scholarly journals Movement of Voltage Sensor S4 in Domain 4 Is Tightly Coupled to Sodium Channel Fast Inactivation and Gating Charge Immobilization

1999 ◽  
Vol 114 (2) ◽  
pp. 167-184 ◽  
Author(s):  
Frank J.P. Kühn ◽  
Nikolaus G. Greeff
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Time Constant ◽  
Voltage Sensor ◽  
Functional Coupling ◽  
Fast Inactivation ◽  
Voltage Sensors ◽  
Steady State Inactivation ◽  
Arginine Residues ◽  
Domain 4

The highly charged transmembrane segments in each of the four homologous domains (S4D1–S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

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

Abstract 20627: Modulation of Cardiac Sodium Channel by Palmitoylation and Its Association With Cardiac Disease Mutations

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Zifan Pei ◽  
Andy Hudmon ◽  
Theodore R Cummins
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Functional Activity ◽  
Site Directed Mutagenesis ◽  
Significant Shift ◽  
Biophysical Properties ◽  
Disease Mutations ◽  
Cardiac Sodium Channel ◽  
Steady State Inactivation

Cardiac sodium channel (Nav1.5) is responsible for the generation and propagation of the cardiac action potential, which underlies cardiac excitability. It can be modified by a variety of post-translational modifications. Palmitoylation is one of the most common post-translational lipid modifications that can dynamically regulate protein life cycle and functional activity. In our study, we identified palmitoylation on Nav1.5 and its alteration in channel biophysical properties. Nav1.5 palmitoylation was identified in both HEK 293 cells stably expressing Nav1.5 and cardiac tissues using acyl-biotin exchange assay. Nav1.5 palmitoylation was inhibited by pre-incubating the cells with the inhibitor 2-Br-Palmitate (2BP, 25uM, 24hrs). Biophysically, 2BP treatment drastically shifted the channel steady-state inactivation to more hyperpolarized voltages, suggesting palmitoylation altering channel functional activity. In addition, four predicted endogenous palmitoylation sites were identified using CSS-Palm 3.0. Site-directed mutagenesis method was used to generate a cysteine removing background of wt Nav1.5 to study the role of predicted sites. Patch clamp analysis of wt and cysteine-removed Nav1.5 revealed a significant change in channel biophysics. 2BP treatment significantly shifted steady-state inactivation of wt Nav1.5 while not affecting cysteine-removed Nav1.5 significantly, indicating the important role of these four cysteine sites in modulating channel palmitoylation. Moreover, several LQT disease mutations were identified to potentially add or remove palmitoylation sites. Further analysis of these disease mutations revealed a significant shift in channel steady-state inactivation and this alteration cannot be seen with the substitution of other residues on the same site, suggesting the specific role of cysteine residue in causing the functional alteration. For the LQT mutation that removes potential palmitoylation site, 2BP treatment did not affect channel biophysical properties, indicating the essential role of this cysteine in channel palmitoylation. These results suggest that palmitoylation on Nav1.5 regulates channel functional activity and its modulation may contribute to new cardiac channelopathies.

Modification of Synaptic Transmission and Sodium Channel Inactivation by the Insect-Selective Scorpion Toxin LqhαIT

2000 ◽  
Vol 83 (3) ◽  
pp. 1181-1187 ◽  
Author(s):  
Daewoo Lee ◽  
Michael Gurevitz ◽  
Michael E. Adams
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Transmitter Release ◽  
House Fly ◽  
The Body ◽  
Scorpion Toxin ◽  
Nerve Endings ◽  
Channel Inactivation ◽  
Sodium Channel Inactivation ◽  
Steady State Inactivation

The peptide LqhαIT is an α-scorpion toxin that shows significant selectivity for insect sodium channels over mammalian channels. We examined the symptoms of LqhαIT-induced paralysis and its neurophysiological correlates in the house fly ( Musca domestica). Injection of LqhαIT into fly larvae produced hyperactivity characterized by continuous, irregular muscle twitching throughout the body. These symptoms were correlated with elevated excitability in motor units caused by two physiological effects of the toxin: 1) increased transmitter release and 2) repetitive action potentials in motor nerves. Increased transmitter release was evident as augmentation of neurally evoked synaptic current, and this was correlated with an increased duration of action potential–associated current (APAC) in loose patch recordings from nerve terminals. Repetitive APACs were observed to invade nerve endings. The toxin produced marked inhibition of sodium current inactivation in fly central neurons, which can account for increased duration of the APAC and elevated neurotransmitter release at the neuromuscular junction. Steady-state inactivation was shifted significantly to more positive potentials, whereas voltage-dependent activation of the channels was not affected. The shift in steady-state inactivation provides a mechanism for inducing repetitive activity in motoneurons. The effects of LqhαIT on sodium channel inactivation in motor nerve endings can account both for increased transmitter release and repetitive activity leading to hyperactivity in affected insects.

scholarly journals p.L1612P, a Novel Voltage-gated Sodium Channel Nav1.7 Mutation Inducing a Cold Sensitive Paroxysmal Extreme Pain Disorder

2015 ◽  
Vol 122 (2) ◽  
pp. 414-423 ◽  
Author(s):  
Marc R. Suter ◽  
Zahurul A. Bhuiyan ◽  
Cédric J. Laedermann ◽  
Thierry Kuntzer ◽  
Muriel Schaller ◽  
...  
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Cold Exposure ◽  
Pain Disorder ◽  
Clinical Genetic ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Extreme Pain

Abstract Background: Mutations in the SCN9A gene cause chronic pain and pain insensitivity syndromes. We aimed to study clinical, genetic, and electrophysiological features of paroxysmal extreme pain disorder (PEPD) caused by a novel SCN9A mutation. Methods: Description of a 4-generation family suffering from PEPD with clinical, genetic and electrophysiological studies including patch clamp experiments assessing response to drug and temperature. Results: The family was clinically comparable to those reported previously with the exception of a favorable effect of cold exposure and a lack of drug efficacy including with carbamazepine, a proposed treatment for PEPD. A novel p.L1612P mutation in the Nav1.7 voltage-gated sodium channel was found in the four affected family members tested. Electrophysiologically the mutation substantially depolarized the steady–state inactivation curve (V1/2 from −61.8 ± 4.5 mV to −30.9 ± 2.2 mV, n = 4 and 7, P < 0.001), significantly increased ramp current (from 1.8% to 3.4%, n = 10 and 12) and shortened recovery from inactivation (from 7.2 ± 5.6 ms to 2.2 ± 1.5 ms, n = 11 and 10). However, there was no persistent current. Cold exposure reduced peak current and prolonged recovery from inactivation in wild-type and mutated channels. Amitriptyline only slightly corrected the steady–state inactivation shift of the mutated channel, which is consistent with the lack of clinical benefit. Conclusions: The novel p.L1612P Nav1.7 mutation expands the PEPD spectrum with a unique combination of clinical symptoms and electrophysiological properties. Symptoms are partially responsive to temperature but not to drug therapy. In vitro trials of sodium channel blockers or temperature dependence might help predict treatment efficacy in PEPD.

scholarly journals S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

2013 ◽  
Vol 141 (5) ◽  
pp. 601-618 ◽  
Author(s):  
James R. Groome ◽  
Vern Winston
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Negative Charge ◽  
Critical Role ◽  
Voltage Sensor ◽  
Mammalian Skeletal Muscle ◽  
Fast Inactivation ◽  
Domain Specific ◽  
Sensor Module ◽  
Charge Cluster

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

scholarly journals Paramyotonia congenita mutations reveal different roles for segments S3 and S4 of domain D4 in hSkM1 sodium channel gating.

1996 ◽  
Vol 107 (2) ◽  
pp. 183-194 ◽  
Author(s):  
S Ji ◽  
A L George ◽  
R Horn ◽  
R L Barchi
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Molecular Mechanisms ◽  
Dominant Role ◽  
Channel Gating ◽  
Paramyotonia Congenita ◽  
Double Mutation ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Kinetics Of

Mutations in the gene encoding the voltage-gated sodium channel of skeletal muscle (SkMl) have been identified in a group of autosomal dominant diseases, characterized by abnormalities of the sarcolemmal excitability, that include paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP). We previously reported that PC mutations cause in common a slowing of inactivation in the human SkMl sodium channel. In this investigation, we examined the molecular mechanisms responsible for the effects of L1433R, located in D4/S3, on channel gating by creating a series of additional mutations at the 1433 site. Unlike the R1448C mutation, found in D4/S4, which produces its effects largely due to the loss of the positive charge, change of the hydropathy of the side chain rather than charge is the primary factor mediating the effects of L1433R. These two mutations also differ in their effects on recovery from inactivation, conditioned inactivation, and steady state inactivation of the hSkMl channels. We constructed a double mutation containing both L1433R and R1448C. The double mutation closely resembled R1448C with respect to alterations in the kinetics of inactivation during depolarization and voltage dependence, but was indistinguishable from L1433R in the kinetics of recovery from inactivation and steady state inactivation. No additive effects were seen, suggesting that these two segments interact during gating. In addition, we found that these mutations have different effects on the delay of recovery from inactivation and the kinetics of the tail currents, raising a question whether this delay is a reflection of the deactivation process. These results suggest that the S3 and S4 segments play distinct roles in different processes of hSkM1 channel gating: D4/S4 is critical for the deactivation and inactivation of the open channel while D4/S3 has a dominant role in the recovery of inactivated channels. However, these two segments interact during the entry to, and exit from, inactivation states.

Properties of two voltage-activated potassium currents in acutely isolated juvenile rat dentate gyrus granule cells

1992 ◽  
Vol 68 (6) ◽  
pp. 2086-2099 ◽  
Author(s):  
H. Beck ◽  
E. Ficker ◽  
U. Heinemann
Keyword(s):  
Steady State ◽  
Dentate Gyrus ◽  
Time Constant ◽  
Decay Time ◽  
Granule Cells ◽  
Delayed Rectifier ◽  
Pharmacological Properties ◽  
Age Group ◽  
Boltzmann Function ◽  
Steady State Inactivation

1. The properties of outward currents were investigated in acutely isolated dentate gyrus granule cells at postnatal ages of day 5-7, 10-14, 18-24 (P5-7, P10-14, P18-24) and at adulthood (2-3 mo), with the use of the whole-cell patch-clamp technique. 2. Kinetic analysis and pharmacological properties showed that an A-type K+ current (IA) and a delayed rectifier current (IK) were present in these cells. 3. IA in P10-14 cells activated and inactivated rapidly with a decay time constant of 7.5 +/- 2.1 (SD) ms with command pulses to +30 mV. The removal of inactivation was monoexponential with a time constant of 23.1 ms (holding potential, -50 mV; conditioning voltage steps of varying duration to -110 mV). V 1/2 of the Boltzmann function describing steady-state inactivation was -65.1 +/- 1.8 mV with a slope factor of -6.0. IA was sensitive to 5 mM 4-aminopyridine (4-AP) but not to 10 mM tetraethylammonium (TEA). 4. IK in P10-14 cells displayed a voltage-dependent activation time constant (4.3 +/- 0.8 ms for command pulses to +30 mV and 16.2 +/- 2.4 for command pulses to -10 mV) and a double-exponential decay (time constants 194 +/- 21 and 1,625 +/- 254 ms). The rate constant of removal of inactivation was 332.1 ms. IK showed a reduction by 61.4 +/- 5.3% with 10 mM TEA and was partially blocked by 5 mM 4-AP in a subpopulation of cells. 5. Whereas IA remained stable over time, IK showed a substantial reduction of current amplitude by 67% after 30 min of cell perfusion through the patch pipette. The time course of this reduction was monoexponential with a time constant of 6.9 min and was partly due to a shift in V1/2 of the steady-state inactivation from -79.2 to -99.6 mV. 6. IA and IK remained stable with respect to kinetic properties during ontogenesis. However, the relative contribution and pharmacological properties of the investigated K+ currents varied with age. Although IA dominated in P5-7 cells, IK was prominent in most older cells. Five millimolars 4-AP reduced IA by 40.7 +/- 26.7% in P5-7 cells and blocked IA completely in 80% of investigated P10-14 cells. Similar changes were observed for the effects of 4-AP on IK (18.7% depression in the age group P5-8, 46.1% in the age group P10-14, and 45.7% in adult animals).

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.

Transient and delayed potassium currents in the Retzius cell of the leech, Macrobdella decora

1986 ◽  
Vol 56 (3) ◽  
pp. 812-822 ◽  
Author(s):  
J. Johansen ◽  
A. L. Kleinhaus
Keyword(s):  
Steady State ◽  
Time Constant ◽  
Time Course ◽  
Substantial Part ◽  
Macrobdella Decora ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
K Current ◽  
Retzius Cell ◽  
T Tau

The properties of a quickly inactivating transient K current (IA) and a slowly inactivating delayed K current (IK) were investigated with two-electrode voltage-clamp techniques in the isolated soma of the Retzius cell of the leech, Macrobdella decora. The two currents could be pharmacologically separated according to their different sensitivities to tetraethylammonium ions (TEA) and 4-aminopyridine (4-AP). IA was totally blocked by 3 mM 4-AP but not affected by 25 mM TEA. IK was suppressed almost completely by 25 mM TEA, whereas its peak amplitude only decreased by 10-15% in 3 mM 4-AP. IA was activated at membrane potentials more positive than -35 to -30 mV, whereas the threshold for IK was at more positive potentials of approximately -20 to -15 mV. The activation of IA was rapid with a voltage-dependent time constant [tau m(A)] that varied from 6 to 2 ms for command potentials between -20 and 10 mV (at 22-24 degrees C). The inactivation, which was independent of voltage, was somewhat slower with a time constant (tau A) of approximately 90-110 ms. The time constants for activation [tau m(K)] and the early inactivation phase (tau K) of IK were both voltage dependent. In the range of potential steps from 0 to 30 mV, tau m(K) varied from 12 to 4.5 ms and tau K from 1,500 to 700 ms. The steady-state inactivation of IA varied with holding potential and was complete at potentials more positive than -30 mV. IA was fully available from potentials more negative than -70 mV. IK did not show steady-state inactivation below its threshold of activation. The time course of IA during a maintained depolarization could be reasonably described by the expression IA(t) = IA(infinity) [1-exp(-t/tau m(A))]2 exp(-t/tau A). The time course of activation of IK without allowance for inactivation was approximated by the expression IK(t) = IK(infinity) [1-exp(-t/tau m(K))]2. The reversal potentials and magnitude of both IA and IK were dependent on extra-cellular K concentration, which suggest that a substantial part of the two currents was carried by K ions.

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