Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation

Rosa, Víctor De la; Bennett, Ashley; Ramsey, I. Scott

doi:10.1085/jgp.201711822

Cited by 21 publications

(28 citation statements)

References 51 publications

(169 reference statements)

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“…The bulk of evidence supports the idea that Zn 2+ inhibits WT hH V 1 currents by binding to the closed WT hH V 1 channel and preventing opening (59–61). In a recent proposal, protons permeate hH V 1 by binding consecutively to 3 sites, each with 2 acidic groups; the internal site includes E153 and D174, the central site D112 and D185, and the outer site E119 and D123 (62).…”

Section: Discussionmentioning

confidence: 53%

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

Banh

Cherny

Morgan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Discussionmentioning

confidence: 53%

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

Banh

Cherny

Morgan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Cells treated with Optate showed a significant increase in green fluorescent intensity (n = 4, p = 0.039) and decrease in red fluorescent intensity (n = 4, p = 0.001) compared to those treated with placebo, indicating an increase in intracellular pH (Figure 1, A and B). ZnCl 2 , which is known to acidify the intracellular space 21 , had an effect opposite Optate (n = 3 and 5, p = 0.036 and 0.001, Figure 1B).…”

Section: Resultsmentioning

confidence: 95%

A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects

et al. 2020

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“…A recent study showed that E153A mutation at the highly conserved intracellular Coulombic network (ICN) of the H V 1 channel reduces Zn 2+ potency as an inhibitor of the channel (De La Rosa et al, ). As the AGAP/W38F binding pocket partly overlapped with that of Zn 2+ , we evaluated the effects of AGAP/W38F on the currents of the E153A mutant H V 1 channel and found that the mutation reduced toxin potency, with 10‐μM toxin inhibiting the currents by 29.9 ± 5.2% at +30 mV (Figure b,c,e, n = 6).…”

Section: Resultsmentioning

confidence: 99%

“…All these interactions were well conserved in MD simulations, locking the channel in the resting state structure by suppressing the upward motion of the S4 helix during activation. Moreover, toxin insertion at the extracellular entrance of H V 1 channel triggered certain conformational changes in the pore (Figure b), especially the highly conserved ICN (De La Rosa et al, ). The interaction between R205 and D112 was conserved.…”

Section: Resultsmentioning

confidence: 99%

“…For Zn 2+ and guanidine derivatives, their mechanisms of inhibiting H V 1 channel have been extensively studied, but shortcomings in their affinity and selectivity as H V 1 channel antagonists have also been observed. Recently, De La Rosa et al demonstrated that Zn 2+ inhibits H V 1 channel using a long‐range conformational coupling mechanism (De La Rosa, Bennett, & Ramsey, ). However, it remains unknown whether other extracellularly bound H V 1 channel antagonists use similar mechanism.…”

Section: Introductionmentioning

confidence: 99%

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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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Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation

Cited by 21 publications

References 51 publications

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects

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

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Coupling between an electrostatic network and the Zn2+ binding site modulates Hv1 activation

Cited by 21 publications

References 51 publications

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects

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