pathway, and does not seem to be compatible with the idea of a long water wire across membrane. Furthermore, the Hv1 represents a resting state VSD at 0 mV according to its G-V curve . The structural details from Hv1 provide unique insight to not only the proton conduction, but also the mechanism of voltage sensing.

Modulation of the Voltage-Gated Proton Channel Hv1 by Small Gating-Modifier Ligands

The human voltage-gated proton channel Hv1 is activated by depolarization and intracellular acidification to maintain the cell's neutral pH. It belongs to voltage-gated cation channels, consisting of a typical voltage sensor domain (VSD) but lacking a canonical pore domain. These channels assemble as dimers and recent data indicate strong cooperativity between the two subunits. Recently, guanidine derivatives were found to act as open channel blockers on the Hv1 VSD. Here we show that the gating modifiers NH29 and NH34, two diphenylamine carboxylate derivatives, act as Hv1 channel openers. NH29 was recently shown to exert opposite gating-modifier effects on the VSD of Kv7.2 potassium channels and TRPV1 cationic channels operating as opener and blocker of Kv7.2 and TRPV1 currents, respectively. Both compounds increase the Hv1 current amplitude by more than 2.5-fold at + 50 mV and produce a left-shift in the voltage dependence of channel activation by about −15 mV. We found that NH29 and NH34 change the proton selectivity of Hv1 channels and produce a left-shift of about −10 mV in the reversal potentials both in symmetrical (pH 7.0) and asymmetrical pH solutions (pHi 5.5/pHo 7.0). Our results also show that the mutation R211S in S4 profoundly affects the modulatory properties of the compounds. The S4 residue R211 was previously suggested to be crucial for Hv1 proton selectivity by interacting with D112 in S1. In mutant R211S, NH29 does not act anymore as an opener but rather inhibits Hv1 currents. Other Hv1 mutations are currently tested in order to determine how Hv1 channels interact with these small ligands and to elucidate Hv1 selectivity and gating mechanisms.