Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

Abstract Background Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Results We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. Conclusions The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

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Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

10.1101/2020.07.22.215491 ◽

2020 ◽

Author(s):

Kazuki Matsumura ◽

Takushi Shimomura ◽

Yoshihiro Kubo ◽

Takayuki Oka ◽

Naohiro Kobayashi ◽

...

Keyword(s):

Potassium Channel ◽

Resting State ◽

Mutational Analysis ◽

Functional Characterization ◽

Structural Basis ◽

Related Gene ◽

Wild Type ◽

Hydrophobic Residues ◽

Gating Modifier ◽

Voltage Sensing Domain

AbstractHuman ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. Although it is known that APETx1 inhibits hERG by stabilizing the resting state, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Here, we prepared a recombinant APETx1, which is structurally and functionally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, in addition to a previously reported acidic hERG residue, play key roles in the inhibition of hERG by APETx1. Docking models of the APETx1-VSD complex that satisfy the results of mutational analysis suggest a molecular recognition mode between APETx1 and the resting state of hERG; this would provide a structural basis for designing ligands that control hERG function by binding to the VSD.

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APETx1 from Sea Anemone Anthopleura elegantissima Is a Gating Modifier Peptide Toxin of the Human Ether-a-go-go- Related Potassium Channel

Molecular Pharmacology ◽

10.1124/mol.107.035840 ◽

2007 ◽

Vol 72 (2) ◽

pp. 259-268 ◽

Cited By ~ 37

Author(s):

M. Zhang ◽

X.-S. Liu ◽

S. Diochot ◽

M. Lazdunski ◽

G.-N. Tseng

Keyword(s):

Potassium Channel ◽

Sea Anemone ◽

Anthopleura Elegantissima ◽

Gating Modifier ◽

Peptide Toxin

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Variation of Two S3b Residues in KV4.1–4.3 Channels Underlies Their Different Modulations by Spider Toxin κ-LhTx-1

Frontiers in Pharmacology ◽

10.3389/fphar.2021.692076 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhen Xiao ◽

Piao Zhao ◽

Xiangyue Wu ◽

Xiangjin Kong ◽

Ruiwen Wang ◽

...

Keyword(s):

Mutation Analysis ◽

Voltage Dependence ◽

Underlying Mechanism ◽

Voltage Sensor ◽

Action Mode ◽

Gating Modifier ◽

Peptide Toxin ◽

Peptide Toxins ◽

Key Residues ◽

Animal Venoms

The naturally occurred peptide toxins from animal venoms are valuable pharmacological tools in exploring the structure-function relationships of ion channels. Herein we have identified the peptide toxin κ-LhTx-1 from the venom of spider Pandercetes sp (the Lichen huntsman spider) as a novel selective antagonist of the KV4 family potassium channels. κ-LhTx-1 is a gating-modifier toxin impeded KV4 channels’ voltage sensor activation, and mutation analysis has confirmed its binding site on channels’ S3b region. Interestingly, κ-LhTx-1 differently modulated the gating of KV4 channels, as revealed by toxin inhibiting KV4.2/4.3 with much more stronger voltage-dependence than that for KV4.1. We proposed that κ-LhTx-1 trapped the voltage sensor of KV4.1 in a much more stable resting state than that for KV4.2/4.3 and further explored the underlying mechanism. Swapping the non-conserved S3b segments between KV4.1(280FVPK283) and KV4.3(275VMTN278) fully reversed their voltage-dependence phenotypes in inhibition by κ-LhTx-1, and intensive mutation analysis has identified P282 in KV4.1, D281 in KV4.2 and N278 in KV4.3 being the key residues. Furthermore, the last two residues in this segment of each KV4 channel (P282/K283 in KV4.1, T280/D281 in KV4.2 and T277/N278 in KV4.3) likely worked synergistically as revealed by our combinatorial mutations analysis. The present study has clarified the molecular basis in KV4 channels for their different modulations by κ-LhTx-1, which have advanced our understanding on KV4 channels’ structure features. Moreover, κ-LhTx-1 might be useful in developing anti-arrhythmic drugs given its high affinity, high selectivity and unique action mode in interacting with the KV4.2/4.3 channels.

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Structural basis for voltage-sensor trapping of the cardiac sodium channel by a deathstalker scorpion toxin

10.1101/2020.12.28.424592 ◽

2020 ◽

Author(s):

Daohua Jiang ◽

Lige Tonggu ◽

Tamer M. Gamal El-Din ◽

Richard Banh ◽

Régis Pomès ◽

...

Keyword(s):

Ion Pairs ◽

Voltage Sensor ◽

Structural Basis ◽

Fast Inactivation ◽

Toxin Binding ◽

Cardiac Sodium Channel ◽

Activated State ◽

Nav Channels ◽

Gating Modifier ◽

Voltage Sensing Domain

AbstractVoltage-gated sodium (NaV) channels initiate action potentials in excitable cells, and their function is altered by potent gating-modifier toxins. The α-toxin LqhIII from the deathstalker scorpion inhibits fast inactivation of cardiac NaV1.5 channels with IC50=11.4 nM. Here we reveal the structure of LqhIII bound to NaV1.5 at 3.3 Å resolution by cryo-EM. LqhIII anchors on top of voltage-sensing domain IV, wedged between the S1-S2 and S3-S4 linkers, which traps the gating charges of the S4 segment in a unique intermediate-activated state stabilized by four ion-pairs. This conformational change is propagated inward to weaken binding of the fast inactivation gate and favor opening the activation gate. However, these changes do not permit Na+ permeation, revealing why LqhIII slows inactivation of NaV channels but does not open them. Our results provide important insights into the structural basis for gating-modifier toxin binding, voltage-sensor trapping, and fast inactivation of NaV channels.

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Structural basis for voltage-sensor trapping of the cardiac sodium channel by a deathstalker scorpion toxin

Nature Communications ◽

10.1038/s41467-020-20078-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Daohua Jiang ◽

Lige Tonggu ◽

Tamer M. Gamal El-Din ◽

Richard Banh ◽

Régis Pomès ◽

...

Keyword(s):

Ion Pairs ◽

Voltage Sensor ◽

Structural Basis ◽

Fast Inactivation ◽

Excitable Cells ◽

Toxin Binding ◽

Cardiac Sodium Channel ◽

Activated State ◽

Gating Modifier ◽

Voltage Sensing Domain

AbstractVoltage-gated sodium (NaV) channels initiate action potentials in excitable cells, and their function is altered by potent gating-modifier toxins. The α-toxin LqhIII from the deathstalker scorpion inhibits fast inactivation of cardiac NaV1.5 channels with IC50 = 11.4 nM. Here we reveal the structure of LqhIII bound to NaV1.5 at 3.3 Å resolution by cryo-EM. LqhIII anchors on top of voltage-sensing domain IV, wedged between the S1-S2 and S3-S4 linkers, which traps the gating charges of the S4 segment in a unique intermediate-activated state stabilized by four ion-pairs. This conformational change is propagated inward to weaken binding of the fast inactivation gate and favor opening the activation gate. However, these changes do not permit Na+ permeation, revealing why LqhIII slows inactivation of NaV channels but does not open them. Our results provide important insights into the structural basis for gating-modifier toxin binding, voltage-sensor trapping, and fast inactivation of NaV channels.

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Structural insight into Marburg virus nucleoprotein-RNA complex formation

10.1101/2021.07.13.452116 ◽

2021 ◽

Author(s):

Yoko Fujita-Fujiharu ◽

Yukihiko Sugita ◽

Yuki Takamatsu ◽

Kazuya Houri ◽

Manabu Igarashi ◽

...

Keyword(s):

Rna Synthesis ◽

Ebola Virus ◽

Mutational Analysis ◽

Marburg Virus ◽

Viral Rna ◽

Structural Basis ◽

Close Relative ◽

Rna Complex ◽

Key Residues ◽

Viral Genomic Rna

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 angstrom. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

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