scholarly journals A unique role for the S4 segment of domain 4 in the inactivation of sodium channels.

1996 ◽  
Vol 108 (6) ◽  
pp. 549-556 ◽  
Author(s):  
L Q Chen ◽  
V Santarelli ◽  
R Horn ◽  
R G Kallen
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Na Channel ◽  
H Infinity ◽  
Unique Role ◽  
Transmembrane Segments ◽  
Gating Charge ◽  
Steady State Inactivation ◽  
And Shifts

Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-S6). The highly charged S4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in S4 segments of each domain of the human heart Na+ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na+ current, consistent with a role of S4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (eo) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h infinity) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in S4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

Electrophysiological characteristics of cloned skeletal and cardiac muscle sodium channels

1996 ◽  
Vol 271 (2) ◽  
pp. H498-H506 ◽  
Author(s):  
M. Chahine ◽  
I. Deschene ◽  
L. Q. Chen ◽  
R. G. Kallen
Keyword(s):  
Steady State ◽  
Sodium Channels ◽  
H Infinity ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Current Decay ◽  
Voltage Gated Sodium Channels ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Kinetics Of

The alpha-subunit encoding for voltage-gated sodium channels rSkM1 (rat skeletal muscle subtype 1) and hH1 (human heart subtype 1) has been cloned and expressed by various groups under various conditions in Xenopus oocytes and the tsA201 (HEK 293) mammalian cell line derived from human embryonic kidney cells. In this study, we have expressed hH1 and rSkM1 in tsA201 cells for comparison under the same conditions using patch-clamp methods. Our results show significant differences in the current-voltage (I-V) relationship, kinetics of current decay, voltage dependence of steady-state inactivation, and the time constant for recovery from inactivation. We studied several rSkM1/hH1 chimeric sodium channels to identify the structural regions responsible for the different biophysical behavior of the two channel subtypes. Exchanging the interdomain (ID3-4) loops, thought to contain the inactivation particle, between rSkM1 and hH1 had no effect on the electrophysiological behaviors, including inactivation, indicating that the differences in channel subtype characteristics are determined by parts of the channel other than the ID3-4 segment. The data on a chimeric channel in which D1 and D4 are derived from hH1 while D2 and D3 and the ID1-2, ID2-3, and ID3-4 loops are from rSkM1 show that D1 and/or D4 seem to be responsible for the slower kinetics of inactivation of hH1 while D2 and/or D3 appear to contain the determinants for the differences in the I-V relationship, steady-state inactivation (h infinity) curve, and the kinetics of the recovery from inactivation.

scholarly journals Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification.

1992 ◽  
Vol 99 (1) ◽  
pp. 1-20 ◽  
Author(s):  
G K Wang ◽  
S Y Wang
Keyword(s):  
Time Course ◽  
Cell Voltage ◽  
Gh3 Cells ◽  
Na Channel ◽  
Na Channels ◽  
H Infinity ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Pharmacological Modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.

Differences in Na+ Conductance Density and Na+ Channel Functional Properties Between Dopamine and GABA Neurons of the Rat Substantia Nigra

2010 ◽  
Vol 103 (6) ◽  
pp. 3099-3114 ◽  
Author(s):  
Vincent Seutin ◽  
Dominique Engel
Keyword(s):  
Substantia Nigra ◽  
Functional Properties ◽  
Voltage Dependence ◽  
Na Channel ◽  
Inactivation Curve ◽  
Voltage Gated ◽  
Gaba Neurons ◽  
Steady State Inactivation ◽  
The Mean ◽  
Firing Properties

Dopamine (DA) neurons and GABA neurons of the substantia nigra (SN) promote distinct functions in the control of movement and have different firing properties and action potential (AP) waveforms. APs recorded from DA and GABA neurons differed in amplitude, maximal rate of rise, and duration. In addition, the threshold potential for APs was higher in DA neurons than in GABA neurons. The activation of voltage-gated Na+ channels accounts largely for these differences as the application of a low concentration of the voltage-gated Na+ channel blocker TTX had an effect on all of these parameters. We have examined functional properties of somatic Na+ channels in nucleated patches isolated from DA and GABA neurons. Peak amplitudes of macroscopic Na+ currents were smaller in DA neurons in comparison to those in GABA neurons. The mean peak Na+ conductance density was 24.5 pS μm−2 in DA neurons and almost twice as large, 41.6 pS μm−2, in GABA neurons. The voltage dependence of Na+ channel activation was not different between the two types of SN neurons. Na+ channels in DA and GABA neurons, however, differed in the voltage dependence of inactivation, the mean mid-point potential of steady-state inactivation curve being more positive in DA neurons than in GABA neurons. The results suggest that specific Na+ channel gating properties and Na+ conductance densities in the somatic membrane of SN neurons may have consequences on synaptic signal integration in the soma of both types of neurons and on somatodendritic release of dopamine by DA neurons.

Operantly conditioned motoneuron plasticity: possible role of sodium channels

1995 ◽  
Vol 73 (2) ◽  
pp. 867-871 ◽  
Author(s):  
J. A. Halter ◽  
J. S. Carp ◽  
J. R. Wolpaw
Keyword(s):  
Voltage Dependence ◽  
Myelinated Axon ◽  
H Reflex ◽  
Na Channel ◽  
Channel Activation ◽  
Positive Shift ◽  
Fiber Properties ◽  
Kinase C ◽  
Experimental Findings

1. Learning is traditionally thought to depend on synaptic plasticity. However, recent work shows that operantly conditioned decrease in the primate H reflex is associated with an increase in the depolarization needed to fire the spinal motoneuron (VDEP) and a decrease in its conduction velocity (CV). Furthermore, the increase in VDEP appears to be largely responsible for the H-reflex decrease. The conjunction of these changes in VDEP and CV suggests that an alteration in Na+ channel properties throughout the soma and axon could be responsible. 2. A mathematical model of the mammalian myelinated axon was used to test whether a positive shift in the voltage dependence of Na+ channel activation, a decrease in Na+ channel peak permeability, or changes in other fiber properties could have accounted for the experimental findings. 3. A positive shift of 2.2 mV in Na+ channel activation reproduced the experimentally observed changes in VDEP and CV, whereas a reduction in Na+ channel permeability or changes in other fiber properties did not. 4. These results are consistent with the hypothesis that operantly conditioned decrease in the primate H reflex is largely due to a positive shift in the voltage dependence of Na+ channel activation. Recent studies suggest that change in activation of protein kinase C may mediate this effect.

scholarly journals Neutralization of Gating Charges in Domain II of the Sodium Channel α Subunit Enhances Voltage-Sensor Trapping by a β-Scorpion Toxin

2001 ◽  
Vol 118 (3) ◽  
pp. 291-302 ◽  
Author(s):  
Sandrine Cestèle ◽  
Todd Scheuer ◽  
Massimo Mantegazza ◽  
Hervé Rochat ◽  
William A. Catterall
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Voltage Dependence ◽  
Membrane Potentials ◽  
Voltage Sensor ◽  
Scorpion Toxin ◽  
Channel Activation ◽  
Α Subunit ◽  
Gating Charges

β-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.

Differential Effects of Anesthetic and Nonanesthetic Cyclobutanes on Neuronal Voltage-gated Sodium Channels

2000 ◽  
Vol 92 (2) ◽  
pp. 529-529 ◽  
Author(s):  
Lingamaneni Ratnakumari ◽  
Tatyana N. Vysotskaya ◽  
Daniel S. Duch ◽  
Hugh C. Hemmings
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Voltage Dependence ◽  
Glutamate Release ◽  
Dorsal Root Ganglion Neurons ◽  
Na Channel ◽  
Na Channels ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Ganglion Neurons

Background Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods. Methods Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording. Results F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [approximately 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [approximately 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50 = 0.5 mM [approximately 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70+/-9% block at 0.6 mM [approximately 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21+/-9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16+/-2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation. Conclusions The anesthetic cyclobutane F3 significantly inhibited Na+ channel-mediated glutamate release and increases in [Ca2+]i. In contrast, the nonanesthetic cyclobutane F6 had no significant effects at predicted anesthetic concentrations. F3 inhibited dorsal root ganglion neuron Na+ channels with a potency and by mechanisms similar to those of conventional volatile anesthetics; F6 was less effective and did not produce voltage-dependent block. This concordance between anesthetic activity and Na+ channel inhibition supports a role for presynaptic Na+ channels as targets for general anesthetic effects and suggests that shifting the voltage-dependence of Na+ channel inactivation is an important property of volatile anesthetic compounds.

scholarly journals Single rat muscle Na+ channel mutation confers batrachotoxin autoresistance found in poison-dart frog Phyllobates terribilis

2017 ◽  
Vol 114 (39) ◽  
pp. 10491-10496 ◽  
Author(s):  
Sho-Ya Wang ◽  
Ging Kuo Wang
Keyword(s):  
Defense Mechanism ◽  
Na Channel ◽  
Single Nucleotide ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Naturally Occurring ◽  
Resistant Phenotype ◽  
Nucleotide Mutation ◽  
Channel Mutation

Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 µM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC).

scholarly journals Role of Amino Acid Residues in Transmembrane Segments IS6 and IIS6 of the Na+Channel α Subunit in Voltage-dependent Gating and Drug Block

2002 ◽  
Vol 277 (38) ◽  
pp. 35393-35401 ◽  
Author(s):  
Vladimir Yarov-Yarovoy ◽  
Jancy C. McPhee ◽  
Diane Idsvoog ◽  
Caroline Pate ◽  
Todd Scheuer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Voltage Dependent

scholarly journals Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7

Biomedicines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 37
Author(s):  
Kathleen Yin ◽  
Jennifer R. Deuis ◽  
Zoltan Dekan ◽  
Ai-Hua Jin ◽  
Paul F. Alewood ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Rational Design ◽  
Voltage Dependence ◽  
Spider Venom ◽  
Fast Inactivation ◽  
Peak Current ◽  
Native Peptide ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
And Shifts

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at NaV channels in the future.

Sign in / Sign up

Export Citation Format

Share Document