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 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 Interactions between Multiple Phosphorylation Sites in the Inactivation Particle of a K+ Channel

1998 ◽  
Vol 112 (1) ◽  
pp. 71-84 ◽  
Author(s):  
Edward J. Beck ◽  
Roger G. Sorensen ◽  
Simon J. Slater ◽  
Manuel Covarrubias
Keyword(s):  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
K Channel ◽  
Phosphorylation Sites ◽  
Free Energies ◽  
Channel Inactivation ◽  
Recovery From Inactivation ◽  
Pkc Isoforms ◽  
Inactivation Gating ◽  
Rate Of Recovery

Protein kinase C inhibits inactivation gating of Kv3.4 K+ channels, and at least two NH2-terminal serines (S15 and S21) appeared involved in this interaction (Covarrubias et al. 1994. Neuron. 13:1403–1412). Here we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine → alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by ∼85%, whereas S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large effects of individual S → A mutations, simultaneous mutation of the four sites was necessary to completely abolish inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was phosphorylated by specific PKC isoforms, but the mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S → D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosphoserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had little effect on the onset of inactivation but accelerated the recovery from inactivation. Interestingly, the effects of single S → D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple additive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest that the NH2-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and possible interactions that can explain the action of PKC on inactivation gating.

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.

Structural basis of α-scorpion toxin action on Nav channels

Science ◽  
2019 ◽  
Vol 363 (6433) ◽  
pp. eaav8573 ◽  
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.

Characterization of the isoform-specific differences in the gating of neuronal and muscle sodium channels

1998 ◽  
Vol 76 (10-11) ◽  
pp. 1041-1050 ◽  
Author(s):  
Michael E O'Leary
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Cultured Cells ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Rate Of Recovery

Human heart (hH1), human skeletal muscle (hSkM1), and rat brain (rIIA) Na channels were expressed in cultured cells and the activation and inactivation of the whole-cell Na currents measured using the patch clamp technique. hH1 Na channels were found to activate and inactivate at more hyperpolarized voltages than hSkM1 and rIIA. The conductance versus voltage and steady state inactivation relationships have midpoints of -48 and -92 mV (hH1), -28 and -72 mV (hSkM1), and -22 and -61 mV (rIIA). At depolarized voltages, where Na channels predominately inactivate from the open state, the inactivation of hH1 is 2-fold slower than that of hSkM1 and rIIA. The recovery from fast inactivation of all three isoforms is well described by a single rapid component with time constants at -100 mV of 44 ms (hH1), 4.7 ms (hSkM1), and 7.6 ms (rIIA). After accounting for differences in voltage dependence, the kinetics of activation, inactivation, and recovery of hH1 were found to be generally slower than those of hSkM1 and rIIA. Modeling of Na channel gating at hyperpolarized voltages where the channel does not open suggests that the slow rate of recovery from inactivation of hH1 accounts for most of the differences in the steady-state inactivation of these Na channels.Key words: cardiac, neuronal, skeletal muscle, sodium channel.

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.

pH and voltage dependence of INa recovery kinetics in atrial cells exposed to lidocaine

1988 ◽  
Vol 255 (6) ◽  
pp. H1554-H1557
Author(s):  
K. R. Courtney
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Ph Dependence ◽  
Channel Inactivation ◽  
Atrial Cells ◽  
Recovery From Inactivation ◽  
Electrode Voltage ◽  
Rate Of Recovery ◽  
Myelinated Nerves ◽  
Drug Free

Lidocaine blocks sodium channels during depolarizations. The rate of recovery (repriming) of drug-blocked channels between depolarizations is slowed by both membrane depolarization and by acidification. This modulation of recovery kinetics was studied using a single-electrode voltage clamp on atrial cells isolated from the bullfrog. The pH dependence of recovery from inactivation, in drug-free conditions, is opposite to that observed in myelinated nerves; recovery occurs faster at higher pH levels in these cardiac preparations. The combined pH dependence and voltage dependence of repriming kinetics during lidocaine treatment can be explained by assuming that channels occupied by neutral drug can reactivate most readily at a rate that appears to be coupled to recovery from channel inactivation.

scholarly journals Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels

1997 ◽  
Vol 109 (5) ◽  
pp. 589-605 ◽  
Author(s):  
Stephan Kellenberger ◽  
James W. West ◽  
Todd Scheuer ◽  
William A. Catterall
Keyword(s):  
Amino Acid ◽  
Single Channel ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
Fast Inactivation ◽  
Hydrophobic Cluster ◽  
Recovery From Inactivation ◽  
The Stability ◽  
Single Channel Currents

Fast Na+ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in LIII-IV of the rat brain type IIA Na+ channel. We have analyzed macroscopic and single channel currents from Na+ channels with mutations within and adjacent to hydrophobic clusters in LIII-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in LIII-IV also affected the latency to first opening, indicating coupling between LIII-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na+ channels.

scholarly journals Extracellular Zinc Ion Inhibits ClC-0 Chloride Channels by Facilitating Slow Gating

1998 ◽  
Vol 112 (6) ◽  
pp. 715-726 ◽  
Author(s):  
Tsung-Yu Chen
Keyword(s):  
Chloride Channels ◽  
Zinc Ion ◽  
Forward Rate ◽  
Temperature Sensitive ◽  
Recovery From Inactivation ◽  
Wide Range ◽  
Rate Of Recovery ◽  
The Difference ◽  
Two State Model ◽  
Gating Process

Extracellular Zn2+ was found to reversibly inhibit the ClC-0 Cl− channel. The apparent on and off rates of the inhibition were highly temperature sensitive, suggesting an effect of Zn2+ on the slow gating (or inactivation) of ClC-0. In the absence of Zn2+, the rate of the slow-gating relaxation increased with temperature, with a Q10 of ∼37. Extracellular Zn2+ facilitated the slow-gating process at all temperatures, but the Q10 did not change. Further analysis of the rate constants of the slow-gating process indicates that the effect of Zn2+ is mostly on the forward rate (the rate of inactivation) rather than the backward rate (the rate of recovery from inactivation) of the slow gating. When ClC-0 is bound with Zn2+, the equilibrium constant of the slow-gating process is increased by ∼30-fold, reflecting a 30-fold higher Zn2+ affinity in the inactivated channel than in the open-state channel. As examined through a wide range of membrane potentials, Zn2+ inhibits the opening of the slow gate with equal potency at all voltages, suggesting that a two-state model is inadequate to describe the slow-gating transition. Following a model originally proposed by Pusch and co-workers (Pusch, M., U. Ludewig, and T.J. Jentsch. 1997. J. Gen. Physiol. 109:105–116), the effect of Zn2+ on the activation curve of the slow gate can be well described by adding two constraints: (a) the dissociation constant for Zn2+ binding to the open channel is 30 μM, and (b) the difference in entropy between the open state and the transition state of the slow-gating process is increased by 27 J/ mol/°K for the Zn2+-bound channel. These results together indicate that extracellular Zn2+ inhibits ClC-0 by facilitating the slow-gating process.

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