The Hv1 proton channel responds to mechanical stimuli.
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ABSTRACT:
SUBMITTER: Pathak MM
PROVIDER: S-EPMC5089936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Hv1 proton channel shares striking structural homology with fourth transmembrane helical segment-type voltage-sensor (VS) domains but manifests distinctive functional properties, including a proton-selective "aqueous" conductance and allosteric control of voltage-dependent gating by changes in the transmembrane pH gradient. The mechanisms responsible for Hv1's functional properties remain poorly understood, in part because methods for measuring gating currents that directly report VS activation have not yet been described. Here, we describe an approach that allows robust and reproducible measurement of gating-associated charge movements in Hv1. Gating currents reveal that VS activation and proton-selective aqueous conductance opening are thermodynamically distinct steps in the Hv1 activation pathway and show that pH changes directly alter VS activation. The availability of an assay for gating currents in Hv1 may aid future efforts to elucidate the molecular mechanisms of gating cooperativity, pH-dependent modulation, and H+ selectivity in a model VS domain protein.
| S-EPMC6026397 | biostudies-literature
Project description:The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: It is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute, but the nature of transmembrane packing is unclear. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, we find that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. Our results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.
| S-EPMC7456152 | biostudies-literature
Project description: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 AspH(0)-H2O(0)-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.
| S-EPMC4429351 | biostudies-literature
Project description:The NADPH oxidase reaction produces protons. In the case of the NADPH oxidase, NOX2, activity depends on secretion of these protons and is inhibited by blockade of the voltage-gated proton channel (Hv1). Duox1 and Duox2 activities similarly produce intracellular protons but synthesize hydrogen peroxide directly instead of superoxide. Hv1 contributes to acid secretion in some epithelia that express Duox. To test the hypothesis that Duox activity is also sensitive to Hv1 channel blockers, Duox was assayed in the presence of either Zn2+ or 5-chloro-2-guanidinobenzimidazole (ClGBI). Both compounds inhibited Duox activity in normal human bronchial epithelial cells but with an IC50 over 10-fold higher than that reported for Hv1 (IC50 Zn2+?=?0.68?mM; IC50 ClGBI?=?0.07-0.14?mM). Homogenized HEK293T cells expressing either Duox1 or Duox2 showed similar IC50 values for ClGBI suggesting these compounds inhibit the enzymes through alternate mechanisms independent of Hv1 proton secretion. Inclusion of superoxide dismutase did not restore Duox hydrogen peroxide synthesis. Addition of nigericin to eliminate any possible transmembrane pH gradients in intracellular membrane-localized Duox did not alter activity in HEK293T homogenates. Extracellular Zn2+ blocked intracellular Ca2+ increases needed for Duox activity. Together the data suggest that Duox enzyme activities in epithelia are inhibited by compounds that block Hv1 but inhibition occurs through Hv1-independent mechanisms and support the idea that Hv1 is not required for Duox activity.
| S-EPMC6920136 | biostudies-literature
Project description:The voltage-gated proton channel Hv1 plays important roles in proton extrusion, pH homeostasis, and production of reactive oxygen species in a variety of cell types. Excessive Hv1 activity increases proliferation and invasiveness in cancer cells and worsens brain damage in ischemic stroke. The channel is composed of two subunits, each containing a proton-permeable voltage-sensing domain (VSD) and lacking the pore domain typical of other voltage-gated ion channels. We have previously shown that the compound 2-guanidinobenzimidazole (2GBI) inhibits Hv1 proton conduction by binding to the VSD from its intracellular side. Here, we examine the binding affinities of a series of 2GBI derivatives on human Hv1 channels mutated at positions located in the core of the VSD and apply mutant cycle analysis to determine how the inhibitor interacts with the channel. We identify four Hv1 residues involved in the binding: aspartate 112, phenylalanine 150, serine 181, and arginine 211. 2GBI appears to be oriented in the binding site with its benzo ring pointing to F150, its imidazole ring inserted between residue D112 and residues S181 and R211, and the guanidine group positioned in the proximity of R211. We also identify a modified version of 2GBI that is able to reach the binding site on Hv1 from the extracellular side of the membrane. Understanding how compounds like 2GBI interact with the Hv1 channel is an important step to the development of pharmacological treatments for diseases caused by Hv1 hyperactivity.
| S-EPMC4103315 | biostudies-literature
Project description: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.
| S-EPMC6187172 | biostudies-literature
Project description:The Hv1 proton channel is unique among voltage-gated channels for containing the pore and gate within its voltage-sensing domain. Pore opening has been proposed to include assembly of the selectivity filter between an arginine (R3) of segment S4 and an aspartate (D1) of segment S1. We determined whether gating involves motion of S1, using Ciona intestinalis Hv1. We found that channel opening is concomitant with solution access to the pore-lining face of S1, from the cytoplasm to deep inside the pore. Voltage- and patch-clamp fluorometry showed that this involves a motion of S1 relative to its surroundings. S1 motion and the S4 motion that precedes it are each influenced by residues on the other helix, thus suggesting a dynamic interaction between S1 and S4. Our findings suggest that the S1 of Hv1 has specialized to function as part of the channel's gate.
| S-EPMC4385474 | biostudies-literature
Project description:The voltage-gated proton (Hv1) channel, a voltage sensor and a conductive pore contained in one structural module, plays important roles in many physiological processes. Voltage sensor movements can be directly detected by measuring gating currents, and a detailed characterization of Hv1 charge displacements during channel activation can help to understand the function of this channel. We succeeded in detecting gating currents in the monomeric form of the Ciona-Hv1 channel. To decrease proton currents and better separate gating currents from ion currents, we used the low-conducting Hv1 mutant N264R. Isolated ON-gating currents decayed at increasing rates with increasing membrane depolarization, and the amount of gating charges displaced saturates at high voltages. These are two hallmarks of currents arising from the movement of charged elements within the boundaries of the cell membrane. The kinetic analysis of gating currents revealed a complex time course of the ON-gating current characterized by two peaks and a marked Cole-Moore effect. Both features argue that the voltage sensor undergoes several voltage-dependent conformational changes during activation. However, most of the charge is displaced in a single central transition. Upon voltage sensor activation, the charge is trapped, and only a fast component that carries a small percentage of the total charge is observed in the OFF. We hypothesize that trapping is due to the presence of the arginine side chain in position 264, which acts as a blocking ion. We conclude that the movement of the voltage sensor must proceed through at least five states to account for our experimental data satisfactorily.
| S-EPMC6140481 | biostudies-literature
Project description:The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H(+)-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (?pH). Allosteric control of Hv1 channel opening by ?pH (V-?pH coupling) is manifested by a characteristic shift of approximately 40 mV per ?pH unit in the activation. To further understand the mechanism for V-?pH coupling in Hv1, H(+) current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pHI). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.
| S-EPMC4190649 | biostudies-literature
Project description:Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH oxidase activity.
| S-EPMC2990768 | biostudies-literature