structure, functions, receptor sites and modulators of voltage-gated sodium channels

1. Voltage-gated sodium channels
presented by : nasim pourjalili

2. Voltage-gated ion channels
▰ Cell membranes are generally impermeable to ions
▰ Voltage-gated ion channels (VGICs) are transmembrane proteins that play important roles
in the electrical signaling of cells
▰ Voltage-gated ion channels are responsible for generation of electrical signals in cell
membranes.
▰ The activity of VGICs is regulated by the membrane potential of a cell
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3. Voltage-gated ion channels
▰ Depending on the ions conducted, VGICs can be classified as voltage-gated sodium,1
potassium, calcium, or chloride channels
▰ They have a crucial role in excitable cells such as neuronal and muscle tissues, allowing a
rapid and co-ordinated depolarization in response to triggering voltage change.
▰ Upon depolarization, permeability to sodium, calcium, or potassium increases dramatically
over a period of 0.5 to hundreds of msec and then decreases to the baseline level over a
period of 2 msec to a few seconds.
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4. Voltage-gated sodium channels
▰ VGSCs (or Navs) are heteromeric transmembrane proteins that are activated in response to
membrane depolarization and have a fundamental role in the generation and propagation of
action potentials in neurons and other electrically excitable cells via control of the flow of Na+
ions through cell membranes.
▰ VGSCs belong to the voltage-gated ion channel (VGIC) superfamily
▰ Each eukaryotic VGSC comprises a single polypeptide chain of α-subunit that folds into four
homologous but nonidentical domains (DI to DIV) and one or more auxiliary β-subunits
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5. Voltage-gated Sodium Channels
▰ More than 1000 disease related mutations of nine VGSC-encoding genes implicated in
channel dysfunctions and channelopathies have been identified.
▰ VGSCs are involved in a variety of diseases, including epilepsy, cardiac arrhythmias, and
neuropathic pain, and therefore have been regarded as appealing therapeutic targets for
the development of anticonvulsant, antiarrhythmic, and local anesthetic drugs.
▰ For a long time, the design of isoform-selective VGSC modulators was challenging owing to
the lack of co-crystallized protein–ligand structures and high sequence conservation in the
channel pores across VGSC isoforms. However, X-ray and electron microscopy structures
of VGSCs have been gradually resolved.
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6. The structures and functions of VGSCs
▰ The α-subunit is the core subunit of the VGSC, with a molecular weight as large as 240–260
kDa. It is composed of the following three parts:
a) four highly homologous transmembrane domains
b) three intracellular loops (2 long loops, L1 and L2, and 1 short loop, L3)
c) N-terminus and C-terminus (NT and CT)
▰ The sequence homology of the mammalian α-subunits is >70%,
▰ There are ten different isoforms of α-subunits, Nav1.1–Nav1.9; Nax.
▰ Nav1.2, Nav1.3, and Nav1.6 are predominantly expressed in the central nervous system
(CNS), whereas Nav1.4 is mainly expressed in skeletal muscle. Nav1.5 is primarily
expressed in cardiac muscle, and Nav1.7, Nav1.8 and Nav1.9 are generally abundant in the
peripheral nervous system.
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▰ To date, five β-subunits have been identified in vertebrates (β1, β1B, β2, β3, and β4), which
have a prominent role in modulating the expression and membrane trafficking of the α-
subunit, channel activation and inactivation, and ligand binding.
▰ Each β-subunit comprises an N-terminal extracellular immunoglobulin (Ig) domain, a single
transmembrane α helix, and a small intracellular C-terminal region.

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▰ Each transmembrane domain contains six hydrophobic α-helical transmembrane segments
(S1- S6).
▰ The S1–S4 transmembrane segments act as a voltage sensor, and a number of positively
charged amino acids (usually arginine’s) on S4 are important for such voltage sensing.
▰ S5 and S6 form the pore.
▰ The loops between S5 and S6 from each domain form the outer vestibule of the channel,
which houses the ion selectivity filter.
▰ The intracellular loop connecting DIIIS6 and DIVS1 functions as an inactivation gate, closing
the channel pore during fast inactivation.

10. The typical open–closed–inactivated cycle
▰ There are at least three primary states for a typical VGSC : resting (closed), activated (open),
and inactivated (closed).
Upon membrane depolarization, the voltage sensors in the S4 helices move toward the
extracellular surface, which stimulates the channel gate to open briefly (<1 ms). Then, Na+ ions
move into the cell, subsequently depolarizing the membrane, which is responsible for the rising
phase of an action potential.
After a few milliseconds, the sodium channels are rapidly inactivated
Fast inactivation occurs over a timescale of ∼1–5 ms and the channel stops ion conduction
while the voltage sensor is still in an active conformation. The channel then enters a slow
inactivated state in response to prolonged depolarization or rapid repetitive stimulations, which
occur on the timescale of seconds to minutes. When the gating charged residues of each S4
return to their resting positions upon membrane repolarization, the sodium channel recovers
from inactivation and deactivates (i.e., the activation gate closes). Therefore, the outward and
inward movements of S4 lead to the opening and closing states of the pore domain.
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12. Toxin and drug receptor sites of VGSCs
▰ Natural toxins and synthesized molecular modulators of VGSCs can be roughly categorized into
two classes: pore blockers and gating modifiers.
▰ Pore blockers inhibit channel conductance by binding to the extracellular loops and/or the pore.
▰ Gating modifiers stabilize the channel in a particular functional state and alter the generation
and propagation of action potentials, which include toxins binding to intramembrane or
extracellular receptor sites .
▰ To date, eight binding sites of VGSCs for toxins and small molecules have been identified.
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▰ Site 1: extracellular pore blocker
Binding site 1 is formed by the four P-loops. This site can be occupied by water-soluble
heterocyclic guanidinium toxins (TTX and STX) and peptide μ-contoxins from the marine cone
snail.
▰ Site 2: intracellular pore gating activator (state dependent)
Binding site 2 is generally considered to be formed by DI-S6 and DIV-S6, and can be occupied by
lipid-soluble toxins with diverse chemical structures, such as batrachotoxin (BTX), veratridine
(VTD), antillatoxin (ATX), aconitine (ACT), and grayanotoxin (GTX). These modulators contribute
to activating the channel or impeding inactivation.
▰ Site 3: extracellular gating activator
Binding site 3 mainly locates at the extracellular S3–S4 loops of DIV, and is occupied by δ-
atracotoxins from spiders, α-scorpion toxins, and anthopleurin from sea anemones.These toxins
impair inactivation and induce a prolonged opening of VGSCs by blocking the conformational
changes of DIV-S4 during fast inactivation.
▰ Site 4: extracellular gating blocker
Binding site 4 mainly localizes in the extracellular loops that connect the S1–S2 and S3–S4
segments of DII. Site 4 can be recognized by long-chain peptide toxins, such as β-scorpion toxins
and several spider toxins. These toxins reduce the peak sodium current amplitude and shift the
voltage-dependent activation toward more negative.

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▰ Site 5: intracellular pore gating activator (state dependent)
binding site 5 scattered in DI-S6 and DIV-S5. This site can be occupied by highly lipophilic cyclic
polyether toxins, such as ciguatoxins (CTX) and brevetoxins (PbTxs).These toxins preferentially
bind to the activated channel and exert a multitude of electrophysiological effects by suppressing
activation and shifting the activation potential toward hyperpolarized potentials
▰ Site 6: extracellular gating activator
Site 6 might be occupied by δ-conotoxins. Although site 6 is close to site 3, their activators do
not compete with each other.
▰ Site 7: intracellular gating activator
Binding site 7 locates at DIII-S6 and can be occupied by some insecticides, including pyrethroids
and DDT. These insecticidal agents can prolong the opening state of VGSCs by suppressing
channel deactivation and inactivation, resulting in firing and depolarization of the nerve
membrane in the insect nervous system
▰ Site 8: local anesthetic (LA) binding site
Binding site 8 positions in the inner cavity of the channel pore and comprises residues in S6 of
DI, DIII, and DIV, which exhibits significant overlap with site 2. This site has been targeted by
most VGSC drugs for the treatment of sodium channelopathies, such as local anesthetics, class
I cardiac antiarrhythmics, anticonvulsants, and antidepressants, which share common structural
features with a lipophilic aromatic ring and a hydrophilic tertiary amine group. However, the site
is almost identical across all VGSCs. As a consequence, these drugs are nonselective VGSC
blockers with state and frequency dependence.

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16. Small-molecule VGSC modulators
▰ By inhibiting the conduction of action potential through a simple pore blocking mechanism or
through preferential binding to and stabilization of the channels in nonconducting inactivated
states, VGSC-modulating drugs have therapeutic significance as LAs (lidocaine), general
anesthetics (sevoflurane and isoflurane), anticonvulsants (carbamazepine and lamotrigine), and
antiarrhythmic drugs (mexiletine).
▰ most of these drugs bind to the highly conserved LA binding site of VGSCs; thus, they are first-
generation nonselective modulators of VGSCs.
▰ Given the low isoform selectivity, limited therapeutic indices, and dose-limiting adverse effects
of first-generation VGSC drugs, extensive efforts have been dedicated to search for more
isoform-selective and disease-specific VGSC modulators.
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17. Effects of Curcumin on VGSC
▰ Curcumin was able to improve diabetes mellitus and its complications such as diabetic
neuropathic pain through voltage-gated sodium channels and by increasing sodium content in
dorsal rootganglion neurons.As the most active ingredient of the plant Curcuma longa,
curcumin is a lipophilic polyphenol substance that has been reported to exhibit several
pharmacological actions including wound healing, anti-inflammatory, antimicrobial, antioxidant,
immunomodulatory, lipid-modifying, antitumor, antiplatelet aggregation,and antiangiogenic
effects . It can also be used as an encouraging treatment for multiple sclerosis, cancer,
diabetes, metabolic, autoimmune, Alzheimer, human immunodeficiency virus (HIV),
cardiovascular, neurological, and liver and lung disease
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18. Channelopathies & Mutation
▰ Research of the molecular properties of sodium channels has elucidated mutations that cause
multiple inherited hyperexcitabilty diseases in humans. These disorders are termed
channelopathies. The first channelopathy involving sodium channels was found in skeletal
muscle. Other channelopathies have been reported in cardiac and neuronal subtypes.
▰ Epilepsy Associated with the SCN1A and SCN2A Genes
SCN1A, encodes the Nav1.1 channel. SCN1A is responsible for several types of epilepsy:
generalized epilepsy with febrile seizure (GEFS+) and severe myoclonic epilepsy (Dravet)
syndrome.
 Similar to SCN1A, SCN2A is also involved in Dravet syndrome. The first mutation in this gene
was identified in patients with GEFS+ . SCN2A is also implicated in benign familial neonatal-
infantile seizure (BFNIS) . In a recent study, a North Indian population with SCN1A and
SCN2A polymorphic genes showed various responses to antiepileptic drugs, such as
phenytoin, carbamazepine, phenobarbital and valproate .
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▰ SCN4A Mutation-Associated Skeletal Muscle Sodium Channelopathy
The Nav1.4 sodium channel, which is encoded by the SCN4A gene is responsible for
channelopathies in skeletal muscle.
Numerous SCN4A mutations cause abnormalities, such as familial periodic paralysis
syndromes.
The four major human channelopathies that are associated with SCN4A are as follows:
potassium-aggravated myotonia (PAM), including myotonia fluctuans, myotonia
permanens and painful myotonia, as well as paramyotonia congenita (PC) , hyperkalemic
periodic paralysis (HyperPP) and hypokalemic periodic paralysis 2 (HypoPP2).

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▰ Cardiac Sodium (SCN5A) Channelopathies
The cardiac sodium channel is responsible for the fast depolarization phase in
cardiomyocytes.
 Impulse conduction in the atria, His Purkinje system and the ventricle is sustained by the
transient increase in sodium permeability .
Disturbances in conduction and lifethreatening arrhythmias are caused by decreased
sodium channel function .
Mutations in SCN5A, which encodes the primary sodium channel in cardiac tissues, cause
sodium channel dysfunction and are associated with a number of unrelated arrhythmic
syndromes, such as long QT syndrome , Brugada syndrome , conduction dysfunction ,
sinus node dysfunction , sudden infant death syndrome (SIDS) and atrial fibrillation.
▰ Sodium Channel and Neuropathic Pain
 Of the nine sodium channel subtypes, four genes, SCN3A, SCN9A, SCN10A and
SCN11A, encode for the Nav1.3, Nav1.7, Nav1.8 and Nav1.9 channels, respectively, and
are involved in neuropathic pain.

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