Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1

The hydrophobic gasket (HG), 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 “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

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Voltage-dependent structural models of the human Hv1 proton channel from long-timescale molecular dynamics simulations

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920943117 ◽

2020 ◽

Vol 117 (24) ◽

pp. 13490-13498 ◽

Cited By ~ 3

Author(s):

Andrew D. Geragotelis ◽

Mona L. Wood ◽

Hendrik Göddeke ◽

Liang Hong ◽

Parker D. Webster ◽

...

Keyword(s):

Molecular Dynamics ◽

Membrane Potential ◽

Conformational Changes ◽

Structural Model ◽

Ph Regulation ◽

Conformational Dynamics ◽

Structural Models ◽

Dynamics Simulation ◽

Proton Channel ◽

Voltage Dependent

The voltage-gated Hv1 proton channel is a ubiquitous membrane protein that has roles in a variety of cellular processes, including proton extrusion, pH regulation, production of reactive oxygen species, proliferation of cancer cells, and increased brain damage during ischemic stroke. A crystal structure of an Hv1 construct in a putative closed state has been reported, and structural models for the channel open state have been proposed, but a complete characterization of the Hv1 conformational dynamics under an applied membrane potential has been elusive. We report structural models of the Hv1 voltage-sensing domain (VSD), both in a hyperpolarized state and a depolarized state resulting from voltage-dependent conformational changes during a 10-μs-timescale atomistic molecular dynamics simulation in an explicit membrane environment. In response to a depolarizing membrane potential, the S4 helix undergoes an outward displacement, leading to changes in the VSD internal salt-bridge network, resulting in a reshaping of the permeation pathway and a significant increase in hydrogen bond connectivity throughout the channel. The total gating charge displacement associated with this transition is consistent with experimental estimates. Molecular docking calculations confirm the proposed mechanism for the inhibitory action of 2-guanidinobenzimidazole (2GBI) derived from electrophysiological measurements and mutagenesis. The depolarized structural model is also consistent with the formation of a metal bridge between residues located in the core of the VSD. Taken together, our results suggest that these structural models are representative of the closed and open states of the Hv1 channel.

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Coupling of Voltage Sensing to Channel Opening Reflects Intrasubunit Interactions in Kv Channels

The Journal of General Physiology ◽

10.1085/jgp.200409194 ◽

2004 ◽

Vol 125 (1) ◽

pp. 71-80 ◽

Cited By ~ 11

Author(s):

Alain J. Labro ◽

Adam L. Raes ◽

Dirk J. Snyders

Keyword(s):

Voltage Dependence ◽

Voltage Sensing ◽

Α Subunit ◽

K Channels ◽

Voltage Gated ◽

Kv Channels ◽

Channel Opening ◽

Subunit Stoichiometry ◽

Voltage Dependent ◽

Tetrameric Structure

Voltage-gated K+ channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same α-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction.

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Conserved Dimeric Subunit Stoichiometry of SLC26 Multifunctional Anion Exchangers

Journal of Biological Chemistry ◽

10.1074/jbc.m704924200 ◽

2007 ◽

Vol 283 (7) ◽

pp. 4177-4188 ◽

Cited By ~ 69

Author(s):

Silvia Detro-Dassen ◽

Michael Schänzler ◽

Heike Lauks ◽

Ina Martin ◽

Sonja Meyer zu Berstenhorst ◽

...

Keyword(s):

Anion Exchangers ◽

Subunit Stoichiometry ◽

Dimeric Subunit

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Biophysical Studies of the Voltage-Dependent Proton Channel

Biophysical Journal ◽

10.1016/j.bpj.2012.11.226 ◽

2013 ◽

Vol 104 (2) ◽

pp. 34a

Author(s):

Roderick MacKinnon ◽

Joel Butterwick ◽

James Letts Letts

Keyword(s):

Proton Channel ◽

Biophysical Studies ◽

Voltage Dependent

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

The Journal of General Physiology ◽

10.1085/jgp.201711968 ◽

2018 ◽

Vol 150 (6) ◽

pp. 851-862 ◽

Cited By ~ 4

Author(s):

Vladimir V. Cherny ◽

Deri Morgan ◽

Sarah Thomas ◽

Susan M.E. Smith ◽

Thomas E. DeCoursey

Keyword(s):

Single Point ◽

Species Difference ◽

Transmembrane Helix ◽

Proton Channel ◽

Single Point Mutation ◽

Ph Sensing ◽

Electrophysiological Properties ◽

Voltage Gated ◽

Voltage Dependent ◽

Kinetics Of

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.

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Interactions between Electron and Proton Currents in Excised Patches from Human Eosinophils

The Journal of General Physiology ◽

10.1085/jgp.200308891 ◽

2003 ◽

Vol 122 (6) ◽

pp. 713-726 ◽

Cited By ~ 19

Author(s):

Gábor L. Petheö ◽

Andrés Maturana ◽

András Spät ◽

Nicolas Demaurex

Keyword(s):

Proton Channel ◽

Resting Cells ◽

Long Term Stability ◽

Combined Application ◽

Bath Application ◽

Diphenylene Iodonium ◽

Voltage Dependent ◽

Excised Patches ◽

H Channels ◽

Proton Currents

The NADPH–oxidase is a plasma membrane enzyme complex that enables phagocytes to generate superoxide in order to kill invading pathogens, a critical step in the host defense against infections. The oxidase transfers electrons from cytosolic NADPH to extracellular oxygen, a process that requires concomitant H+ extrusion through depolarization-activated H+ channels. Whether H+ fluxes are mediated by the oxidase itself is controversial, but there is a general agreement that the oxidase and H+ channel are intimately connected. Oxidase activation evokes profound changes in whole-cell H+ current (IH), causing an approximately −40-mV shift in the activation threshold that leads to the appearance of inward IH. To further explore the relationship between the oxidase and proton channel, we performed voltage-clamp experiments on inside-out patches from both resting and phorbol-12-myristate-13-acetate (PMA)-activated human eosinophils. Proton currents from resting cells displayed slow voltage-dependent activation, long-term stability, and were blocked by micromolar internal [Zn2+]. IH from PMA-treated cells activated faster and at lower voltages, enabling sustained H+ influx, but ran down within minutes, regaining the current properties of nonactivated cells. Bath application of NADPH to patches excised from PMA-treated cells evoked electron currents (Ie), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both IH and Ie was delayed, and sometimes prevented, by cytosolic ATP and GTP-γ-S. A good correlation was observed between the amplitude of Ie and both inward and outward IH when a stable driving force for e− was imposed. Combined application of NADPH and DPI reduced the inward IH amplitude, even in the absence of concomitant oxidase activity. The strict correlation between Ie and IH amplitudes and the sensitivity of IH to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.

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Subunit Stoichiometry of Human Muscle Chloride Channels

The Journal of General Physiology ◽

10.1085/jgp.109.1.93 ◽

1997 ◽

Vol 109 (1) ◽

pp. 93-104 ◽

Cited By ~ 75

Author(s):

Christoph Fahlke ◽

Timothy Knittle ◽

Christina A. Gurnett ◽

Kevin P. Campbell ◽

Alfred L. George

Keyword(s):

Chloride Channels ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Multimeric Complex ◽

Subunit Stoichiometry ◽

Voltage Dependent ◽

Cl Channels ◽

Homogenous Population

Voltage-gated Cl− channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl− channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl− channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.

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The low-resolution 3D-structure of porin, a channel-forming protein in E. coliouter membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075932 ◽

1983 ◽

Vol 41 ◽

pp. 440-441

Author(s):

A. Engel ◽

D.L. Dorset ◽

A. Massalski ◽

J.P. Rosenbusch

Keyword(s):

Crystal Packing ◽

3D Structure ◽

Gram Negative Bacteria ◽

Protein Ratio ◽

Crystal Forms ◽

Large Molecules ◽

Functional Studies ◽

Voltage Dependent ◽

Planar Membranes ◽

Hexagonal Arrays

Porins represent a group of channel forming proteins that facilitate diffusion of small solutes across the outer membrane of Gram-negative bacteria, while excluding large molecules (>650 Da). Planar membranes reconstituted from purified matrix porin (OmpF protein) trimers and phospholipids have allowed quantitative functional studies of the voltage-dependent channels and revealed concerted activation of triplets. Under the same reconstitution conditions but using high protein concentrations porin aggregated to 2D lattices suitable for electron microscopy and image processing. Depending on the lipid-to- protein ratio three different crystal packing arrangements were observed: a large (a = 93 Å) and a small (a = 79 Å) hexagonal and a rectangular (a = 79 Å b = 139 Å) form with p3 symmetry for the hexagonal arrays. In all crystal forms distinct stain filled triplet indentations could be seen and were found to be morphologically identical within a resolution of (22 Å). It is tempting to correlate stain triplets with triple channels, but the proof of this hypothesis requires an analysis of the structure in 3 dimensions.

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Cryo-Electron Microscopy and Correlation Averaging of Frozen-Hydrated, Polymorphic 2D Crystals of the Mitochondrial Outer Membrane Channel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179257 ◽

1990 ◽

Vol 48 (1) ◽

pp. 100-101

Author(s):

Xiao-Wei Guo

Keyword(s):

Outer Membrane ◽

Hexagonal Lattice ◽

Mitochondrial Outer Membrane ◽

Rectangular Lattice ◽

Electron Microscopic ◽

Cryo Electron Microscopy ◽

Voltage Dependent ◽

Outer Membrane Channel ◽

2D Crystals ◽

Vdac Channel

Voltage-dependent, anion-selective channels (VDAC) are formed in the mitochondrial outer membrane (mitOM) by a 30-kDa polypeptide. These channels form ordered 2D arrays when mitOMs from Neurospora crassa are treated with soluble phospholipase A2. We obtain low-dose electron microscopic images of unstained specimens of VDAC crystals preserved in vitreous ice, using a Philips EM420 equipped with a Gatan cryo-transfer stage. We then use correlation analysis to compute average projections of the channel crystals. The procedure involves Fourier-filtration of a region within a crystal field to obtain a preliminary average that is subsequently cross-correlated with the entire crystal. Subregions are windowed from the crystal image at coordinates of peaks in the cross-correlation function (CCF, see Figures 1 and 2) and summed to form averages (Figure 3).The VDAC channel forms several different types of crystalline arrays in mitOMs. The polymorph first observed during phospholipase treatment is a parallelogram array (a=13 run, b=11.5 run, θ==109°) containing 6 water-filled pores per unit cell. Figure 1 shows the CCF of a sub-field of such an “oblique” array used to compute the correlation average of Figure 3A. With increased phospholipase treatment, other polymorphs are observed, often co-existing within the same crystal. For example, two distinct (but closely related) types of lattices occur in the field corresponding to the CCF of Figure 2: a “contracted” version of the parallelogram lattice (a=13 run, b=10 run, θ=99°), and a near-rectangular lattice (a=8.5 run, b=5 nm). The pattern of maxima in this CCF suggests that a third, near-hexagonal lattice (a=4.5 nm) may also be present. The correlation averages of Figures 3B-D were computed from polycrystalline fields, using peak coordinates in regions of CCFs corresponding to each of the three lattice types.

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