Human Nav channel subunits’ gene information.
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Open access peer-reviewed chapter
By Tianbo Li and Jun Chen
Submitted: November 13th 2017Reviewed: May 2nd 2018Published: October 10th 2018
Voltage-gated sodium channels (Nav) control the initiation and propagation of action potential, and thus mediate a broad spectrum of physiological processes, including central and peripheral nervous systems’ function, skeletal muscle contraction, and heart rhythm. Recent advances in elucidating the molecular basis of channelopathies implicating Nav channels are the most appealing druggable targets for pain and many other pathology conditions. This chapter overviews Nav super family from genetic evolution, distribution, human diseases/pathology association, highlighting the most recent structure function breakthrough. The second section will discuss current small and large Nav modulators, including traditional nonselective pore blockers, intracellular modulators, and extracellular modulators.
Voltage-gated sodium channels (Nav) are large transmembrane proteins that conduct the flow of sodium ions down the electrochemical gradient through cell membranes. In excitable and nonexcitable cells, these channels control action potential initiation/propagation, cell motility, and proliferation. Na+ currents were firstly discovered in 1949 by Hodgkin and Huxley in their study of action potentials in squid giant axon [1, 2]. This early work demonstrated that the resting membrane potential mostly depends on potassium permeability, whereas action potential is directly shaped by sodium permeability, which allows transient influx of Na+ to raise membrane potentials and is followed by rapid inactivation within milliseconds. From 1950s to 1970s, studies from many laboratories established conceptual models and equations conceptualizing sodium channel function .
In a typical cell, sodium, potassium, chloride, and other membrane permeable ions are in unequal distribution across plasma membrane, Figure 1A. This unequal distribution and its resultant electrical gradient can be explained by Donnan equilibrium. For a specific ion, the electrical potential difference that exactly counterbalances diffusion due to the concentration difference is called the equilibrium potential for that specific ion. To use Na+ as an example, at equilibrium, the chemical force moving Na+ into the cell is balanced by the electrical force moving Na+ out of the cell (Figure 1B).
For each ion, the equilibrium (or reversal) potential, , can be calculated by the Nernst equation (Eq. (1)), where R = gas constant, 8.135 J K−1 mol−1; T = temperature in K (273 + temp in oC); z = valency of ion (Na+ = 1, Ca2+ = 2, and Cl− = −1); F = Faraday’s constant, 9.684 × 104 C mol−1. In a typical mammalian neuron with [Na+], [K+], and [Cl−] described in Figure 1A, based on Nernst equation, we can calculate = 67 mV, = 83 mV, and = 61 mV. In cell resting state, the experimental measured membrane potential = 65 mV. As indicated in Figure 2, Na+ has a tendency to flow into the cell due to , while K+ flow out of the cell (), and Cl− near equilibrium (). These concentration and electrical gradients are maintained by the dynamic equilibrium of ion channels and active ion transporters, most importantly by sodium pump (Na/K-ATPase).
The whole cell membrane potential can be calculated by all permeable ions’ equilibrium potentials and their relative permeability P, which is described by Goldman-Hodgkin-Katz equation (GHK equation) (Eq. (2)). This equation explained the experimental finding that resting membrane potential is more depending on PK, which is about 25-folds to PNa.
The Hodgkin-Huxley model describes how action potentials in neurons are initiated and propagated . Originally developed to fit action potential dynamics of squid giant axon, this model has been successfully applied to a wide range of neurons. It describes the electrical properties of excitable membranes as typical electrical circuit components. For instance, the cell membrane is modeled as a capacitor with capacitance (Cm) and ion channels are resistors with conductance of Na channel (, K channel (and leak channel (.
From Hodgkin-Huxley model, the total cell membrane current, I, can be calculated by Eq. (4), where membrane potential , ion conductances and , sodium activation variable m, sodium inactivation variable h, potassium activation variable n are variable functions of time, whereas , ,, and are constants.
Hodgkin-Huxley model provides a relatively simple and experimentally testable equation to deduce Na+ conductance change with time and voltages (Figure 2B), and nerve action potential (Figure 2C). It also embodies the three key features of Nav channels: voltage-dependent activation (submillisecond scale), rapid inactivation (millisecond scale), and selective Na+ conductance.
Electrophysiology was originated by Luigi Galvani in studying “animal electricity” in 1780s. In 1903, an extracellular recording technique, electrocardiography, was invented by Willem Einthoven (1924 Nobel Laureate); in 1952, intracellular recording technique was developed by Alan Hodgkin and Andrew Huxley (1963 Nobel Laureates); and in 1976, Erwin Neher and Bert Sackmann (1991 Noble Laureates) succeeded in measuring the ionic current of single channels in the cell membrane by developing patch clamp technique. These works were fundamental in revealing the physiological function of ion channels. Since then, the field of electrophysiology had undergone rapid evolution, especially after the introduction of automated patch clamp in the early 2000s.
Manual patch clamp technique employs glass microelectrode(s) with desired filling solution and tip diameter to perform either voltage or current clamp. For larger cells, such as Xenopus oocytes, two-electrode voltage clamp (TEVC) is performed using two electrodes: one to measure membrane potential and the other to apply the current, with each tip diameter <1 μm resulting in 10–100 MΩ resistances. For most other circumstances, a single electrode with an open tip diameter 1–3 μm (1–3 MΩ resistances) is used for whole cells or small patches of cell membrane recording. A typical patch-clamp recording starts with cell attaching procedures including: placing glass tip next to a cell, using gentle suction, drawing a piece of the cell membrane to the microelectrode tip, and then letting glass tip to form a high-resistance seal with the cell membrane (ideally >1GΩ, so-called “gigaseal”). By applying different following-up manipulations, the patch-clamp can be achieved in four configurations: cell-attached patch, whole-cell patch, inside-out patch, and outside-out patch, as shown in Figure 3.
Many other electrophysiological techniques have been developed for various applications, including extracellular recordings such as electroencephalography (EEG), electrocardiography (ECG or EKG), and electromyography (EMG) for clinical diagnosis; artificial lipid bilayer recording for studying activities of reconstituted ion channel proteins; automated patch clamp recording to enable high-throughput recordings; and optogenetics to employ light to switch on and off ion channel activities. The technology evolution has brought the ion channel research and drug discovery to a new era, which will be discussed in later part of this chapter.
Nav channels were the first ion channel family discovered back in 1952 [1, 4] and cloned in 1984 . Its pedigree spans across prokaryotic and eukaryotic kingdoms . To date, ∼500 Nav channels from bacteria (BacNavs) [8, 9, 10], and even more Nav channels from other species including fly , jellyfish , electric eel , cockroach , teleost fishes , and mammals have been cloned . The BacNavs regulate the survival response to extreme pH, electrophiles, and hypoosmotic shock. Despite their markedly difference in physiology function, voltage dependence and kinetics, BacNavs share common features of mammalian Nav channels, thus serving as surrogates in the study of molecular evolution and channel architecture. Eukaryotic Navs display ultrafast kinetics, with milliseconds activation to inactivation, and high sodium ion selectivity, Na+ (l): K+ (0.14): Rb+ (0.02): Cs+ (0.005), which together enable them being responsible to initiate and transduce fast action potential firing in the vast electrical signaling pathways throughout the cardiovascular and nervous systems.
Nav family belongs to the voltage-gated ion channel (VGIC) superfamily, with less intrafamily variation comparing to the other two VGIC families, voltage-gated potassium channels (Kvs) and voltage-gated calcium channels (Cavs). The core functional unit of Nav channel is a tetrameric complex composed of four homologous domains (DI/II/III/IV), with each domain containing six transmembrane segments (S1–S6), an intracellular N-terminus and C-terminus. The first four transmembrane segments form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). The central PD is responsible for ion-transduction through the structural top funnel, selectivity filter and gate, and all other domains are served as regulatory modlues for activation, fast and slow inactivation, albeit with different natures and structures.
Each eukaryotic Nav channel is composed of a single macromolecular α subunit (∼2000 amino acid residues, ∼260 kDa), forming a pseudoheterotetrameric core functional unit, in association with one or more auxiliary β subunits (β1, β2, β3, and/or β4, ∼35 kDa)  (Figure 4). While the prokaryotic Navs are formed by four separate α subunit, similar to Kv channels, representing a simpler construction than eukaryotic Navs. A typical Nav channel has at least three distinct states, resting (closed), activated (open), inactivated (closed), which itself includes fast-inactivated (within milliseconds) and slow-inactivated (seconds), and recovering from inactivation (repriming), which is a period in which the channel is not available to open in response to a depolarization. Each Nav channel can be characterized by these different voltage-dependent biophysiological preporties, and pharmacological perporties according to its expression pattern and modulation.
In human, there are 9 Nav channels (Nav1.1-1.9), which are encoded by the genes SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A, respectively. A tenth isoform, Nax, is considered as atypical, as it contains key difference in DI/III/IV S4 VSDs and DIII-IV inactivation linker, also it is activated by augmentation of extracellular sodium (over 150 mM) instead of voltage. Thus, Nax was classified as a different Nav subfamily (type 2) [16, 17]. Based on the timeline of gene cloining, each α subunit gene was assigned as SCN1A to SCN11A, and likewise, auxiliary β subunit genes were assigned as SCN1B to SCN4B, respectively (Table 1).
|hNav isoform||Gene||Variant(s)||Human chromosome locus||UniProt||Homology*||TTX IC50 (nM)||Primary tissue location||Therapeutic relevance|
|Nine voltage-gated α subunits|
|Nav1.2||SCN2A||2||2q23–24||Q99250||90.8%||12||CNS, glia||Epilepsy, autism|
|Nav1.5||SCN5A||6||3p21||Q14524||79.4%||2000||Cardiac muscle||Cardiac rhythm disorders|
|Nav1.6||SCN8A||5||12q13||Q9UQD0||85.9%||1||CNS, PNS, glia||Ataxia, motor neuron disease|
|One nonvoltage-gated α subunits|
|Navx||SCN7A**||1||2p21-23||Q01118||71.2%||PNS, DRG, epithelia|
|Four β subunits|
|Navβ1||SCN1B||2||19q13||Q07699||100%||CNS, PNS, muscle||Epilepsy|
Due to their fundamental role in regulating central and peripheral nervous systems function, skeletal muscle contraction and heart rhythm, much of the early works on Navs involved characterizing their expression patterns, biophysiological properties, structure-function, and molecular pharmacology.
Compared to Cav and Kv channel family, the rise of Nav family is relatively recent . The study of intron/exon organization suggested that Navs were evolved from the similarly structured Cav channels. This is supported by the finding that the four domains of Navs have higher similarity to their corresponding domains in the Cav channels than to each other. The ancestral Navs and Cavs genes might have evolved by two rounds of gene duplication, i.e., from an ancestral, single-domain Kv-like gene to a two-domain protein, then from a two-domain protein to a four-domain protein. This hypothesis is also in line with the observation that among the four domians of Navs, DI shares higher similarity with DIII, and DII shares higher similarity to DIV .
In choanoflagellate (the sister group of animals), the rapid long-distance communication among excitable cells is achieved at the emergence of Metazoa (represented by bilaterian animals and cnidarians) through the use of Nav channel [20, 21]. The gene organization, biophysical, and pharmacological properties of invertebrate sodium channels are largely similar to their mammalian counterparts, suggesting that the primordial Nav channels were established before the evolutionary separation of the invertebrates from the vertebrates, and evolution of Navs played a critical role in the emergence of nervous systems in animals [19, 22]. Of note, sodium selectivity might be acquired independently in BacNav and mammalian Nav channels as indicated by phylogenetic analysis . Therefore, BacNav channels can serve as models for studying Navs structure function, but evolutionary variation should be taken into consideration.
Historically, the tissue distribution of mammalian Nav isoforms was obtained by methods such as quantitative PCR, expressed sequence tag (EST) profiling, and pharmacology study using isoform selective toxins. The more precise expression data were recently obtained by microarray [24, 25] and mRNA sequencing from Genotype-Tissue Expression (GTEx) project  (Figure 5). Now, we know that Nav1.1, Nav1.2, and Nav1.3 are predominantly expressed in central nervous system (CNS). Nav1.1 is the predominant channel in the caudal regions and the spinal cord, though relatively low-level expression of Nav1.1 has been shown in the peripheral nervous system (PNS). Nav1.2 is the highest expression isoform in the rostral regions. Nav1.3 peaks at birth but remains detectable at a lower level in adulthood. Interestingly, all of these CNS-enriched isoforms are sensitive to tetrodotoxin (TTX) at nanomolar concentrations, and their genes are clustered on chromosome 2 in both mice and humans.
Nav1.4 and Nav1.5 exhibit strikingly high-level expression in muscle and heart, respectively. More sensitive approaches have detected Nav1.5 in the piriform cortex and limbic regions of the brain but at relatively low level . These two Nav isoforms can be distinguished from each other and from the CNS isoforms on the basis of toxin sensitivity. Adult skeletal muscle-enriched Nav1.4 is sensitive to TTX and μ conotoxin GIIIA at nanomolar concentrations, while the CNS-enriched channels are only sensitive to TTX, and heart-specific Nav1.5 is resistant to TTX. In addition, Nav1.4 is inhibited by nanomolar concentrations of μ conotoxin PIIIA, whereas Nav1.2 is approximately 15-fold less sensitive, and Nav1.7 is resistant [28, 29, 30].
Nav1.6 appears to be abundantly expressed in both PNS and CNS tissues. Nav1.7, Nav1.8, and Nav1.9 are primarily expressed in PNS, including nociceptive neurons, Aβ-fibers, C-fibers, and both large and small diameter dorsal root ganglion (DRG) . This PNS-specific localization of Nav1.7, Nav1.8, and Nav1.9 make them attractive targets for developing isoform selective modulators to treat many PNS-related diseases and pathologic conditions.
The core functional unit of Nav channels is α subunit. Each α subtype is composed of a single polypeptide chain with ∼2000 amino acid residues forming four pseudoheteromeric domains designated as DI to DIV. Nine α subunits’ protein sequence homology is greater than 70% (Table 1). The structure complexity and high-sequence homology make it difficult to design subtype-selective drugs. In 2011, the first X-ray crystal structure of bacterial Arcobacter butzleri Nav channels (NavAb) was determined . Subsequently, structures for several BacNavs and a BacNav-human Nav chimeric channel have been resolved, representing closed [33, 34], open [34, 35], and potentially inactive states of the channels [36, 37]. In 2017, the first two eukaryotic Nav structures for American cockroach and electric eel (Ee) Nav1.4-β1 complex were determined by using cryogenic electron microscopy (CryoEM) . Findings from crystal and cryo-EM structures are mostly consistent, and collectively provided important insights into Nav channel structure-function and structure-based drug design.
Voltage-sensing domains (VSDs). The S1 to S4 segments form voltage-sensing domain. There are four VSDs in a sodium channel. Each VSD is featured by repetitively occurring positively charged Lys or Arg residues at every third position in S4. These charge clusters move toward the extracellular surface upon membrane depolarization and return to their resting positions upon membrane repolarization. Each VSD is connected to an intracellular S4-S5 linker, which transfers the movement of S4 segment to the central pore domain formed by S5-S6. Thus, the outward and inward movements of S4 result in channel opening and closing, respectively. Unlike homotetrameric Kvs and BacNavs, mammalian Nav channel’s four VSDs possess distinctive sequence signature, conformation, and role in channel gating [12, 13]. For examples, the four VSDs have nonconserved intra- and extracellular loops, and the different charge clusters in S4, i.e., RRRR in DI, RRRRK in DII, KRRRR in DIII, and RRRRR in DIV. Also, the S2 in each VSD also makes asymmetric functional contributions to Nav channel activation and inactivation .
Pore domain (PD): the S5 to S6 segments from the four domains of the α subunits enclose the central pore of the channel. The extracellular linker connecting S5-S6 is defined as pore-loop (P-loop), which can be separated into two α helices named P1 and P2. The functional entities along the ion permeation pathway in PD include the selectivity filter (SF), the central cavity, and the intracellular activation gate, as shown in Figure 4E.
The outer vestibule and selectivity filter are formed in P-loop reentering membrane segments, designated as P1-SF-P2 funnel. Early study by comparing Navs with Cavs found that one residue, Asp/Glu/Lys/Ala (DEKA), at the corresponding locus in the middle of P1-P2 determines Na+ selectivity . Structure studies confirmed that the asymmetric selectivity filter vestibule is constituted by the side chains of the signature DEKA residues and the carbonyl oxygens atoms of the two preceding residues in each domain, Thr/Gln (DI), Cys/Gly (DII), Thr/Phe (DIII), and The/Ser (DIV). Mutational study identified an additional outer ring above the selectivity filter, Glu/Glu/Met/Asp (EEMD), which significantly interfered the tetrodotoxin (TTX) binding [40, 41]. The outer vestibule and SF structures were further discerned by using bacteria KcsA channel X-ray structure as template and guanidinium toxins (TTX and saxitoxin, STX), which successfully defined the first pharmacological relevant site on Nav channels, site 1 . After that, local anesthetic binding site was determined within the four fenestrations in PD, each with distinct shape and size [13, 43]. From studying a group of activators, including batrachotoxin (BTX), veratridine (VTD), grayanotoxin (GTX), and aconitine (ACD), the neurotoxin site 2 was determined in the inner cavity of PD [44, 45].
Activation gate: the activation gate of Nav channels was originally predicted to be at the inner end of the pore based on the study of local anesthetics, which exhibit usage-dependent blockage [46, 47]. From thiol-modifying reagent accessibility study, a ∼3.8 Å diameter constriction formed by a ring of conserved hydrophobic residues at the end of S6 (represented as DI-V440, DII-L795, DIII-I1287, DIV-I1590 in Nav1.4 and DI-Y405, DII-F960, DIII-F1449, DIV-F1752 in Nav1.7) was found to occlude only the pore at closed state but not at open state . Recent cryo-EM structures confirmed that this activation gate is located at the cytoplasmic boundary level of the membrane . Channelopathy study found that Nav1.7 DII S6 L955 deletion cause F960 side change conformational change in the activation gate. This mutation produces radial shift of the channel open at 25 mV more hyperpolarizing voltage, which renders hyperexcitability of DRG neurons to cause inherited erythromelalgia (IEM) .
Fast inactivation: while the channel opening is controlled by VSD and activation gate, the fast inactivation is regulated by a highly conserved Ile/Phe/Met (IFM) motif, which is localized in an intracellular loop connecting the domain DIII and DIV . The IFM motif was originally discovered as a particle segment attached to the inner end of the pore. Study showed that pronase and N-bromoacetamide removed inactivation only when applied from intracellular side , and acetyl-KIFMK-amide peptides restored fast inactivation . In the eukaryotic EeNav1.4-β1 structure at open and resting states, the LFM motif (equivalent to IFM) in DIII-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in DIII and DIV. Once the channel opens, the LFM motif acts as a hinged lid and folds into the intracellular mouth of the open pore to produce fast inactivation.
Slow inactivation: in contrast to fast inactivation (milliseconds scale), slow inactivation occurs in seconds during prolonged depolarization or rapid repetitive stimulations. Slow inactivation determines channel availability for action potential generation; thus, it endows neuronal tissues with memory of previous excitation, prevents excitation of skeletal muscle by mild hyperkalemia, and affects the conduction velocity and excitability of cardiac tissue. For the cardiac Nav1.5 channel, two putative proton sensor residuals in P-loop, C373 and H880, were responsible of tissue-acidification-induced slow inactivation underlying cardiac arrhythmia . Other mutations, including DII S4-S5 linker L689I in Nav1.4, DII S6 Del-L955 in Nav1.7, have also been identified to impair slow inactivation, causing hyperkalemic periodic paralysis and inherited erythromelalgia, respectively [53, 54]. Voltage-clamp fluorimetry (VCF) study of Nav1.4 channels showed that that immobilization of DI and DII VSDs is involved in the development of slow inactivation, while DIII VSD is involved in the recovery from slow inactivation . However, the structural basis for slow inactivation remains undefined.
In vertebrates, five Nav auxiliary β subunits, β1, β1B, β2, β3, and β4, have been identified (SCN1B to SCN4B), with molecular weight ranging from 30 to 40 kD (Table 1) [57, 58, 59, 60]. In invertebrates, auxiliary subunits such as tipE and Vssc β in drosophila bear no homology to vertebrate β subunits, suggesting a separate evolutionary pathway [61, 62, 63]. All β subunits comprise an amino terminal immunoglobulin (Ig) domain, a single transmembrane (TM) segment, and an intracellular carboxyl-terminal, except for β 1B (previously named β1A), which is a β1 splice variant that lacks TM segment . β1 and β3 interact with α subunit noncovalently, whereas β2 and β4 bind to the α subunit via a disulfide bond. All β isoforms are expressed in CNS, PNS, and cardiovascular systems, including excitable and nonexcitable cells, where they are part of the V-set immunoglobulin superfamily of cell adhesion molecules facilitating cell adhesion and cell migration. β1 is highly expressed in skeletal and cardiac muscles, and the expression patterns of all β isoforms vary during development . β subunits play broad role in modulating Nav function. They regulate expression and membrane trafficking of α subunits, modulate channel activation and inactivation, and interfere with toxin binding [66, 67, 68].
In 2017, the cryo-EM strucutre for EeNav1.4 in complex with the β1 subunit was determined . This structure provided a first glimpse into the interaction between α and β subunits. The β1 subunit interacts with the α subunits as an ax, wherein its TM interacts with VSDIII within the membrane as the handle, while its Ig domain as the head interacts with DI-L5 and DIV-L6 extracellular loops and the intervening segment between DIII S1 and S2 (Figure 4C and D). Mutations in β subunits have been linked to many human diseases, including epilepsy, and cardiac arrhythmia, and sudden death syndromes. Although β subunit-specific drugs have not yet been developed, the Nav β subunit family remains a potential therapeutic target .
Nav channels are fundamentally important in a broad spectrum of physiological processes. Not surprisingly, genetic mutations of Nav channels result in many debilitating to severe phenotypes in CNS, PNS, cardiac, and neuromuscular systems. To date, at least ∼50 human diseases have been attributed to aberrant activities of Nav channels; and hundreds of diseased related mutations of α and the β subunits have been identified. These mutations lead to channel dysfunctions called channelopathies (summarized in Table 2) and suggest the disease association of respective channels.
|Isoform||Channelopathies||Discovered mutant number||Discovery publication number|
|SCN1A||GEFS+2||Generalized epilepsy with febrile seizures plus 2||37||30|
|EIEE6||Epileptic encephalopathy, early infantile, 6||317||41|
|ICEGTC||Intractable childhood epilepsy with generalized tonic-clonic seizures||23||5|
|FHM3||Migraine, familial hemiplegic, 3||5||4|
|FEB3A||Febrile seizures, familial, 3A||2||2|
|SCN2A||BFIS3||Seizures, benign familial infantile 3||18||13|
|EIEE11||Epileptic encephalopathy, early infantile, 11||50||21|
|ASD||Autism spectrum disorders||16||1|
|SCN3A||CPE||Cryptogenic partial epilepsy||4||1|
|ADNSHL||Autosomal dominant nonsyndromic hearing loss||14||1|
|SCN4A||PMC||Paramyotonia congenita of von Eulenburg||16||16|
|HOKPP2||Periodic paralysis hypokalemic 2||10||12|
|HYPP||Periodic paralysis hyperkalemic||13||4|
|NKPP||Periodic paralysis normokalemic||3||3|
|MYOSCN4A||Myotonia SCN4A-related CMS16||14||13|
|CMS16||Myasthenic syndrome, congenital, 16||3||3|
|SCN5A||PFHB1A||Progressive familial heart block 1A||4||6|
|LQT3||Long QT syndrome 3||123||30|
|BRGDA1||Brugada syndrome 1||197||33|
|SSS1||Sick sinus syndrome 1 VF1||2||3|
|VF1||Familial paroxysmal ventricular fibrillation 1||1||1|
|SIDS||Sudden infant death syndrome||2||1|
|ATRST1||Atrial standstill 1||1||2|
|CMD1E||Cardiomyopathy, dilated 1E||1||2|
|ATFB10||Atrial fibrillation, familial, 10||9||2|
|MEPPC||Multifocal ectopic Purkinje-related premature contractions||3||4|
|SCN8A||EIEE13||Epileptic encephalopathy, early infantile, 13||44||15|
|BFIS5||Seizures, benign familial infantile, 5||1||2|
|SCN9A||IEM||Inherited erythermalgia (primary erythermalgia) pain||23||11|
|CIP||Congenital insensitivity to pain||13||1|
|PEPD||Paroxysmal extreme pain disorder||9||3|
|GEFS+7||Generalized epilepsy with febrile seizures plus 7||2||1|
|FEB3B||Febrile seizures, familial, 3B||2||1|
|SFN||Small fiber neuropathy||7||1|
|DS (SMEI)||Dravet syndrome (severe myoclonic epilepsy of infancy)||13||1|
|SCN10A||FEPS2||Episodic pain syndrome, familial, 2||2||1|
|SFN||Small fiber neuropathy||4||3|
|SCN11A||HSAN7||Neuropathy, hereditary sensory, and autonomic, 7||2||2|
|FEPS3||Episodic pain syndrome, familial, 3||7||4|
|SFN||Small fiber neuropathy||4||4|
|SCN1B||ATFB13||Atrial fibrillation, familial, 13||2||1|
|BRGDA5||Brugada syndrome 5||1||1|
|EIEE52||Epileptic encephalopathy, early infantile, 52||2||2|
|GEFS+1||Generalized epilepsy with febrile seizures plus 1||2||3|
|SCN2B||ATFB14||Atrial fibrillation, familial, 14||2||1|
|SCN3B||ATFB16||Atrial fibrillation, familial, 16||4||2|
|BRGDA7||Brugada syndrome 7||1||1|
|SCN4B||ATFB17||Atrial fibrillation, familial, 17||2||1|
|LQT10||Long QT syndrome 10||1||1|
Nav1.7, 1.8, and 1.9 are highly expressed in sensory neurons. They control the excitability of nociceptive neurons, and thus are considered as therapeutic targets for pain relief [31, 71, 72, 73, 74]. Among them, Nav1.7 was the first gene linked to human pain. In 2004, two missense mutations, I848T and L858H in SCN9A (Nav1.7), were associated with edema, redness, warmth, and bilateral pain in human inherited erythromelalgia (IEM) patients . In 2006, three nonsense mutations, S459X, I767X, and W897X, were identified in congenital insensitivity to pain (CIP) patients . Since then, additional gain-of-function mutations are associated with IEM, paroxysmal extreme pain disorder (PEPD), small fiber neuropathy (SFN), and additional loss-of-function mutations that are associated with CIP (Figure 6). The mechanism underlying these conditions was unraveled by characterizing biophysical properties of disease mutations  and nociceptor-specific Nav1.7 knockout . Together, these studies have established that Nav1.7 is an essential and nonredundant requirement for nociception in humans.
While Nav1.7 is responsible for setting threshold for generation of action potentials, Nav1.8 and Nav1.9 contribute to the rising phase of action potentials in nociceptive neurons. Both channels are expressed in small-diameter DRG neurons, which include the C fibers that transmit nociceptive impulses . Four gain-of-function Nav1.8 mutations (L554P, I1706V, A1304T, G1662S) have been identified, which lead to an increase in excitability in small-diameter neurons, underlying pain in small fiber neuropathy (SFN) [79, 80, 81]. In Nav1.9, gain-of-function mutations were recently reported to be associated with pain, albeit with opposing effects . So far, naturally occurring loss-of-function mutations of Nav1.8 and Nav1.9 are yet to be described in humans; Nav1.8 and 1.9 are clearly important in the pain pathology and worth exploring as potential pain targets.
Nav channel dysfunction is central to the pathophysiology of epileptic seizures, and many of the most widely used antiepileptic drugs, including phenytoin, carbamazepine, and lamotrigine, are Nav inhibitors. Many types of general epilepsy are resulted from mutations primarily in Nav1.1, and also in other Nav α isoforms, including Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav β1. GEFS+ type 1 results from Nav β1 mutation C387G, which destroys extracellular immunoglobulin domain and thus indirectly decreases rate of channel inactivation . GEFS+ type 2 results from Nav1.1 mutations, such as T875 M (in DII-S4) and R1648H (in DIV-S4), which decrease Nav1.1 inactivation rate directly . Worth noting, some epilepsy-assicated Nav1.2 and Nav1.6 mutations cause a gain-of-function when characterized in transfected cells [85, 86]. Better understanding of clinical genetics and channel structure-function will facilitate the drug development for Nav-associated neurological diseases.
Mutations of Nav1.5 have been linked to long QT syndrome (LQTS). Recent study suggests that even modest depression of Nav1.5 expression may promote pathologic cardiac remodeling and progression of heart failure . Since the first LQT-related Nav1.5 mutation being discovered in 1995 , more than a hundred Nav1.5 mutations that associate with distinct cardiac rhythm disorders, such as LQT syndrome subtype 3, Brugada syndrome, and cardiac conduction disease, have been identified . These Nav1.5 mutations are spread out the whole protein. Most Nav1.5 mutations change biophysical property by increasing persistent Na+ current or gain-of-function, while in Brugada syndrome and cardiac conduction disease, most of the mutations are missense loss-of-function mutations. Because many mutations produce overlapping clinical phenotypes, it is crucial to understand genotype-phenotype correlations in Nav1.5 channelopathies for drug development.
Mutations of Nav1.4 channel are associated with inherited neuromuscular diseases. For examples, the M1592V mutation causes hyperkalemic periodic paralysis (HYPP), in which increased levels of serum potassium lead to muscle hypoexcitability and paralysis; the R1448C mutation causes paramyotonia congenita (PMC), which is induced by cold and aggravated by increased muscle activity; and the G1306A mutation causes potassium-aggravated myotonias (PAM). These mutations affect either voltage-dependent activation or inactivation of Nav1.4, and are inherited in an autosomal dominant manner . In contrast, most Nav1.6 mutations are recessive. An allelic mutation A107T in DIII S4-S5 caused ataxic phenotype by shifting Nav1.6 activation and inactivation to about 14 mV in the depolarizing direction, suggesting Nav1.6 is required for the complex spiking of cerebellar Purkinje cells and for persistent sodium current in several classes of neurons [91, 92, 93].
Nav channels have long been recognized as targets for treating pain, neurological disorders, and cardiac arrhythmias. In nature, Nav channels are the molecular targets of a broad range of neurotoxins including tetrodotoxin (TTX), saxitoxin (STX), veratridine (VTD), and batrachotoxin (BTX) from marine bacteria and plants, as well as peptide toxins such as ProToxin (ProTX), Huwentoxin (HwTX and α-ScTxs) from the venoms of scorpions, spiders, sea anemones, and cone snails. Additionally, many Nav-targeting drugs have been developed, including local anesthesia (LA), antiarrhythmics (e.g., lidocaine, mexiletine), anticonvulsants (e.g., carbamazepine), and antidepressants (e.g., amitriptyline). In general, these drugs do not have subtype selectivity and have small therapeutic index. Recently, two series of highly isoform-selective compounds, aryl sulfonamides for Nav1.7 and phenyl imidazole for Nav1.8, have been reported [94, 95]. Besides small molecules, monoclonal antibody has also been proposed as an alternative strategy. Nonetheless, due to the high-sequence homology among all Nav isoforms, subtype selective targeting remains a challenge.
All nature or synthesized small and large modulators for Nav channels can be classified as pore blockers or gating modifiers. Pore blockers (e.g., TTX) physically occlude the pore, thereby inhibiting channel conductance. Often the blockade is tonic, or independent of states of the channels. Gating modifiers (aryl sulfonamides as example) preferentially modify activated or inactivated states, thus reducing currents progressively with increased stimulation duration and frequency.
Site 1: extracellular pore blocker. This site is formed by the four P-loops and represents the binding site of two known groups of pore blockers, including small molecular guanidinium toxins from marine bacteria (TTX and STX), and 17–25 amino acids’ peptide μ-conotoxins from marine cone snail toxins [96, 97, 98]. These toxins physically plug the pore and thereby inhibit the sodium conductance.
Site 2: intracellular pore gating activator (state dependent). The binding site is the fenestration site formed between DI-S6 and DIV-S6, which binds to many small lipid-soluble toxins including batrachotoxin (BTX) from frog, antillatoxin (ATX) from marine species, and veratridine (VTD), aconitine (ACT), and grayanotoxin (GTX) from plants. These modulators facilitate channel activation or prevent inactivation, thereby increasing channel conductance. They often have much higher affinity to the open and inactivated states of Nav channel as the binding fenestration exposed.
Site 3: extracellular gating activator. The binding site is localized in the DIV S3-S4 extracellular loop for two groups of peptide toxin activators: scorpion α-toxins (α-ScTxs) and sea. Upon binding, these modulators prevent the movement of DIV-S4, thus inhibiting transition to fast inactivation.
Site 4: extracellular gating blocker. The binding site is the DII S1-S2 and S3-S4 extracellular loop, which binds to four groups of peptide toxin blockers, including β-scorpion toxins (β-ScTxs), β-spider toxins, μO-conotoxins, and ι-conotoxins. In general, these toxins block the DII-VSD conformational change and shift the voltage dependence of activation toward more hyperpolarized potentials, though the structural-based mechanism of action is not clear.
Site 5: intracellular pore gating activator (state dependent). The fenestration site between DI-S6 and DIV-S5 binds two classes of cyclic polyether toxins from dinoflagellate: brevetoxins (PbTx) and ciguatoxins (CTX). These modulators have higher affinity to activated channel. Upon binding, they shift the activation and inactivation both to more hyperpolarizing voltage, thus keeping the channel hyperactive.
Site 6: extracellular gating activator. The DIV-S4 fenestration binds to δ-conotoxins causing similar effects as site 3 toxins by slowing or inhibiting inactivation. Although sites 6 and 3 are structurally close to each other, however, the two categories activators do not compete with each other . Site 6 activators trap DIV-S4 in outward conformation, thus leading to a persistent activation and prolongation of action potential.
Site 7: local anesthetic (LA) binding site. The inner cavity of channel pore, consisting of amino acid residues in S6 of DI, DIII, and DIV, forms the LA binding site. The LA binding site is highly conserved across Nav channels and accounts for the lack of subtype selectivity for most clinically used sodium channel blockers.
Site 8: extracellular gating blocker. The site was identified by studying a class of cone snail μO§-conotoxins blockers, such as μO§-GVIIJ. The putative binding site is close to a cysteine near DII P-loop (C910 in rNav1.2), which is responsible of the antagonist effect .
Site 9: aryl sulfonamide site. The unique Nav1.7 VSD4 binding site for the new series of aryl sulfonamide compounds, such as ICA-121431, PF-04856264 .
Site 10: phenyl imidazole site. The unique fenestration-selectivity filter site in Nav1.8 for the new series of phenyl imidazole compounds, such as A-803467, PF-04531083 .
Voltage-gated sodium channels play essential roles in physiological function, and historically, sodium channel blockers have been developed as local anesthetics and anticonvulsants. However, these early generation of sodium channel drugs were developed decades ago without the exact understanding of their molecular targets and mechanisms of action; their general lack of on-target potency and off-target selectivity renders narrow therapeutic windows. In recent years, several scientific frontiers have been rapidly evolving. First, the physiological functions of each sodium channel have been determined. Second, their associations to human diseases have been revealed in the form of “channelopathies.” Often both gain-of-function and loss-of-function mutations have been linked to human diseases, therefore pinpointing exactly the molecular targets. Third, the structural determination of sodium channels provides opportunities for structural-based drug design. Together, these progresses have ushered in a new, exciting era of sodium channel drug discovery.
We gratefully acknowledge contributions from Genentech Inc. to support this publication.
The authors are all employees of Genentech Inc. (a Roche Group Company) and declare no financial and conflict of interest in this chapter.
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Edited by Fatima Shad Kaneez
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Edited by Fatima Shad Kaneez
By K. Fatima-Shad and K. Bradley
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