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 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.

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 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 Possible Interactions of Extracellular Loop IVP2-S6 With Voltage-Sensing Domain III in Cardiac Sodium Channel

2021 ◽  
Vol 12 ◽  
Author(s):  
Anastasia K. Zaytseva ◽  
Aleksandr S. Boitsov ◽  
Anna A. Kostareva ◽  
Boris S. Zhorov
Keyword(s):  
Steady State ◽  
Hot Spot ◽  
Salt Bridge ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Extracellular Loop ◽  
Voltage Sensing ◽  
Cardiac Sodium Channel ◽  
Domain Iii ◽  
Voltage Sensing Domain

Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson–Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.

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.

Abstract 17379: Atrial Fibrillation Mutations in β3 Subunits Enhance DIII Voltage Sensing Domain Coupling to Channel Activation Which Inhibits Lidocaine Blockade

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Wandi Zhu ◽  
Eric J Hsu ◽  
Bicong Li ◽  
Angela R Schubert ◽  
Zoltan Varga ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Channel Gating ◽  
Ionic Current ◽  
Fluorescence Emission ◽  
Post Translational Modification ◽  
Voltage Sensing ◽  
Channel Activation ◽  
Α Subunit ◽  
Heart Chamber ◽  
Voltage Sensing Domain

Background: Co-assembly of cardiac Na + channels (Na v 1.5) with β subunits modifies channel gating, expression, and post-translational modification. β subunit mutations have been linked to the Brugada and Long QT Syndromes, and atrial fibrillation (AF). Hypothesis: We tested whether β 3 subunits regulate Nav.1.5 ionic current and drug response by modulating the voltage sensing domains (VSDs). Methods: The Na V 1.5 α subunit contains four domains (DI-DIV), each with its own voltage sensing domain (VSD). We previously created four DNA constructs that carried a cysteine within a single VSD. Channels expressed in Xenopus oocytes and these cysteines were labeled with TAMRA-MTS fluorophores. Ionic current and fluorescence emission that tracked VSD conformation were simultaneously recorded using the cut-open configuration with and without β 3 . Results: Steady state inactivation is significantly right shifted by β 3 (V 1/2 = -88.9 ± 1.1 SEM (with, +β 3 ) and -97.8 ± 1.5 (without, -β 3 ), p=0.002, n=4). β 3 also right shifts DIII-VSD activation (V 1/2 = -93.0±2.3 +β 3 , V 1/2 = --114.8±0.8 -β 3 , p=0.001, n=4), while modestly left-shifting channel activation, suggesting enhanced DIII-VSD to pore coupling (V 1/2GV -V 1/2FV =55.0 ± 4.0 +β 3 , V 1/2GV -V 1/2FV =73.6 ± 2.4 -β 3 , n=4). DI and DII were not affected, while DIV was modestly shifted, consistent with DIII/DIV cooperativity. Extracellular domain AF-linked β 3 mutations, R6K and L10P, further enhance DIII-VSD to pore coupling (V 1/2GV -V 1/2FV =34.1 ± 5.8, (R6K), V 1/2GV -V 1/2FV =41.5 ± 4.7 (L10P), n=4). β 3 nearly abolishes stabilization of the DIII-VSD by lidocaine (DIII FV shift by lidocaine: ΔV 1/2 lido=-27.71 ± 12.23 (+β 3 ), ΔV 1/2 lido=-65.44 ± 3.83 (-β 3 ), n=3). The conservative R6K mutation exacerbates this effect, suggesting a cation-pi interaction with Na V 1.5. W1684 is co-localized with the DIII-VSD, and W1684A disrupted β 3 modification of channel gating and the DIII lidocaine interaction. Conclusions: β 3 modifies Nav1.5 gating by increasing DIII-VSD coupling to the pore via interaction with W1684. AF β 3 mutants further enhance DIII-VSD to pore coupling. The differential lidocaine response caused by WT and AF β 3 mutants suggests a molecular mechanism whereby the lidocaine response is patient and heart-chamber specific.

New Molecular Scaffolds for Fluorescent Voltage Indicators

2018 ◽  
Author(s):  
Steven Boggess ◽  
Shivaani Gandhi ◽  
Brian Siemons ◽  
Nathaniel Huebsch ◽  
Kevin Healy ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potentials ◽  
Induced Pluripotent Stem Cell ◽  
Molecular Wire ◽  
Voltage Sensing ◽  
Design Synthesis ◽  
Cardiac Action ◽  
Action Potential Waveform ◽  
Induced Pluripotent ◽  
Voltage Sensing Domain

<div> <p>The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicator that makes use of a fluorene-based molecular wire as a voltage sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2) readily reports on action potentials in mammalian neurons, detects perturbations to cardiac action potential waveform in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlights the utility of fVF 2 for interrogating membrane potential dynamics.</p> </div>

Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

2019 ◽  
Author(s):  
Alisher M Kariev ◽  
Michael Green
Keyword(s):  
Charge Transfer ◽  
Energy Charge ◽  
Quantum Calculation ◽  
Quantum Calculations ◽  
Gating Current ◽  
Transmembrane Segment ◽  
Voltage Sensing ◽  
Standard Models ◽  
Charge Displacement ◽  
Voltage Sensing Domain

Quantum calculations on 976 atoms of the voltage sensing domain of the K<sub>v</sub>1.2 channel, with protons in several positions, give energy, charge transfer, and other properties. Motion of the S4 transmembrane segment that accounts for gating current in standard models is shown not to occur; there is H<sup>+ </sup>transfer instead. The potential at which two proton positions cross in energy approximately corresponds to the gating potential for the channel. The charge displacement seems approximately correct for the gating current. Two mutations are accounted for (Y266F, R300cit, cit =citrulline). The primary conclusion is that voltage sensing depends on H<sup>+</sup> transfer, not motion of arginine charges.

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