Proton Channel Hv1 Research Articles

Functional insights into voltage gated proton channel (Hv1) in bull spermatozoa

Acid extrusion and intracellular alkalisation are the key events during sperm capacitation and these are mediated through proton gated channels (Hv1). Role of Hv1 in regulating sperm motility, capacitation and acrosome reaction has been documented in human spermatozoa; but no such data is available in bull spermatozoa; therefore, the present study was undertaken in Hariana bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls to investigate the functional involvement of Hv1 in regulation of sperm motility, capacitation and acrosome reaction in bull spermatozoa. Immunoblotting revealed the presence of a single band of 31.8 kDa corresponding to Hv1 in Hariana bull spermatozoa and immunoflourescence confirmed the positive immune-reactivity at principal piece of spermatozoa for Hv1. Functional study was carried out using 200 μM 2-Guanidinobenzimidazole (2-Guanidinobenzimidazole,selective Hv1 blocker) and 1 mM zinc chloride (potent Hv1 blocker), and 0.3 μM Anandamide (AEA), an activator of Hv1. Blocking of Hv1 resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM). Activation of Hv1 with AEA also resulted in significant (P < 0.05) reduction in PSM till 1 h and thereafter, the PSM was restored and reduction was almost similar to that in control. However, during activation and inactivation of Hv1, per cent spermatozoa showing hyperactive motility were found to be markedly increased (20–30%) compared to 10–20% in control. 2-Guanidinobenzimidazole, zinc and AEA treated spermatozoa revealed significant (P < 0.05) increase in B-pattern of spermatozoa indicating induction of capacitation. Downstream signalling of Hv1 activation or inactivation seemed to be mediated through cAMP and PKA pathways. Catsper channels were found to be intimately associated with Hv1 function in regulating sperm motility. Blocking as well as activation of Hv1 resulted in significant (P < 0.05) reduction in sperm livability, spermatozoa having intact membrane, intact acrosome, and high mitochondrial transmembrane potential (MTP). Our findings evidently suggest that Hv1 channels are present in bull spermatozoa and these regulate sperm functions like hypermotility, capacitation and acrosome reaction through complex interplay between different pathways involving cAMP, PKC, and Catsper. Further studies are required to find out the possible relationship between Hv1 channels and other channels in regulating spermatozoa functions.

Hv1-deficiency protects β cells from glucotoxicity through regulation of NOX4 level

High glucose (HG)-induced oxidative stress contributes to the dysfunction of pancreatic β cells in diabetes. The voltage-gated proton channel Hv1 has been proposed to support reactive oxygen species (ROS) production during respiratory bursts. However, the effect of Hv1 on glucotoxicity in pancreatic β cells is not clear yet. In this study, we examined the protective effects of Hv1-deficiency in HG cultured β cells. Following 48 h of treatment with 30 mM high glucose, Hv1 KO β cells showed higher cell viability, lower cell apoptosis and a more stable insulin gene expression level compared to WT β cells. In both control and HG cultured β cells, deficiency of Hv1 decreased the glucose- and PMA-induced ROS production. Finally, HG incubation led to NOX4 upregulation in WT β cells, which could be inhibited by HV1 deficiency. In conclusion, Hv1-deficiency prevents the HG treatment-induced NOX4 upregulation and protects β cells from glucotoxicity.

Real-time functional analysis of Hv1 channel in neutrophils: a new approach from zebrafish model.

Voltage-gated proton channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken an in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos (Danio rerio). Hvcn1-deficiency (hvcn1-/-) in zebrafish completely abolished voltage-gated proton current, which is typically observed in wild-type neutrophils. Importantly, hvcn1-deficiency significantly reduced reactive oxygen species production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild-type and hvcn1-/- zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in hvcn1-/- neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach toward the understanding of Hv1 in a physiological context.

PH Dependence of a Monomeric Non-Conducting Voltage-Gated Proton Channel (HV1)

The voltage-gated proton channel (Hv1) is a transmembrane protein made of a dimer of four transmembrane segments. This structure is capable of sensing the voltage and conforms the permeation pathway to protons. One of the most striking biophysical properties of Hv1 is its pH dependence of gating. The conductance-voltage (G-V) curve of Hv1 shifts depending on the pH gradient established across the membrane, but the molecular mechanism of this property is still poorly understood. Recently, gating currents of Hv1 were characterized using the low-conducting N264R mutant, giving us a new tool to understand the mechanism behind the pH dependence of Hv1. We measured currents produced by the monomeric N264R mutant of Ciona intestinalis Hv1 at different internal and external pH in membrane patches of Xenopus laevis oocytes. Although results showed a clear pH dependence of the gating currents, the presence of proton currents and charge trapping produced by the N264R mutation prevent a detailed analysis of the data. To eliminate proton currents, we measured gating currents produced by a monomeric non-conducting Hv1. Gating currents of the non-conducting mutant are free of proton current contamination and charge trapping. They preserve all the characteristics of Hv1 gating currents described previously. The charge-voltage (Q-V) curve is shifted to the left at lower ΔpH (pHin-pHout). Additionally, the ON-gating current decay is faster when ΔpH decreases. Our results show that the non-conducting mutant is a better choice to study pH dependence of Hv1 and understand the molecular mechanism behind this biophysical property. Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt 1180464 to C.G. and 1150273 to R.L. The Centro Interdisciplinario de Neurociencia de Valparaiso is a Millennium Institute supported by the Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (P029-022-F).

Two-Pronged Aromatic Arginine-Mimics as Hv1 Proton Channel Inhibitors

The voltage-gated proton channel Hv1 regulates cellular pH homeostasis and the production of reactive oxygen species by the NADPH oxidase. The channel is made of two identical subunits, which gate cooperatively. Each subunit contains a voltage-sensing domain (VSD) that conducts protons and is inhibited by the arginine mimic 2-guanidinobenzimidazole (2GBI). 2GBI operates from the intracellular side and gains access to its binding site when the channel is open. The blocker must leave the binding site for the channel gate to close. We previously found that mutation F150A strongly increases the binding affinity. We now show that this increase in affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150 and use this information to design new arginine mimics with improved affinity for the non-rearranged binding site of the wild type channel. The new compounds consist of two “prongs”, an aminoimidazole ring and a fluorinated aromatic group connected by extended linkers. We find that Hv1 inhibition by the new compounds presents fast and slow binding modalities, which are affected by distinct types of perturbations of the binding site. The conservative amino acid substitution D112E in the Hv1 selectivity filter abolishes the fast component, leaving the slow component unaltered, whereas the F150A substitution affects primarily the slow component. Based on kinetic analysis of the inhibition process, we propose that the new arginine mimics are able to bind the open channel like 2GBI. However, unlike 2GBI, the new compounds become “trapped” inside the VSD when the gate closes, and they can slowly reach the binding site in the trapped conformation even from the closed state. These pharmacological properties are desirable features for the treatment of diseases associated with Hv1 gain of function.

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner.

Sperm function and male fertility are closely related to pH dependent K+ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

On the control of the proton current in the voltage-gated proton channel Hv1

The nature of the action of voltage-activated proton transport proteins is a conundrum of great current interest. Here we approach this issue by exploring the action of Hv1, a voltage-gated proton channel found in different cells in humans and other organisms. Our study focuses on evaluating the free energy of transporting a proton through the channel, as well as the effect of the proton transfer through D112, in both the closed and open channel conformations. It is found that D112 allows a transported proton to bypass the electrostatic barrier of the open channel, while not being able to help in passing the barrier in the closed form. This reflects the change in position of the gating arginine residues relative to D112, upon voltage activation. Significantly, the effect of D112 accounts for the observed trend in selectivity by overcoming the electrostatic barrier at its highest point. Thus, the calculations provide a structure/function correlation for the Hv1 system. The present work also clarifies that the action of Hv1 is not controlled by a Grotthuss mechanism but, as is always the case, by the protein electrostatic potential at the rate-limiting barriers.

Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation

Ion channels control sperm navigation within the female reproductive tract and, thus, are critical for their ability to find and fertilize an egg. The flagellar calcium channel CatSper controls sperm hyperactivated motility and is dependent on an alkaline cytoplasmic pH. The latter is accomplished by either proton transporters or, in human sperm, via the voltage-gated proton channel Hv1. To provide concerted regulation, ion channels and their regulatory proteins must be compartmentalized. Here, we describe flagellar regulatory nanodomains comprised of Hv1, CatSper, and its regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 is distributed asymmetrically within bilateral longitudinal lines and that inhibition of this channel leads to a decrease in sperm rotation along the long axis. We suggest that specific distribution of flagellar nanodomains provides a structural basis for the selective activation of CatSper and subsequent flagellar rotation. The latter, together with hyperactivated motility, enhances the fertility of sperm.

Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H+-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the KD-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters. Graphical abstract ᅟ.

Sodium bicarbonate loading limits tubular cast formation independent of glomerular injury and proteinuria in Dahl salt-sensitive rats.

Sodium bicarbonate (NaHCO3) slows the decline in kidney function in patients with chronic kidney disease (CKD), yet the mechanisms mediating this effect remain unclear. The Dahl salt-sensitive (SS) rat develops hypertension and progressive renal injury when fed a high salt diet; however, the effect of alkali loading on kidney injury has never been investigated in this model. We hypothesized that NaHCO3 protects from the development of renal injury in Dahl salt-sensitive rats via luminal alkalization which limits the formation of tubular casts, which are a prominent pathological feature in this model. To examine this hypothesis, we determined blood pressure and renal injury responses in Dahl SS rats drinking vehicle (0.1 M NaCl) or NaHCO3 (0.1 M) solutions as well as in Dahl SS rats lacking the voltage-gated proton channel (Hv1). We found that oral NaHCO3 reduced tubular NH4+ production, tubular cast formation, and interstitial fibrosis in rats fed a high salt diet for 2 weeks. This effect was independent of changes in blood pressure, glomerular injury, or proteinuria and did not associate with changes in renal inflammatory status. We found that null mutation of Hv1 also limited cast formation in Dahl SS rats independent of proteinuria or glomerular injury. As Hv1 is localized to the luminal membrane of TAL, our data suggest that alkalization of the luminal fluid within this segment limits cast formation in this model. Reduced cast formation, secondary to luminal alkalization within TAL segments may mediate some of the protective effects of alkali loading observed in CKD patients.

Hv 1 Proton Channels in Dinoflagellates: Not Just for Bioluminescence?

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.

The acid test for pH-dependent gating in cloned HV1 channels.

Islas evaluates two papers that provide mechanistic insight into pH-dependent gating in HV1 proton channels.

Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation.

The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H+-selective "aqueous" conductance (GAQ) that is potently modulated by extracellular Zn2+ Although two conserved His residues are required for Zn2+ effects on GAQ gating, the atomic structure of the Zn2+ coordination site and mechanism by which extracellular Zn2+ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn2+ potency and are therefore likely to participate in first solvation shell interactions with Zn2+ Experimental Zn2+-mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn2+ coordination spheres in Zn2+-bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn2+-interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg "gating charges" in S4 as well as acidic "counter-charges" in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn2+ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting GAQ activation to more negative voltages, E153A also decreases Zn2+ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.

Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced β-cell damage

Reactive oxygen species (ROS) impairs pancreatic β-cells and plays an important role in development of diabetes. Streptozotocin (STZ) can lead to β-cell dysfunction via inducing ROS production. The voltage-gated proton channel Hv1 contributes a majority of the charge compensation required for ROS production. Here, we investigated the effects of Hv1 on STZ-induced β-cell damage. We found that deficiency of Hv1 obviously inhibits STZ-induced glucose intolerance in mice, and prevents the decrease in β-cell mass and pancreatic insulin content from STZ-treatment. Further studies showed that loss of Hv1 significantly attenuates STZ-induced β-cell damage and ROS production in pancreatic β-cells. Our results suggest that Hv1 might contribute to development of diabetes through producing ROS.

Gating Currents in Hv1 Proton Channels

Gating Currents in Hv1 Proton Channels

Properties of the Voltage-Gated Proton Channel Gating Currents

The voltage-gated proton channel (Hv1) is a dimer of subunits containing four transmembrane domains (S1-S4) which play the double role of harboring the voltage-sensing charges and forming the proton conduction pathway. Some biophysical properties of Hv1, as the coupling between voltage-sensing and permeation pathway opening, the pH dependence of gating, the cooperativity between each subunit in the dimer, and the steps of charge movement during activation and deactivation can be better understood by measuring and characterizing the Hv1 gating currents. However, to measure gating currents, ionic currents needs to be eliminated or greatly reduced, a feat that is particularly challenging for Hv1 for which there is no effective blocker and proton concentration cannot be reduced more than 10 fold. Here, we expressed the low-conducting N264R Hv1 mutant from Ciona intestinalis in Xenopus laevis oocytes and succeeded in recording gating currents membrane patches with both, the monomeric and dimeric channels. In agreement with previous fluorescence measurements (Qiu et al., Neuron 77:288, 2013), the analysis of the gating currents kinetics and charge movement revealed that the ON component has a double exponential decay. Additionally, changes in pH modulate Hv1 gating currents kinetics and Q-V curves. This suggests that the pH sensors in Hv1 are located in the voltage-sensing domain. Finally, in our experimental conditions we observed charge immobilization after activation, which could be an intrinsic characteristic of Hv1 voltage-sensing domain. Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt grants ACT 1104, Fondecyt 1160261 to C.G. and 1150273 to R.L.

Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation

The ability of sperm to fertilize an egg is controlled by ion channels, one of which is the pHdependent calcium channel of sperm CatSper. For CatSper to be fully activated, the cytoplasmic pH must be alkaline, which is accomplished by either proton transporters, or a faster mechanism, such as the voltage-gated proton channel Hv1. To ensure effective regulation, these channels and regulatory proteins must be tightly compartmentalized. Here, we characterize human sperm nanodomains that are comprised of Hv1, CatSper and regulatory protein ABHD2. Superresolution microscopy revealed that Hv1 forms asymmetrically positioned bilaterally distributed longitudinal lines that span the entire length of the sperm tail. Such a distribution provides a direct structural basis for the selective activation of CatSper, and subsequent flagellar rotation along the long axis that, together with hyperactivated motility, enhances sperm fertility. Indeed, Hv1 inhibition leads to a decrease in sperm rotation. Thus, sperm ion channels are organized in distinct regulatory nanodomains that control hyperactivated motility and rotation.

Photocontrol of the Hv1 Proton Channel.

The voltage-gated proton channel Hv1 is expressed in various human cell types, including macrophages, epithelial cells, and sperm. Hv1 opening leads to proton efflux that alkalizes the cytosol. Here, we describe light-activated Hv1 inhibitors (photoswitches) that allow controlling its activity with high spatiotemporal precision. The photoswitches comprise a light-sensitive azobenzene moiety and 2-guanidinobenzimidazole (2GBI), a known Hv1 inhibitor. In the dark, photoGBI inhibits heterologously expressed Hv1 channels. Blue light, which isomerizes the azobenzene group from trans to cis conformation, releases inhibition. We demonstrate photocontrol of native proton currents in human macrophages and sperm using photoGBI, underlining their use as valuable optochemical tools to study the function of Hv1 channels.

Proton pump inhibitors have pH-dependent effects on the thermostability of the carboxyl-terminal domain of voltage-gated proton channel Hv1.

The voltage-gated proton channel Hv1 is highly selective for H+ and is activated by membrane depolarization and pH gradient. An increased external and decreased internal pH opens the Hv1 channel. The intracellular C-terminal domain of Hv1 is responsible for channel dimerization, cooperative, and thermosensitive gating. Here, we found that proton pump inhibitors (PPIs) interact with the C-terminal domain of human Hv1. The interaction between PPIs and the C-terminal domain, which is pH-dependent, lowered the thermal and structural stability of the protein at pH 4, but enhanced the thermal and structural stability at pH 8. Furthermore, we investigated in vitro the interaction of PPIs with the C-terminal domain of Hv1 by fluorescence and micro-Raman spectra. Fluorescence quenching measurements revealed that the interaction between the C-terminal domain and PPIs is a mainly hydrophobic interaction. The micro-Raman spectra showed that PPIs did not form stable disulfide bonds with the unique thiol group within this domain (Cys249 residue). The preferential interaction of PPIs with the inactive form of Hv1 stabilizes the high pH inactive state of the C-terminal domain, indicating a mechanism by which PPIs might act explicitly on the stabilization of a closed state of the proton channel.

Na+-H+ exchanger and proton channel in heart failure associated with Becker and Duchenne muscular dystrophies.

Cardiomyopathy is found in patients with Duchenne (DMD) and Becker (BMD) muscular dystrophies, which are linked muscle diseases caused by mutations in the dystrophin gene. Dystrophin defects are not limited to DMD but are also present in mild BMD. The hereditary cardiomyopathic hamster of the UM-X7.1 strain is a particular experimental model of heart failure (HF) leading to early death in muscular dystrophy (dystrophin deficiency and sarcoglycan mutation) and heart disease (δ-sarcoglycan deficiency and dystrophin mutation) in human DMD. Using this model, our previous work showed a defect in intracellular sodium homeostasis before the appearance of any apparent biochemical and histological defects. This was attributed to the continual presence of the fetal slow sodium channel, which was also found to be active in human DMD. Due to muscular intracellular acidosis, the intracellular sodium overload in DMD and BMD was also due to sodium influx through the sodium-hydrogen exchanger NHE-1. Lifetime treatment with an NHE-1 inhibitor prevented intracellular Na+ overload and early death due to HF. Our previous work also showed that another proton transporter, the voltage-gated proton channel (Hv1), exists in many cell types including heart cells and skeletal muscle fibers. The Hv1 could be indirectly implicated in the beneficial effect of blocking NHE-1.