Voltage‐gated proton channels in polyneopteran insects

Chaves, Gustavo; Derst, Christian; Jardin, Christophe; Franzen, Arne; Musset, Boris

doi:10.1002/2211-5463.13361

Cited by 8 publications

(28 citation statements)

References 54 publications

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“…We found a threshold potential around – 70 mV (Fig. 2A) in symmetrical pH conditions using our previously published protocol ( 27, 28 ). In contrast, the wild type is mainly closed at 0 mV which is its threshold potential (Fig.…”

Section: Resultsmentioning

confidence: 80%

“…We overexpressed wild-type H V 1 and D174A mutant in tsa201 cells. Using our previous protocol under symmetrical pH conditions ( 28, 29 ), we detected threshold potentials of activation, V thres , of – 70 mV for the mutant and 0 mV for the wild type (Fig. 2).…”

Section: Resultsmentioning

confidence: 96%

“…Page 4 of 15 published protocol (27,28). In contrast, the wild type is mainly closed at 0 mV which is its threshold potential (Fig.…”

Section: Trapped Water In Hv1mentioning

confidence: 99%

“…Constructs were later sub-cloned into a pQBI25-fC3 or pcDNA3.1, using 5′BamHI and 3′ EcoRI restriction sites and GFP fused to N-terminal (27,28).…”

Section: Heterologous Expression In Mammalian Cells and Electrophysio...mentioning

confidence: 99%

“…Constructs were later sub-cloned into the vector pQBI25-fC3 or pcDNA3.1, using 5′BamHI and 3′ EcoRI restriction sites, and GFP laws fused to HV1's N-terminal (28,29). tSA201 cells (human kidney cell line) were cultivated to 85% confluency in 35 mm culture dishes and transfected with 1.0 μg plasmid DNA using polyethyleneimine (Sigma, St. Louis, MO, USA).…”

Section: Heterologous Expression In Mammalian Cells and Electrophysio...mentioning

confidence: 99%

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Trapped pore waters in the open proton channel H_V1

Boytsov

Brescia

Chaves

et al. 2022

Preprint

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The voltage-gated proton channel, HV1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, we estimate the water mobility for the putative case of an uninterrupted wire. The predicted single-channel water permeability 3x10-12cm3s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the prediction, i.e., it is negligible. Osmotic deflation of reconstituted lipid vesicles reveals trapped water inside the HV1 wild-type channel and D174A mutant open at 0 mV. The conductance of 1400 H+s-1 per wild-type channel agrees with the calculated diffusion limit for a ~0.2nm capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance, conceivably by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while blocking water flow.

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 96%

“…Page 4 of 15 published protocol (27,28). In contrast, the wild type is mainly closed at 0 mV which is its threshold potential (Fig.…”

Section: Trapped Water In Hv1mentioning

confidence: 99%

“…Constructs were later sub-cloned into a pQBI25-fC3 or pcDNA3.1, using 5′BamHI and 3′ EcoRI restriction sites and GFP fused to N-terminal (27,28).…”

Section: Heterologous Expression In Mammalian Cells and Electrophysio...mentioning

confidence: 99%

Section: Heterologous Expression In Mammalian Cells and Electrophysio...mentioning

confidence: 99%

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Trapped pore waters in the open proton channel H_V1

Boytsov

Brescia

Chaves

et al. 2022

Preprint

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Trapped Pore Waters in the Open Proton Channel H_V1

Boytsov

Brescia

Chaves

et al. 2023

Small

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to H V 1 gating. [3] Its structure is homologous to classical voltage-gated channels' voltage-sensing domain (VSD). [4] Experimentally, exclusively closed structures are available -a crystal structure of a chimera between mouse H V 1 and Ciona intestinalis voltage-sensing phosphatase [5] and an NMR structure of the human H V 1. [6] Also, electron paramagnetic resonance (EPR) spectroscopy data decipher the resting structure of H V 1. [7] An AlphaFold structure has been recently added (Figure 1). AlphaFold uses a machine-learning approach. [8] Its deep learning algorithm exploits physical and biological knowledge about protein structure -mainly gained by X-ray crystallography, cryo-electron microscopy, or NMR. None of these technical approaches allows the application of membrane voltage, a prerequisite to attain the open channel configuration of voltage-sensitive channels. Since Hv1 belongs to this class of channels, the AlphaFold structure reflects, in all likelihood, the closed channel structure.The analysis of the AlphaFold structure corroborates the conclusion (Figure 1A,B). It shows three positively charged arginine residues in the membrane-spanning part of the 4th helix (S4) responsible for Hv1's voltage sensitivity. They may interact with three negatively charged aspartate residues on the first (S1) and third helices (S3). The AlphaFold structure suggests the following Coulombic interactions: R205 -D112 (on S1), R211 -D185 (on S3) and R208 -D174 (on S3). There is consensus that i) D174 is part of the intracellular electrostatic network [9,10] and ii) D174's salt bridge with R208 stabilizes the closed, resting-state conformation. [11] The open channel's structure has solely been computed as homology models. [12][13][14][15][16][17][18] One of the most recent models reflects the experimentally estimated gating charge suggesting that the transition from the closed to the open conformation may be correctly captured. [17] Yet, the closed conformations of the homology model (Figure S1, Supporting Information) and AlphaFold (Figure 1A,B) differ in pore diameter (Figure 1C). While the former is large enough to allow water molecules to pass, the latter is much too narrow. The observation suggests that an assessment of H V 1's unitary water permeability, p f , may be used as a diagnostic tool. By undertaking water flux measurements through purified and reconstituted H V 1 channels in The voltage-gated proton channel, H V 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10 −12 cm 3 s −1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does no...

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Identification of a Novel Structural Class of H_V1 Inhibitors by Structure-Based Virtual Screening

Piga,

Varga,

Feher

et al. 2024

J. Chem. Inf. Model.

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The human voltage-gated proton channel, hH V 1, is highly expressed in various cell types including macrophages, B lymphocytes, microglia, sperm cells and also in various cancer cells. Overexpression of H V 1 has been shown to promote tumor formation by highly metastatic cancer cells, and has been associated with neuroinflammatory diseases, immune response disorders and infertility, suggesting a potential use of hH V 1 inhibitors in numerous therapeutic areas. To identify compounds targeting this channel, we performed a structurebased virtual screening on an open structure of the human H V 1 channel. Twenty selected virtual screening hits were tested on Chinese hamster ovary (CHO) cells transiently expressing hH V 1, with compound 13 showing strong block of the proton current with an IC 50 value of 8.5 μM. Biological evaluation of twenty-three additional analogs of 13 led to the discovery of six other compounds that blocked the proton current by more than 50% at 50 μM concentration. This allowed for an investigation of structure−activity relationships. The antiproliferative activity of the selected promising hH V 1 inhibitors was investigated in the cell lines MDA-MB-231 and THP-1, where compound 13 inhibited growth with an IC 50 value of 9.0 and 8.1 μM, respectively. The identification of a new structural class of H V 1 inhibitors contributes to our understanding of the structural requirements for inhibition of this ion channel and opens up the possibility of investigating the role of H V 1 inhibitors in various pathological conditions and in cancer therapy.

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Voltage‐gated proton channels in polyneopteran insects

Cited by 8 publications

References 54 publications

Trapped pore waters in the open proton channel H_V1

Trapped pore waters in the open proton channel H_V1

Trapped Pore Waters in the Open Proton Channel H_V1

Identification of a Novel Structural Class of H_V1 Inhibitors by Structure-Based Virtual Screening

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Voltage‐gated proton channels in polyneopteran insects

Cited by 8 publications

References 54 publications

Trapped pore waters in the open proton channel HV1

Trapped pore waters in the open proton channel HV1

Trapped Pore Waters in the Open Proton Channel HV1

Identification of a Novel Structural Class of HV1 Inhibitors by Structure-Based Virtual Screening

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Trapped pore waters in the open proton channel H_V1

Trapped pore waters in the open proton channel H_V1

Trapped Pore Waters in the Open Proton Channel H_V1

Identification of a Novel Structural Class of H_V1 Inhibitors by Structure-Based Virtual Screening