Thermodynamics and Mechanism of the Membrane Permeation of Hv1 Channel Blockers.
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ABSTRACT:
SUBMITTER: Lim VT
PROVIDER: S-EPMC8549822 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description:Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H(+) flux and are gated by both voltage and pH gradients. Selective H(+) transfer in membrane proteins is commonly achieved by Grotthuss proton 'hopping' in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H(+) currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.
| S-EPMC4035905 | biostudies-literature
Project description:Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1, however, was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom molecular dynamics simulations with a swarm of trajectories with the string method for extensive transition path sampling and evolution. A detailed residue-residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular, it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al. (Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A, Kurokawa T, et al. X-ray crystal structure of voltage-gated proton channel. Nature 2014). The mechanisms governing ion transport in the wild-type and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results, we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport.
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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:Calcium is the most abundant metal in the human body that plays vital roles as a cellular electrolyte as well as the smallest and most frequently used signaling molecule. Calcium uptake in epithelial tissues is mediated by tetrameric calcium-selective transient receptor potential (TRP) channels TRPV6 that are implicated in a variety of human diseases, including numerous forms of cancer. We used TRPV6 crystal structures as templates for molecular dynamics simulations to identify ion binding sites and to study the permeation mechanism of calcium and other ions through TRPV6 channels. We found that at low Ca2+ concentrations, a single calcium ion binds at the selectivity filter narrow constriction formed by aspartates D541 and allows Na+ permeation. In the presence of ions, no water binds to or crosses the pore constriction. At high Ca2+ concentrations, calcium permeates the pore according to the knock-off mechanism that includes formation of a short-lived transition state with three calcium ions bound near D541. For Ba2+, the transition state lives longer and the knock-off permeation occurs slower. Gd3+ binds at D541 tightly, blocks the channel and prevents Na+ from permeating the pore. Our results provide structural foundations for understanding permeation and block in tetrameric calcium-selective ion channels.
| S-EPMC5890290 | biostudies-literature
Project description:The majority of voltage-gated ion channels contain a defined voltage-sensing domain and a pore domain composed of highly conserved amino acid residues that confer electrical excitability via electromechanical coupling. In this sense, the voltage-gated proton channel (Hv1) is a unique protein in that voltage-sensing, proton permeation and pH-dependent modulation involve the same structural region. In fact, these processes synergistically work in concert, and it is difficult to separate them. To investigate the process of Hv1 voltage sensor trapping, we follow voltage-sensor movements directly by leveraging mutations that enable the measurement of Hv1 channel gating currents. We uncover that the process of voltage sensor displacement is due to two driving forces. The first reveals that mutations in the selectivity filter (D160) located in the S1 transmembrane interact with the voltage sensor. More hydrophobic amino acids increase the energy barrier for voltage sensor activation. On the other hand, the effect of positive charges near position 264 promotes the formation of salt bridges between the arginines of the voltage sensor domain, achieving a stable conformation over time. Our results suggest that the activation of the Hv1 voltage sensor is governed by electrostatic-hydrophobic interactions, and S4 arginines, N264 and selectivity filter (D160) are essential in the Ciona-Hv1 to understand the trapping of the voltage sensor.
| S-EPMC10779229 | biostudies-literature
Project description:MLKL is crucial for necroptosis, permeabilizing membranes through its N-terminal region upon phosphorylation of its kinase-like domain by RIP3. However, the mechanism underlying membrane permeabilization is unknown. The solution structure of the MLKL N-terminal region determined by nuclear magnetic resonance spectroscopy reveals a four-helix bundle with an additional helix at the top that is likely key for MLKL function, and a sixth, C-terminal helix that interacts with the top helix and with a poorly packed interface within the four-helix bundle. Fluorescence spectroscopy measurements indicate that much of the four-helix bundle inserts into membranes, but not the C-terminal helix. Moreover, we find that the four-helix bundle is sufficient to induce liposome leakage and that the C-terminal helix inhibits this activity. These results suggest that the four-helix bundle mediates membrane breakdown during necroptosis and that the sixth helix acts as a plug that prevents opening of the bundle and is released upon RIP3 phosphorylation.
| S-EPMC4192069 | biostudies-literature
Project description:Several channels, ranging from TRP receptors to Gap junctions, allow the exchange of small organic solute across cell membrane. However, very little is known about the molecular mechanism of their permeation. Cyclic Nucleotide Gated (CNG) channels, despite their homology with K+ channels and in contrast with them, allow the passage of larger methylated and ethylated ammonium ions like dimethylammonium (DMA) and ethylammonium (EA). We combined electrophysiology and molecular dynamics simulations to examine how DMA interacts with the pore and permeates through it. Due to the presence of hydrophobic groups, DMA enters easily in the channel and, unlike the alkali cations, does not need to cross any barrier. We also show that while the crystal structure is consistent with the presence of a single DMA ion at full occupancy, the channel is able to conduct a sizable current of DMA ions only when two ions are present inside the channel. Moreover, the second DMA ion dramatically changes the free energy landscape, destabilizing the crystallographic binding site and lowering by almost 25 kJ/mol the binding affinity between DMA and the channel. Based on the results of the simulation the experimental electron density maps can be re-interpreted with the presence of a second ion at lower occupancy. In this mechanism the flexibility of the channel plays a key role, extending the classical multi-ion permeation paradigm in which conductance is enhanced by the plain interaction between the ions.
| S-EPMC6091977 | biostudies-literature
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