Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor

Zhao, Ruiming; Kennedy, Kelleigh; Blas, Gerardo A. De; Orta, Gerardo; Pavarotti, Martín; Arias, Rodolfo; Vega-Beltrán, José Luis de la; Li, Qufei; Dai, Hui; Perozo, Eduardo; Mayorga, Luis S.; Darszon, Alberto; Goldstein, Steve A.N.

doi:10.1073/pnas.1816189115

Cited by 47 publications

(68 citation statements)

References 63 publications

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“…Until today, no specific blocker/inhibitor of the channel has been found. However, many interesting inhibitor candidates are under investigation [54][55][56][57][58][59]. The well investigated is the transition metal zinc, which was given its modern name by the sixteenth-century academic Paracelsus.…”

Section: Introductionmentioning

confidence: 99%

Zinc modulation of proton currents in a new voltage‐gated proton channel suggests a mechanism of inhibition

et al. 2020

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The H V 1 voltage-gated proton (H V 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of H V 1 is Zn 2+. Externally applied ZnCl 2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn 2+ and Cd 2+ on H V 1 from Nicoletia phytophila, NpH V 1. We introduced mutations at potential Zn 2+ coordination sites and measured Zn 2+ inhibition in different extracellular pH, with Zn 2+ concentrations up to 1000 lM. Zn 2+ inhibition in NpH V 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn 2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn 2+ in the S3-S4 linker. A simple Hodgkin Huxley model of NpH V 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpH V 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development. Abbreviations Asp145, aspartate at position 145; g H,max , maximal proton conductance; g H,max /10, 10% of the maximal proton conductance; g H-V, proton conductance-voltage relationship; hH V 1, human voltage-gated proton channel; H V 1, voltage-gated proton channel 1; NpH V 1, Nicoletia phytophila voltage-gated proton channel 1; pH i , pH inside the cell; pH o , pH outside the cell; TM, transmembrane domain; TMA, tetramethylammonium; TSA, Transcriptome Shotgun Assembly; V rev , reversal potential; V threshold , threshold potential.

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Section: Introductionmentioning

confidence: 99%

Zinc modulation of proton currents in a new voltage‐gated proton channel suggests a mechanism of inhibition

et al. 2020

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“…Normal tyrosine phosphorylation was observed in spermatozoa capacitated in the presence of TeNT‐CPP, suggesting TeNT‐CPP does not prevent other capacitation‐associated events. In this regard, we have recently published the action of Corza6 (C6), a peptide with inhibitory activity over hHv1 (human voltage‐gated proton channel) (Zhao et al ., ). This channel extrudes H + during capacitation in human spermatozoa.…”

Section: Discussionmentioning

confidence: 97%

“…This channel extrudes H + during capacitation in human spermatozoa. Capacitation in the presence of C6 inhibits acrosomal exocytosis, but does not block tyrosine phosphorylation (Zhao et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Human sperm capacitation is necessary for SNARE assembly in neurotoxin‐resistant complexes

et al. 2019

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Background Capacitation is not a well‐defined process, required for the acrosome reaction triggered by physiological stimuli. In vitro, capacitation is achieved by sperm incubation in artificial media supplemented with HCO3−, Ca2+, and albumin. The role of capacitation in the membrane fusion machinery required for acrosomal exocytosis is not well‐known. SNARE proteins are fundamental for intracellular membrane fusion and acrosomal exocytosis. We have previously shown that in capacitated spermatozoa, the fusion machinery is maintained in an inactive state until the acrosome reaction is initiated. In particular, SNARE proteins are assembled in neurotoxin‐resistant complexes. Objective This work aimed to study the dynamic changes of SNARE complexes during capacitation. Materials and Methods The light chain of tetanus and botulinum neurotoxin has been widely used to study the configuration of SNARE proteins. For this purpose, we developed a recombinant light chain of tetanus neurotoxin linked to a polyarginine peptide. This membrane‐permeant protein was able to cleave cytosolic VAMP2 (a SNARE protein required for acrosome reaction) when present in a monomeric configuration. Results The results show that the VAMP2 is cleaved by the membrane‐permeant tetanus neurotoxin in non‐capacitated spermatozoa, indicating that, before capacitation, SNAREs are not assembled in stable toxin‐resistant complexes. However, 2 h of incubation in a capacitation medium containing albumin was sufficient to render VAMP2 insensitive to the toxin. Discussion We conclude that during capacitation, the SNARE proteins become engaged in stable fully assembled cis‐SNARE complexes. This step is likely essential to prevent untimely activation of the membrane fusion machinery. Conclusion We propose that capacitation promotes the stabilization of the membrane fusion machinery required for acrosomal exocytosis in preparation for the stimulus‐triggered acrosome reaction.

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“…The identification of novel modulators of H V 1 channel would be valuable either as drug candidates or as pharmacological tools to investigate the structure and function of this channel. Recently, Zhao et al identified a novel H V 1 channel toxin, Corza6, from the ICK toxins permutation library using a phage‐display strategy (Zhao et al, ). Both Corza6 and AGAP/W38F inhibit H V 1 currents by trapping the S4 voltage sensor in deactivated states, and these effects could be counteracted by strong depolarization.…”

Section: Discussionmentioning

confidence: 99%

Scorpion toxin inhibits the voltage‐gated proton channel using a Zn²⁺‐like long‐range conformational coupling mechanism

Tang

Yang

Zhang

et al. 2020

British J Pharmacology

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Background and Purpose: Blocking the voltage-gated proton channel H V 1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few H V 1 channel antagonists have been reported. Here, we have identified a novel H V 1 channel antagonist from scorpion venom and have elucidated its action mechanism.Experimental Approach: H V 1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole-cell patch clamp. Sitedirected mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F-H V 1 interaction was modelled by molecular dynamics simulations.Key Results: The scorpion toxin AGAP (anti-tumour analgesic peptide) potently inhibited H V 1 currents. One AGAP mutant has reduced Na V channel activity but intact H V 1 activity (AGAP/W38F). AGAP/W38F inhibited H V 1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited H V 1 currents with less pH dependence than Zn 2+ . Mutation analysis showed that the binding pockets of AGAP/W38F and Zn 2+ in H V 1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in H V 1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn 2+ . Experimental data and MD simulations suggested that AGAP/W38F inhibited H V 1 channel using a Zn 2+ -like long-range conformational coupling mechanism.Conclusion and Implications: Our results suggest that the Zn 2+ binding pocket in H V 1 channel might be a hotspot for modulators and valuable for designing H V 1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study H V 1 channel and a lead compound for drug development.

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Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor

Cited by 47 publications

References 63 publications

Zinc modulation of proton currents in a new voltage‐gated proton channel suggests a mechanism of inhibition

Zinc modulation of proton currents in a new voltage‐gated proton channel suggests a mechanism of inhibition

Human sperm capacitation is necessary for SNARE assembly in neurotoxin‐resistant complexes

Scorpion toxin inhibits the voltage‐gated proton channel using a Zn²⁺‐like long‐range conformational coupling mechanism

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Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor

Cited by 47 publications

References 63 publications

Zinc modulation of proton currents in a new voltage‐gated proton channel suggests a mechanism of inhibition

Zinc modulation of proton currents in a new voltage‐gated proton channel suggests a mechanism of inhibition

Human sperm capacitation is necessary for SNARE assembly in neurotoxin‐resistant complexes

Scorpion toxin inhibits the voltage‐gated proton channel using a Zn2+‐like long‐range conformational coupling mechanism

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Scorpion toxin inhibits the voltage‐gated proton channel using a Zn²⁺‐like long‐range conformational coupling mechanism