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

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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Voltage-Sensing Domain of the Third Repeat of Human Skeletal Muscle NaV1.4 Channel As a New Target for Spider Gating Modifier Toxins

Acta Naturae ◽

10.32607/actanaturae.11425 ◽

2021 ◽

Vol 13 (1) ◽

pp. 134-139

Author(s):

M. Yu. Myshkin ◽

A. S. Paramonov ◽

D. S. Kulbatsky ◽

E. A. Surkova ◽

A. A. Berkut ◽

...

Keyword(s):

Skeletal Muscle ◽

Human Skeletal Muscle ◽

New Drugs ◽

Modular Architecture ◽

Voltage Sensing ◽

Toxin Binding ◽

Voltage Gated Sodium Channels ◽

Nav Channels ◽

Gating Modifier ◽

Voltage Sensing Domain

Voltage-gated sodium channels (NaV) have a modular architecture and contain five membrane domains. The central pore domain is responsible for ion conduction and contains a selectivity filter, while the four peripheral voltage-sensing domains (VSD-I/IV) are responsible for activation and rapid inactivation of the channel. Gating modifier toxins from arthropod venoms interact with VSDs, influencing the activation and/or inactivation of the channel, and may serve as prototypes of new drugs for the treatment of various channelopathies and pain syndromes. The toxin-binding sites located on VSD-I, II and IV of mammalian NaV channels have been previously described. In this work, using the example of the Hm-3 toxin from the crab spider Heriaeus melloteei, we showed the presence of a toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. A developed cell-free protein synthesis system provided milligram quantities of isolated (separated from the channel) VSD-III and its 15N-labeled analogue. The interactions between VSD-III and Hm-3 were studied by NMR spectroscopy in the membrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relatively high affinity to VSD-III (dissociation constant of the complex Kd ~6 M), comparable to the affinity to VSD-I and exceeding the affinity to VSD-II. Within the complex, the positively charged Lys25 and Lys28 residues of the toxin probably interact with the S1S2 extracellular loop of VSD-III. The Hm-3 molecule also contacts the lipid bilayer surrounding the channel.

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Structural basis of α-scorpion toxin action on Nav channels

Science ◽

10.1126/science.aav8573 ◽

2019 ◽

Vol 363 (6433) ◽

pp. eaav8573 ◽

Cited By ~ 43

Author(s):

Thomas Clairfeuille ◽

Alexander Cloake ◽

Daniel T. Infield ◽

José P. Llongueras ◽

Christopher P. Arthur ◽

...

Keyword(s):

Electromechanical Coupling ◽

Receptor Site ◽

Scorpion Toxin ◽

Structural Basis ◽

Fast Inactivation ◽

Gating Charge ◽

Nav Channels ◽

Fast Activation ◽

Electrical Signaling ◽

Voltage Sensing Domain

Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.

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Voltage-Sensing Domain of the Third Repeat of Human Skeletal Muscle NaV1.4 Channel As a New Target for Spider Gating Modifier Toxins

Acta Naturae ◽

10.32607/actanaturae.11279 ◽

2021 ◽

Vol 13 (1) ◽

pp. 134-139

Author(s):

Mikhail Yu. Myshkin ◽

Alexander S. Paramonov ◽

Dmitrii S. Kulbatskii ◽

Yelizaveta A. Surkova ◽

Antonina A. Berkut ◽

...

Keyword(s):

Skeletal Muscle ◽

Human Skeletal Muscle ◽

New Drugs ◽

Modular Architecture ◽

Voltage Sensing ◽

Toxin Binding ◽

Voltage Gated Sodium Channels ◽

Nav Channels ◽

Gating Modifier ◽

Voltage Sensing Domain

Voltage-gated sodium channels (NaV) have a modular architecture and contain five membrane domains. The central pore domain is responsible for ion conduction and contains a selectivity filter, while the four peripheral voltage-sensing domains (VSD-I/IV) are responsible for activation and rapid inactivation of the channel. Gating modifier toxins from arthropod venoms interact with VSDs, influencing the activation and/or inactivation of the channel, and may serve as prototypes of new drugs for the treatment of various channelopathies and pain syndromes. The toxin-binding sites located on VSD-I, II and IV of mammalian NaV channels have been previously described. In this work, using the example of the Hm-3 toxin from the crab spider Heriaeus melloteei, we showed the presence of a toxin-binding site on VSD-III of the human skeletal muscle NaV1.4 channel. A developed cell-free protein synthesis system provided milligram quantities of isolated (separated from the channel) VSD-III and its 15N-labeled analogue. The interactions between VSD-III and Hm-3 were studied by NMR spectroscopy in the membrane-like environment of DPC/LDAO (1 : 1) micelles. Hm-3 has a relatively high affinity to VSD-III (dissociation constant of the complex Kd ~6 M), comparable to the affinity to VSD-I and exceeding the affinity to VSD-II. Within the complex, the positively charged Lys25 and Lys28 residues of the toxin probably interact with the S1S2 extracellular loop of VSD-III. The Hm-3 molecule also contacts the lipid bilayer surrounding the channel.

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Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels

The Journal of General Physiology ◽

10.1085/jgp.201310998 ◽

2013 ◽

Vol 142 (2) ◽

pp. 101-112 ◽

Cited By ~ 116

Author(s):

Deborah L. Capes ◽

Marcel P. Goldschen-Ohm ◽

Manoel Arcisio-Miranda ◽

Francisco Bezanilla ◽

Baron Chanda

Keyword(s):

Sodium Channels ◽

Voltage Sensor ◽

Fast Inactivation ◽

Voltage Range ◽

Voltage Gated Sodium Channels ◽

Channel Opening ◽

The Individual ◽

Electrical Impulses ◽

Voltage Sensing Domain ◽

Rate Limiting

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na+ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K+ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

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Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-Like Toxin Binding Properties

Toxins ◽

10.3390/toxins13100722 ◽

2021 ◽

Vol 13 (10) ◽

pp. 722

Author(s):

Ryan Schroder ◽

Leah Cohen ◽

Ping Wang ◽

Joekeem Arizala ◽

Sébastien Poget

Keyword(s):

Pain Perception ◽

Dissociation Constants ◽

Recombinant Expression ◽

Single Amino Acid ◽

Binding Studies ◽

Voltage Sensing ◽

Toxin Binding ◽

Channel Inhibition ◽

Gating Modifier ◽

Voltage Sensing Domain

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

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

BMC Molecular and Cell Biology ◽

10.1186/s12860-020-00337-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Kazuki Matsumura ◽

Takushi Shimomura ◽

Yoshihiro Kubo ◽

Takayuki Oka ◽

Naohiro Kobayashi ◽

...

Keyword(s):

Potassium Channel ◽

Resting State ◽

Mutational Analysis ◽

Functional Characterization ◽

Structural Basis ◽

Anthopleura Elegantissima ◽

Gating Modifier ◽

Peptide Toxin ◽

Key Residues ◽

Voltage Sensing Domain

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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Proton currents constrain structural models of voltage sensor activation

eLife ◽

10.7554/elife.18017 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 18

Author(s):

Aaron L Randolph ◽

Younes Mokrab ◽

Ashley L Bennett ◽

Mark SP Sansom ◽

Ian Scott Ramsey

Keyword(s):

Structural Models ◽

Voltage Sensor ◽

Proton Channel ◽

Structural Basis ◽

Gating Charge ◽

Activated State ◽

Sensor Activation ◽

Model Structures ◽

Proton Currents ◽

Transfer Mechanisms

The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic ‘aqueous’ H+ conductance (GAQ). Mutation of a highly conserved ‘gating charge’ residue in the S4 helix (R1H) confers a resting-state H+ ‘shuttle’ conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1.

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Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains

The Journal of General Physiology ◽

10.1085/jgp.201611678 ◽

2017 ◽

Vol 149 (3) ◽

pp. 389-403 ◽

Cited By ~ 15

Author(s):

Eric J. Hsu ◽

Wandi Zhu ◽

Angela R. Schubert ◽

Taylor Voelker ◽

Zoltan Varga ◽

...

Keyword(s):

Na Channel ◽

Fast Inactivation ◽

Voltage Sensing ◽

Channel Inactivation ◽

Recovery From Inactivation ◽

Nav Channels ◽

Rate Of Recovery ◽

Membrane Spanning ◽

Voltage Sensing Domain ◽

Gating Process

Functional eukaryotic voltage-gated Na+ (NaV) channels comprise four domains (DI–DIV), each containing six membrane-spanning segments (S1–S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1–S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII–DIV linker, the DIII S4–S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII–DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery.

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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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