scholarly journals Interaction of n-alkylguanidines with the sodium channels of squid axon membrane.

1980 ◽  
Vol 76 (3) ◽  
pp. 315-335 ◽  
Author(s):  
G E Kirsch ◽  
J Z Yeh ◽  
J M Farley ◽  
T Narahashi
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Sodium Current ◽  
Time Dependent ◽  
Squid Axon ◽  
Axon Membrane ◽  
Inactivation Mechanism ◽  
Hydrophobic Binding ◽  
Sodium Inactivation ◽  
Guanidino Group

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.

scholarly journals Analysis of sodium current fluctuations in the cut-open squid giant axon.

1984 ◽  
Vol 83 (2) ◽  
pp. 133-142 ◽  
Author(s):  
I Llano ◽  
F Bezanilla
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Sodium Current ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Statistical Techniques ◽  
Current Fluctuations ◽  
Small Populations ◽  
Squid Axon ◽  
Single Sodium Channel

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.

scholarly journals Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide.

1978 ◽  
Vol 71 (3) ◽  
pp. 227-247 ◽  
Author(s):  
G S Oxford ◽  
C H Wu ◽  
T Narahashi
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Single Channel ◽  
Sodium Current ◽  
Complete Removal ◽  
Specific Protein ◽  
Peptide Bonds ◽  
Voltage Dependent ◽  
Inactivation Gating ◽  
Sodium Inactivation

The group-specific protein reagents, N-bromacetamide (NBA) and N-bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur-containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.

Kinetics of Change in Spike Height During Anodal Polarization of Isolated Single Nerve Fibers

1956 ◽  
Vol 187 (3) ◽  
pp. 549-557 ◽  
Author(s):  
Gordon M. Schoepfle
Keyword(s):  
Membrane Potential ◽  
Time Course ◽  
Sodium Current ◽  
Nerve Fibers ◽  
Time Dependent ◽  
Peak Time ◽  
Axon Membrane ◽  
Membrane Hyperpolarization ◽  
Anodal Polarization ◽  
Sodium Inactivation

The time dependent change in magnitude of the nodal action potential was analyzed as a function of time and of change in membrane potential during brief intervals of anodal polarization. The time course of this function involves an exponential term whose time constant decreases to a limiting value as extent of membrane hyperpolarization is progressively increased. While the time course of change in spike height cannot be correlated with any change in membrane potential induced by the polarization, the kinetics suggest that the sodium inactivation mechanism of Hodgkin and Huxley is sufficient to account for the results obtained. Brief direct current pulses elicit changes of membrane potential during activity which indicate that an augmented change in resistance of the hyperpolarized node is sufficient to account for the time dependent increase in spike height. These findings are consistent with the Hodgkin-Huxley scheme in that hyperpolarization of the squid axon membrane at constant membrane potential inhibits ‘sodium inactivation’ thereby allowing sodium conductance and sodium current to attain greater than normal values at peak time of activity. It is concluded that no change in emf of the membrane need be postulated in order to account for the time dependent increase in spike height during hyperpolarization of normal or depressed nodes.

scholarly journals Kinetic analysis of pancuronium interaction with sodium channels in squid axon membranes.

1977 ◽  
Vol 69 (3) ◽  
pp. 293-323 ◽  
Author(s):  
J Z Yeh ◽  
T Narahashi
Keyword(s):  
Membrane Potential ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Rate Constant ◽  
Time Course ◽  
Voltage Dependence ◽  
Sodium Current ◽  
Sodium Concentration ◽  
Internal Concentration ◽  
Sodium Inactivation

The interaction of pancuronium with sodium channels was investigated in squid axons. Sodium current turns on normally but turns off more quickly than the control with pancuronium 0.1-1mM present internally; The sodium tail current associated with repolarization exhibits an initial hook and then decays more slowly than the control. Pancuronium induces inactivation after the sodium inactivation has been removed by internal perfusion of pronase. Such pancuronium-induced sodium inactivation follows a single exponential time course, suggesting first order kinetics which represents the interaction of the pancuronium molecule with the open sodium channel. The rate constant of association k with the binding site is independent of the membrane potential ranging from 0 to 80 mV, but increases with increasing internal concentration of pancuronium. However, the rate constant of dissociation l is independent of internal concentration of pancuronium but decreases with increasing the membrane potential. The voltage dependence of l is not affected by changine external sodium concentration, suggesting a current-independent conductance block, The steady-state block depends on the membrane potential, being more pronounced with increasing depolarization, and is accounted for in terms of the voltage dependence of l. A kinetic model, based on the experimental observations and the assumption on binding kinetics of pancuronium with the open sodium channel, successfully simulates many features of sodium current in the presence of pancuronium.

scholarly journals Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.

1996 ◽  
Vol 134 (2) ◽  
pp. 499-509 ◽  
Author(s):  
C Paillart ◽  
J L Boudier ◽  
J A Boudier ◽  
H Rochat ◽  
F Couraud ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Amphotericin B ◽  
Sodium Channels ◽  
Time Dependent ◽  
Alpha Subunit ◽  
Dependent Manner ◽  
Down Regulation ◽  
Proteolytic Fragment ◽  
Channel Density ◽  
Specific Distribution

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.

scholarly journals GS-967 and Eleclazine Block Sodium Channels in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

2020 ◽  
Author(s):  
Franck Potet ◽  
Defne E. Egecioglu ◽  
Paul W. Burridge ◽  
Alfred L. George
Keyword(s):  
Stem Cell ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Pluripotent Stem Cell ◽  
Sodium Current ◽  
Induced Pluripotent Stem Cell ◽  
Slow Inactivation ◽  
Molecular Pharmacology ◽  
Recovery From Inactivation ◽  
Induced Pluripotent

ABSTRACTGS-967 and eleclazine (GS-6615) are novel sodium channel inhibitors exhibiting antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current (INaL). Here, we took advantage of a throughput automated electrophysiology platform (SyncroPatch 768PE) to investigate the molecular pharmacology of GS-967 and eleclazine on peak sodium current (INaP) recorded from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We compared GS-967 and eleclazine to the antiarrhythmic drug lidocaine, the prototype INaL inhibitor ranolazine, and the slow inactivation enhancing drug lacosamide. In human induced pluripotent stem cell-derived cardiomyocytes, GS-967 and eleclazine caused a reduction of INaP in a frequency-dependent manner consistent with use-dependent block (UDB). GS-967 and eleclazine had similar efficacy but evoked more potent UDB of INaP (IC50=0.07 and 0.6 μM, respectively) than ranolazine (7.8 μM), lidocaine (133.5 μM) and lacosamide (158.5 μM). In addition, GS-967 and eleclazine exerted more potent effects on slow inactivation and recovery from inactivation compared to the other sodium channel blocking drugs we tested. The greater UDB potency of GS-967 and eleclazine was attributed to the significantly higher association rates (KON) and moderate unbinding rate (KOFF) of these two compounds with sodium channels. We propose that substantial UDB contributes to the observed antiarrhythmic efficacy of GS-967 and eleclazine.SIGNIFICANCE STATEMENTWe investigated the molecular pharmacology of GS-967 and eleclazine on sodium channels in human induced pluripotent stem cell derived cardiomyocytes using a high throughput automated electrophysiology platform. Sodium channel inhibition by GS-967 and eleclazine has unique features including accelerating the onset of slow inactivation and impairing recovery from inactivation. These effects combined with rapid binding and moderate unbinding kinetics explain potent use-dependent block, which we propose contributes to their observed antiarrhythmic efficacy.

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.

Upregulation of a Silent Sodium Channel After Peripheral, but not Central, Nerve Injury in DRG Neurons

1999 ◽  
Vol 82 (5) ◽  
pp. 2776-2785 ◽  
Author(s):  
J. A. Black ◽  
T. R. Cummins ◽  
C. Plumpton ◽  
Y. H. Chen ◽  
W. Hormuzdiar ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Sodium Current ◽  
Sodium Currents ◽  
Drg Neurons ◽  
Adult Rats ◽  
Voltage Range ◽  
Type Iii ◽  
Axonal Projections

After transection of their axons within the sciatic nerve, DRG neurons become hyperexcitable. Recent studies have demonstrated the emergence of a rapidly repriming tetrodotoxin (TTX)-sensitive sodium current that may account for this hyperexcitability in axotomized small (<27 μm diam) DRG neurons, but its molecular basis has remained unexplained. It has been shown previously that sciatic nerve transection leads to an upregulation of sodium channel III transcripts, which normally are present at very low levels in DRG neurons, in adult rats. We show here that TTX-sensitive currents in small DRG neurons, after transection of their peripheral axonal projections, reprime more rapidly than those in control neurons throughout a voltage range of −140 to −60 mV, a finding that suggests that these currents are produced by a different sodium channel. After transection of the central axonal projections (dorsal rhizotomy) of these small DRG neurons, in contrast, the repriming kinetics of TTX-sensitive sodium currents remain similar to those of control (uninjured) neurons. We also demonstrate, with two distinct antibodies directed against different regions of the type III sodium channel, that small DRG neurons display increased brain type III immunostaining when studied 7–12 days after transection of their peripheral, but not central, projections. Type III sodium channel immunoreactivity is present within somata and neurites of peripherally axotomized, but not centrally axotomized, neurons studied after <24 h in vitro. Peripherally axotomized DRG neurons in situ also exhibit enhanced type III staining compared with control neurons, including an accumulation of type III sodium channels in the distal portion of the ligated and transected sciatic nerve, but these changes are not seen in centrally axotomized neurons. These observations are consistent with a contribution of type III sodium channels to the rapidly repriming sodium currents observed in peripherally axotomized DRG neurons and suggest that type III channels may at least partially account for the hyperexcitibility of these neurons after injury.

Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

1989 ◽  
Vol 238 (1290) ◽  
pp. 39-51 ◽  
Keyword(s):  
Optic Nerve ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Node Of Ranvier ◽  
Ultrastructural Localization ◽  
Active Role ◽  
Nodes Of Ranvier ◽  
Electron Microscopic ◽  
Axon Membrane ◽  
Intense Immunoreactivity

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.

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