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.

P1597Two novel mutations in SCN5A gene cause enhanced inactivation and lead to Brugada syndrome

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A Zaytseva ◽  
A V Karpushev ◽  
A V Karpushev ◽  
Y Fomicheva ◽  
Y Fomicheva ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Brugada Syndrome ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Novel Mutations ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Voltage Gated ◽  
Cardiac Sodium Channel

Abstract Background Mutations in gene SCN5A, encoding cardiac potential-dependent sodium channel Nav1.5, are associated with various arrhythmogenic disorders among which the Brugada syndrome (BrS) and the Long QT syndrome (LQT) are the best characterized. BrS1 is associated with sodium channel dysfunction, which can be reflected by decreased current, impaired activation and enhanced inactivation. We found two novel mutations in our patients with BrS and explored their effect on fast and slow inactivation of cardiac sodium channel. Purpose The aim of this study was to investigate the effect of BrS (Y739D, L1582P) mutations on different inactivation processes in in vitro model. Methods Y739D and L1582P substitutions were introduced in SCN5A cDNA using site-directed mutagenesis. Sodium currents were recorded at room temperature in transfected HEK293-T cells using patch-clamp technique with holding potential −100 mV. In order to access the fast steady-state inactivation curve we used double-pulse protocol with 10 ms prepulses. To analyze voltage-dependence of slow inactivation we used two-pulse protocol with 10s prepulse, 20ms test pulse and 25ms interpulse at −100mV to allow recovery from fast inactivation. Electrophysiological measurements are presented as mean ±SEM. Results Y739D mutation affects highly conserved tyrosine 739 among voltage-gated sodium and calcium channels in the segment IIS2. Mutation L1582P located in the loop IVS4-S5, and leucine in this position is not conserved among voltage-gated channels superfamily. We have shown that Y739D leads to significant changes in both fast and slow inactivation, whereas L1582P enhanced slow inactivation only. Steady-state fast inactivation for Y739D was shifted on 8.9 mV towards more negative potentials compare with that for WT, while L1582P did not enhanced fast inactivation (V1/2 WT: −62.8±1.7 mV; Y739D: −71.7±2.3 mV; L1582P: −58.7±1.4 mV). Slow inactivation was increased for both substitutions (INa (+20mV)/INa (−100mV) WT: 0.45±0.03; Y739D: 0,34±0.09: L1582P: 0.38±0.04). Steady-state fast inactivation Conclusions Both mutations, observed in patients with Brugada syndrome, influence on the slow inactivation process. Enhanced fast inactivation was shown only for Y739D mutant. The more dramatic alterations in sodium channel biophysical characteristics are likely linked with mutated residue conservativity. Acknowledgement/Funding RSF #17-15-01292

Effects of Halothane and Isoflurane on Fast and Slow Inactivation of Human Heart hH1a Sodium Channels

1999 ◽  
Vol 90 (6) ◽  
pp. 1671-1683. ◽  
Author(s):  
Anna Stadnicka ◽  
Wai-Meng Kwok ◽  
Hali A. Hartmann ◽  
Zeljko J. Bosnjak
Keyword(s):  
Steady State ◽  
Heterologous Expression ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Human Heart ◽  
Sodium Current ◽  
Volatile Anesthetic ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Cardiac Sodium Channel

Background Cloning and heterologous expression of ion channels allow biophysical and molecular studies of the mechanisms of volatile anesthetic interactions with human heart sodium channels. Volatile anesthetics may influence the development of arrhythmias arising from cardiac sodium channel dysfunction. For that reason, understanding the mechanisms of interactions between these anesthetics and cardiac sodium channels is important. This study evaluated the mechanisms of volatile anesthetic actions on the cloned human cardiac sodium channel (hH1a) alpha subunit. Methods Inward sodium currents were recorded from human embryonic kidney (HEK293) cells stably expressing hH1a channels. The effects of halothane and isoflurane on current and channel properties were evaluated using the whole cell voltage-clamp technique. Results Halothane at 0.47 and 1.1 mM and isoflurane at 0.54 and 1.13 mM suppressed the sodium current in a dose- and voltage-dependent manner. Steady state activation was not affected, but current decay was accelerated. The voltage dependence of steady state fast and slow inactivations was shifted toward more hyperpolarized potentials. The slope factor of slow but not fast inactivation curves was reduced significantly. Halothane increased the time constant of recovery from fast inactivation. The recovery from slow inactivation was not affected significantly by either anesthetic. Conclusions In a heterologous expression system, halothane and isoflurane interact with the hH1a channels and suppress the sodium current. The mechanisms involve acceleration of the transition from the open to the inactivated state, stabilization of the fast and slow inactivated states, and prolongation of the inactivated state by delayed recovery from the fast inactivated to the resting state.

Anticonvulsants modify inactivation but not activation processes of sodium channels in Myxicola axons

1987 ◽  
Vol 65 (6) ◽  
pp. 1220-1225 ◽  
Author(s):  
C. L. Schauf
Keyword(s):  
Steady State ◽  
Time Course ◽  
Internal Surface ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Inactivation Curve ◽  
Kinetics Of ◽  
Enzyme Application ◽  
Voltage Axis ◽  
Pronase Treatment

The effects of pronase and the anticonvulsant drugs diphenylhydantoin, bepridil, and sodium valproate on fast and slow Na+ inactivation were examined in cut-open Myxicola giant axons with loose patch-clamp electrodes applied to the internal surface. Pronase completely eliminated fast Na+ inactivation without affecting the kinetics of Na+ activation or the maximum Na+ conductance. The time and voltage dependences of slow inactivation following pronase treatment were identical to those measured before enzyme application in the same axons. All three anticonvulsants slowed the time course of recovery from fast Na+ inactivation in untreated axons, and shifted the steady-state fast inactivation curve in the hyperpolarizing direction along the voltage axis. Anticonvulsants enhanced steady-state slow inactivation and retarded recovery from slow inactivation in both untreated and pronase-treated axons. Although some quantitative differences were seen, the order of potency of the anticonvulsants on slow Na+ inactivation was the same as that for recovery from fast inactivation.

scholarly journals Molecular Coupling of S4 to a K+ Channel's Slow Inactivation Gate

2000 ◽  
Vol 116 (5) ◽  
pp. 623-636 ◽  
Author(s):  
Eli Loots ◽  
Ehud Y. Isacoff
Keyword(s):  
Transmembrane Domain ◽  
Direct Interaction ◽  
K Channel ◽  
Slow Inactivation ◽  
Domain Interaction ◽  
Voltage Sensing ◽  
Molecular Gates ◽  
Voltage Sensing Domain ◽  
Pore Domain ◽  
Bond Disruption

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K+ channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4–pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.

scholarly journals Refinement of a Cryo-EM Structure of hERG: Bridging Structure and Function

2020 ◽  
Author(s):  
H.M. Khan ◽  
J. Guo ◽  
H.J. Duff ◽  
D. P. Tieleman ◽  
S. Y. Noskov
Keyword(s):  
Molecular Dynamics ◽  
High Resolution ◽  
Salt Bridge ◽  
Structure And Function ◽  
Channel Structure ◽  
Salt Bridges ◽  
Voltage Sensing ◽  
Chain Conformations ◽  
And Function ◽  
Voltage Sensing Domain

AbstractThe human ether-a-go-go-related gene (hERG) encodes the voltage gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using Molecular Dynamics Flexible Fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.Statement of SignificanceCryo-EM has emerged as a major breakthrough technique in structural biology of membrane proteins. However, even high-resolution Cryo-EM structures contain poor side chain conformations and interatomic clashes. A high-resolution cryo-EM structure of hERG1 has been solved in the depolarized (open) state. The state captured by Cryo-EM surprisingly did not feature many functionally important interactions established by previous experiments. Molecular Dynamics Flexible Fitting (MDFF) used to enable refinement of the hERG1 channel structure in complex membrane environment re-establishing key functional interactions in the voltage sensing domain.

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 The Tarantula Venom Peptide Eo1a Binds to the Domain II S3-S4 Extracellular Loop of Voltage-Gated Sodium Channel NaV1.8 to Enhance Activation

2022 ◽  
Vol 12 ◽  
Author(s):  
Jennifer R. Deuis ◽  
Lotten Ragnarsson ◽  
Samuel D. Robinson ◽  
Zoltan Dekan ◽  
Lerena Chan ◽  
...  
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Rational Design ◽  
Voltage Dependence ◽  
Fast Inactivation ◽  
Extracellular Loop ◽  
Peak Current ◽  
Future Studies ◽  
Voltage Gated ◽  
Functional Characterisation

Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (NaV) channels, however relatively few venom-derived peptides with activity at the mammalian NaV1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of β-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates NaV1.8. Eo1a is a 37-residue peptide that increases NaV1.8 peak current (EC50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV50–20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV50–15.5 ± 1.8 mV). At a concentration of 10 μM, Eo1a has varying effects on the peak current and channel gating of NaV1.1–NaV1.7, although its activity is most pronounced at NaV1.8. Investigations into the binding site of Eo1a using NaV1.7/NaV1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of NaV1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at NaV1.8.

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.

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