scholarly journals 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 ◽  
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.

scholarly journals 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 ◽  
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.

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

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.

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

Toxins ◽  
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.

scholarly journals Intrinsic Gating Behavior of Voltage-Gated Sodium Channels Predetermines Regulation by Auxiliary β-subunits

2021 ◽  
Author(s):  
Niklas Brake ◽  
Adamo S Mancino ◽  
Yuhao Yan ◽  
Takushi Shimomura ◽  
Heika Silveira ◽  
...  
Keyword(s):  
Excitable Cells ◽  
Voltage Sensing ◽  
Auxiliary Subunits ◽  
Voltage Gated ◽  
Closed State ◽  
Channel Function ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Regulatory Effects ◽  
Electrical Signaling

AbstractVoltage-gated sodium (Nav) channels mediate rapid millisecond electrical signaling in excitable cells. Auxiliary subunits, β1-β4, are thought to regulate Nav channel function through covalent and/or polar interactions with the channel’ s voltage-sensing domains. How these interactions translate into the diverse and variable regulatory effects of β-subunits remains unclear. Here, we find that the intrinsic movement order of the voltage-sensing domains during channel gating is unexpectedly variable across Nav channel isoforms. This movement order dictates the channel’ s propensity for closed-state inactivation, which in turn modulates the actions of β1 and β3. We show that the differential regulation of skeletal muscle, cardiac, and neuronal Nav channels is explained by their variable levels of closed-state inactivation. Together, this study provides a unified mechanism for the regulation of all Nav channel isoforms by β1 and β3, which explains how the fixed structural interactions of auxiliary subunits can paradoxically exert variable effects on channel function.

scholarly journals The voltage-sensing domain of a phosphatase gates the pore of a potassium channel

2013 ◽  
Vol 141 (3) ◽  
pp. 389-395 ◽  
Author(s):  
Cristina Arrigoni ◽  
Indra Schroeder ◽  
Giulia Romani ◽  
James L. Van Etten ◽  
Gerhard Thiel ◽  
...  
Keyword(s):  
Electromechanical Coupling ◽  
K Channel ◽  
Modular Architecture ◽  
Voltage Sensing ◽  
Mode Shift ◽  
Voltage Gated ◽  
Kv Channels ◽  
Pore Properties ◽  
Voltage Sensing Domain

The modular architecture of voltage-gated K+ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates KvSynth1, a functional voltage-gated, outwardly rectifying K+ channel. KvSynth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

scholarly journals A Na+ Channel Mutation Linked to Hypokalemic Periodic Paralysis Exposes a Proton-selective Gating Pore

2007 ◽  
Vol 130 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Arie F. Struyk ◽  
Stephen C. Cannon
Keyword(s):  
Skeletal Muscle ◽  
Periodic Paralysis ◽  
Proton Leak ◽  
K Channel ◽  
Na Channel ◽  
Missense Mutations ◽  
Ph Homeostasis ◽  
Hypokalemic Periodic Paralysis ◽  
Voltage Sensing ◽  
Voltage Sensing Domain

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca2+ channel) or NaV1.4 (the skeletal muscle voltage-gated Na+ channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice (“gating-pore”) in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K+ channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K+ and Cl− conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of VREST indirectly, for instance, by interfering with intracellular pH homeostasis.

scholarly journals Conformations of voltage-sensing domain III differentially define NaV channel closed- and open-state inactivation

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Paweorn Angsutararux ◽  
Po Wei Kang ◽  
Wandi Zhu ◽  
Jonathan R. Silva
Keyword(s):  
Critical Role ◽  
Voltage Sensing ◽  
Α Subunit ◽  
Functional Studies ◽  
Closed State ◽  
Channel Inactivation ◽  
Nav Channels ◽  
Intracellular Binding ◽  
Domain Iii ◽  
Voltage Sensing Domain

Voltage-gated Na+ (NaV) channels underlie the initiation and propagation of action potentials (APs). Rapid inactivation after NaV channel opening, known as open-state inactivation, plays a critical role in limiting the AP duration. However, NaV channel inactivation can also occur before opening, namely closed-state inactivation, to tune the cellular excitability. The voltage-sensing domain (VSD) within repeat IV (VSD-IV) of the pseudotetrameric NaV channel α-subunit is known to be a critical regulator of NaV channel inactivation. Yet, the two processes of open- and closed-state inactivation predominate at different voltage ranges and feature distinct kinetics. How inactivation occurs over these different ranges to give rise to the complexity of NaV channel dynamics is unclear. Past functional studies and recent cryo-electron microscopy structures, however, reveal significant inactivation regulation from other NaV channel components. In this Hypothesis paper, we propose that the VSD of NaV repeat III (VSD-III), together with VSD-IV, orchestrates the inactivation-state occupancy of NaV channels by modulating the affinity of the intracellular binding site of the IFMT motif on the III-IV linker. We review and outline substantial evidence that VSD-III activates in two distinct steps, with the intermediate and fully activated conformation regulating closed- and open-state inactivation state occupancy by altering the formation and affinity of the IFMT crevice. A role of VSD-III in determining inactivation-state occupancy and recovery from inactivation suggests a regulatory mechanism for the state-dependent block by small-molecule anti-arrhythmic and anesthetic therapies.

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

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.

scholarly journals Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: Wild-type skeletal muscle NaV1.4

2013 ◽  
Vol 141 (3) ◽  
pp. 309-321 ◽  
Author(s):  
Jonathan R. Silva ◽  
Steve A.N. Goldstein
Keyword(s):  
Skeletal Muscle ◽  
Action Potentials ◽  
Time Course ◽  
Voltage Sensor ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Conduction Pathway ◽  
Nav Channels

The number of voltage-gated sodium (NaV) channels available to generate action potentials in muscles and nerves is adjusted over seconds to minutes by prior electrical activity, a process called slow inactivation (SI). The basis for SI is uncertain. NaV channels have four domains (DI–DIV), each with a voltage sensor that moves in response to depolarizing stimulation over milliseconds to activate the channels. Here, SI of the skeletal muscle channel NaV1.4 is induced by repetitive stimulation and is studied by recording of sodium currents, gating currents, and changes in the fluorescence of probes on each voltage sensor to assess their movements. The magnitude, voltage dependence, and time course of the onset and recovery of SI are observed to correlate with voltage-sensor movements 10,000-fold slower than those associated with activation. The behavior of each voltage sensor is unique. Development of SI over 1–160 s correlates best with slow immobilization of the sensors in DI and DII; DIII tracks the onset of SI with less fidelity. Showing linkage to the sodium conduction pathway, pore block by tetrodotoxin affects both SI and immobilization of all the sensors, with DI and DII significantly suppressed. Recovery from SI correlates best with slow restoration of mobility of the sensor in DIII. The findings suggest that voltage-sensor movements determine SI and thereby mediate NaV channel availability.

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

2017 ◽  
Vol 149 (3) ◽  
pp. 389-403 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document