Introduction

Neurons and myocytes communicate by means of electrical signals, action potentials, that are propagated along their cell membranes as controlled deflections of the membrane potential. Action potentials are a result of coordinated alterations in cell-membrane conductance in excitable cells caused by controlled openings and closings of voltage-gated ion channels. Action potentials can reach propagation velocities of more than 100 m/s in the body periphery and are the basis for fast information transfer along neurons and myocytes. Voltage-gated sodium channels (Na_V channels) initiate action potentials and are responsible for their directed propagation (Catterall, 1992). The tailor-made properties of Na_V channels, their voltage-dependent opening, fast inactivation, and high sodium-ion selectivity had been described as early as 1952 by Hodgkin and Huxley, on the basis of electrophysiological experiments in neurons. Owing to the role of Na_Vchannels in electrical signaling it is not surprising, that malfunction of these channel proteins caused by defects in channel-coding genes lead to a variety of serious neurological and muscular diseases (George, 2005; Eijkelkamp et al., 2012).

Defects in Na_V channels predominantly expressed in peripheral afferents have a significant impact on the pain phenotype of affected individuals, the spectrum of which is varying from complete analgesia to chronic pain.

Structure and function of voltage-gated sodium channels

Na_V channels are highly processed tissue-specific protein complexes (Fig. 1), that comprise a large (ca. 260 kDa) pore-forming α-subunit and up to two smaller (ca. 30−40 kDa) accessory β-subunits (Catterall, 2000). The α-subunits alone are sufficient to form functional channels, whereas the β-subunits exhibit regulatory functions. In humans, nine genes (SCN1A−5A, SCN8A−11A) are coding for the homologous α-subunits Na_V1.1–Na_V1.9, and four genes (SCN1B−4B) are coding for the β-subunits Na_Vβ1−Na_Vβ4. According to phylogenetic studies, the individual genes encoding the α- and β-subunits have evolved very early in vertebrate evolution from common precursor genes by repeated gene duplication (Plummer and Meisler, 1999; Goldin, 2002).

The α-subunit of mammalian Na_V channels consist of a single polypeptide chain which can contain more than 2.000 amino acids spanning the cell membrane 24 times (Fig. 1b, c). Six consecutive transmembrane helices each (S1–S6) combine to one of the four homologous domains (DI−DIV), that arrange in a clockwise manner to form a pseudo-tetramer with a central Na⁺-selective pore (Sato et al., 1998). The transmembrane helices S5 and S6 of each domain represent the pore-forming structures. They line the pore of the channel and enclose the Na⁺-selectivity filter, which consists of the conserved amino acids aspartate, glutamate, lysine and alanine (DEKA-motif) within the S5/S6-pore loop (Heinemann et al., 1992). The transmembrane helices S1–S4 of each domain assemble to movable voltage sensors, with the actual gating charges, positively charged amino acids, located in the S4-helices (Stühmer et al., 1989). The voltage sensors translate changes in the membrane potential into conformational changes that open the channel pore in response to a depolarization and close the pore during hyperpolarization. The activation gate of Na_V channels is formed by a bottleneck at the intracellular site of the pore that is gated by voltage sensors (Shen et al., 2017).

The fast inactivation of Na_V channels occurs in the millisecond-range and is mediated by the conserved hydrophobic amino acid motif isoleucine-phenylalanin-methionin (IFM-motif) in the DIII/DIV linker which translocates upon channel activation to occlude the intracellular vestibule of the channel pore (West et al., 1992). A disparate slow type of inactivation involves reversible conformational changes of the channel pore and is characterized by a slow recovery to a resting conformation (Vilin and Ruben, 2001).

The β-subunits of Na_V channels are typical type-1 transmembrane proteins. They possess a large extracellular N-terminus and an intracellular C-terminus both connected to a single transmembrane helix. By means of their extracellular domains that exhibit structural similarities to immunglobulines, they make contact to extracellular matrix components and mediate immobilization and clustering of the channel complexes (Xiao et al., 1999). Depending on the composition of Na_V complexes, β-subunits also enhance membrane localization of Na_V α-subunits and modulate their gating (Chen and Cannon, 1995; Yu and Catterall, 2003; Brackenbury and Isom, 2011).

Na_Vchannels in nociceptive afferents

Painful stimuli are detected by free nerve endings of specialized neurons, so-called nociceptors, that generate action potentials to transmit the information along axons toward the spinal cord and brain. The somata of nociceptors are located in the dorsal root ganglia (DRGs) along the spinal cord, sending their sensory afferents into the respective body segments. Two different types of pain-related afferents can be distinguished: Fast conducting myelinated Aδ-fibers and thin, slowly conducting and unmyelinated C-fibers.

Adult nociceptors mainly express Na_V subtypes Na_V1.7, Na_V1.8, and Na_V1.9 (Fig. 1a). However, Na_V expression profiles differ in Aδ- and C-fibers as Na_V1.7 and Na_V1.8 channels are found in both Aδ- and C-fibers, whereas Na_V1.9 channels are mainly expressed in C-fiber neurons (Akopian et al., 1996; Djouhri et al., 2003).

Despite of their pronounced sequence homology, Na_V1.7−1.9 exhibit distinct properties with respect to their voltage-dependent opening and their gating kinetics (Fig. 1b). Due to their functional differences they serve specific and in part non-redundant functions within nociceptors. Na_V1.7 channels act as threshold channels and are mainly involved in the initial phase of transduction. They open in response to sub-threshold depolarizations and increase the membrane potential to its specific threshold (Rush et al., 2007). After the threshold potential is reached, Na_V1.8 channels open quickly (< 100 µs) and the associated inward Na⁺ current is initiating an action potential (Renganathan et al., 2001; Blair and Bean, 2003). Subsequently, the repolarization phase of the action potential is triggered by the IFM-dependent inactivation of Na_V channels combined with the efflux of K⁺ ions mediated by voltage-gated potassium channels. In contrast to Na_V1.7 and Na_V1.8, Na_V1.9 channels are active near the resting membrane potential (ca. -60 mV) and exhibit a comparatively slow inactivation kinetics. Due to these functional characteristics Na_V1.9 channels can most likely not directly trigger action potentials. It rather seems they regulate the excitability of C-fiber neurons by modulating the level of the resting membrane potential relative to the action potential threshold (Herzog et al., 2001).

Fig. 1

Structure and properties of voltage-gated sodium channels.aThe family of human Na_Vchannels consists of nine tissue-specific α-subunits (Na_V1.1–1.9) which show a high level of sequence conservation. The pain-relevant subtypes Na_V1.7–1.9 are highlighted.bRepresentative current responses of Na_V1.7–1.9 at a membrane potential of -20 mV. As shown, these subtypes differ from each other with respect to their activation and inactivation kinetics.c Cartoon showing the membrane topology of Na_Vchannels with the four domain-structure (DI–DIV) of the channels’ α-subunit. The positive gating charges in the S4 segments as well as the inactivation motif, IFM, are indicated.d Top view of the three-dimensional structure of the sodium channel α-subunit Na_VPaS (5X0M.pdb) fromPeriplaneta americana (Shen et al., 2017). A high degree of sequence conservation between Na_VPaS and mammalian Na_Vchannels suggests a similar structure for human Na_Vchannels. The model contains the 4 homologous domains without the connecting intracellular linkers. Labels in DI mark individual transmembrane segments (S1–S6), colors as in c.

Na_V-associated pain disorders

Gene mutations interfering with the function or expression of channel subtypes Na_V1.7–1.9 cause diseases, that impact impulse generation in peripheral nerves and thus the propagation of painful stimuli. Although these diseases are rare (prevalence < 1 : 10.000), they provide profound insights into human pain physiology and highlight the potential of Na_V channels as target molecules for future treatment strategies of acute and chronic pain.

Na_V1.7

To date, more than 40 mutations were identified in SCN9A, the gene encoding Na_V1.7, and analyzed in view of their consequences for channel function. Most mutations follow an autosomal dominant inheritance pattern and confer gain-of-function properties to Na_V1.7 causing hyperexcitability of nociceptors (Dib-Hajj et al., 2013). Hyperactivity of Na_V1.7 channels is associated with two clinically distinguishable pain disorders, namely primary erythromelalgia (PE, OMIM 133020) and the paroxysmal extreme pain disorder (PEDP, OMIM 167400). Both conditions are characterized by painful attacks but differ with respect to the body regions involved: PE-associated pain attacks occur predominantly symmetrically in both feet and lower legs of patients and are accompanied by flushing and hyperthermia of the affected areas. Exertion, such as standing upright for longer periods of time or exposure of body extremities to elevated temperatures can provoke and aggravate PE-dependent pain. Usually, first symptoms appear in childhood or early adulthood and increase with age. By contrast, PEPD is characterized by painful episodes in rectal, ocular and submandibular regions that are accompanied by flushing of affected body areas. First symptoms of PEPD appear typically during the neonatal phase or early childhood.

Both diseases correlate with specific functional alterations of Na_V1.7 channels (Dib-Hajj et al., 2013). PE is caused by mutations that shift the voltage-dependence of activation of Na_V1.7 to more hyperpolarized potentials, and thus lower the activation threshold of the channels. The result of this gain-of-function is a reduced excitation threshold and increased action potential firing rates of nociceptors. On the other hand, PEPD-associated mutations increase the effective availability of Na_V1.7 channels primarily by impairing channel inactivation. It is not clear yet why these distinct channel malfunctions induce disparate pain phenotypes – even though they both increase nociceptor excitability.

Congenital insensitivity to pain (CIP, OMIM 243000) is another phenotype associated with Na_V1.7 and is caused by the biallelic loss-of-function of SCN9A (Cox et al., 2006). As Na_V1.7-dependent CIP follows a recessive inheritance pattern, it is a rare disorder with less than 30 documented cases worldwide. Affected individuals do not have any cognitive deficits but suffer from repeated and unrecognized trauma, such as bites in lips and tongue, painless fractures, contusions, cuts and scalds. In addition, patients are anosmic suggesting a non-redundant function of Na_V1.7 channels in the olfactory system (Weiss et al., 2011).

Fig. 2

Functional alterations of Na_V1.9 channels in mutant p.V1184A associated with cold-aggravated peripheral pain.aVoltage-dependencies of activation and inactivation of Na_V1.9 and Na_V1.9-V1184A channels. Mutation p.V1184A enhances the activity of Na_V1.9 channels by shifting the voltage-dependence of channel activation to more hyperpolarized voltages and by reducing the voltage-dependence of channel inactivation. Colored areas indicate voltage ranges of basal channel activity (window current) generated by the overlap of channel activation and inactivation.bResting membrane potential (RMP) of murine DRG neurons expressing either human Na_V1.9 or Na_V1.9-V1184A channels.c Representative action potentials obtained from murine DRG neurons expressing either Na_V1.9 or Na_V1.9-V1184A channels in response to current injections of 60 pA for periods of 2 s. As demonstrated, Na_V1.9-V1184A channels increase the firing frequency of the neurons. Data ina-care from Leipold et al. (2015).

Na_V1.8

Na_V1.8 channels are less susceptible to slow inactivation as compared to other Na_V subtypes, i.e. they recover rather quickly from inactivated states. Thus, Na_V1.8 channels support repetitive action potential firing of nociceptors as required for fast transmission of sensory information (Han et al., 2015). Furthermore, the channels are resistant to cold temperatures, ensuring action potential firing and pain transmission along nociceptive fibers at low temperatures (Zimmermann et al., 2007).

All mutations in the Na_V1.8-encoding gene SCN10A known to date are associated with the broad spectrum of small fiber neuropathies (SFN) (Faber et al., 2012; Huang et al., 2013; Dabby et al., 2015). Known mutations confer proexcitatory properties to Na_V1.8 either by shifting the voltage-dependence of channel activation to more hyperpolarized potentials or by enhancing the recovery of the channels from inactivated states. As a result, affected nociceptors are rendered hyperexcitable.

One phenotype associated with hyperactive Na_V1.8 channels is referred to as familial episodic pain syndrome 2 (FEPS2, OMIM 615551) and characterized by adult-onset spontaneous burning pain and itching in the lower extremities but also in the hands of affected individuals. The symptoms can be accompanied by allodynia and hyperalgesia in the extremities, the intraepidermal density of myelinated as well as unmyeilinated nerve fibers might be strongly reduced.

Na_V1.9

The contributions of Na_V1.9 channels to nociception were first studied in mice harboring a global knock-out of the associated scn11a gene and later in rats utilizing a RNA-mediated knock-down strategy (Priest et al., 2005; Amaya et al., 2006; Lolignier et al., 2011). Both approaches led to reduced inflammatory hyperalgesia in the animals and provided evidence for a significant contribution of Na_V1.9 to inflammatory pain states. Apparently, the underlying mechanism depends on inflammatory mediators, such as bradykinin, histamine, norepinephrine and ATP, which increase nociceptor excitability by stimulating the activity of Na_V1.9 via G protein-dependent pathways (Maingret et al., 2008; Ritter et al., 2009). Heterologously expressed human Na_V1.9 channels are regulated in a similar way by G protein-dependent processes suggesting that Na_V1.9 contributes to human inflammatory pain as well (Östman et al., 2008; Vanoye et al., 2013).

Further insights into the role of Na_V1.9 channels in pathological pain states were obtained from patients with gain-of-function mutations in the SCN11A gene. To date, about 10 of these mutations have been functionally characterized.

The familial episodic pain syndrome 3 (FEPS3, OMIM 615552) was associated with mutations identified in the SCN11A gene of several members of two Chinese families (Zhang et al., 2013). The disease is characterized by an onset in early childhood, intense pain episodes that occur predominantly in distal lower extremities and occasionally in distal upper extremities as well. Pain attacks are augmented by fatigue and can last for several days. Pain episodes can be accompanied by autonomous symptoms such as hyperhidrosis and can be partially controlled with anti-inflammatory medication. The pain episodes tend to decline with age.

Gain-of-function mutations in SCN11A were also found in a larger cohort of patients with painful peripheral neuropathy (Huang et al., 2014).Beginning at the age of about 40 years affected individuals showed signs of peripheral neuropathy, such as numbness, prickling and pain in their feet and hands, often accompanied by autonomic dysfunctions like hyperhidrosis, diarrhea or dry mouth and eyes. Intraepidermal nerve density ranged from normal to slightly reduced.

The temperature dependence in pain sensation of affected individuals is distinct from that of PE patients. Na_V1.9-dependent pain episodes can be triggered and aggravated by cold while pain attacks can be alleviated by warming of effected body regions (Huang et al., 2014; Leipold et al., 2015; Okuda et al., 2016). The functional analysis of the gain-of-function mutation p.V1184A in Na_V1.9, identified in patients with cold-aggravated peripheral pain, revealed that hyperactive Na_V1.9 channels can diminish the cold-dependent attenuation of nociceptor activity which is in line with the patients’ temperature dependence of pain sensation (Leipold et al., 2015). Thus, hyperactive Na_V1.9 channels may support cold-dependent pain possibly by provoking overshooting responses of cold-sensitive nociceptors.

In contrast, a second group comprising three known SCN11A mutations causes hereditary sensory and autonomic neuropathy 7 (HSAN 7, OMIM 615548), a disease associated with congenital analgesia (Leipold et al., 2013; Phatarakijnirund et al., 2015; King et al., 2017). All three mutations lead to almost identical symptoms in affected individuals, such as self-mutilation of lips and tongue, skin lesions and malposition of extremities as a consequence of unrecognized painless fractures. Further symptoms include mild muscle weakness, hyperhidrosis as well as disturbances of intestinal motility that can require parenteral nutrition.

The underlying mutations cause complex gain-of-function alterations of Na_V1.9 channels. The analgesia-associated mutation p.L811P enhances the activation of Na_V1.9 channels by hyperpolarizing the voltage-dependence of channel activation and decelerating the channels’ IFM-mediated inactivation (Leipold et al., 2013). Analysis of murine nociceptors expressing mutant channels revealed that p.L811P channels only cause initial hyperexcitability of the cells, but support augmentation of nociceptor excitability during extended activity periods (Leipold et al., 2015). Furthermore, a gain-of-function mechanism on the level of the channel protein was also demonstrated for mutation p.L396P (King et al., 2017). However, the third mutation associated with analgesia, p.L1302F (Phatarakijnirund et al., 2015), has not been characterized functionally so far. It is noteworthy that all three analgesia-associated amino acid substitutions are located in the distal ends of transmembrane helices S6 of DI (p.L396P), DII (p.L811P) and DIII (p.L1302F), and thus are close to the activation gate of Na_V1.9 channels.

Na_Vs as targets in pain therapy

Despite the explicit role of Na_V channels in the pathogenesis of pain in individuals with mutation in channel-associated genes, Na_V channel blockers exhibit only variable and often very limited clinical effects in those and in other pain diseases. Traditional non-selective Na_V blockers like local anesthetics, anti-epileptics, and some anti-depressants bind within the channel pore and show only poor subtype-selectivity due to the high sequence conservation found in this binding region. Thus, the narrow therapeutic window and the risk of severe cerebral and cardiac side effects significantly limit their therapeutic utility.

In general, an adequate target control may well lead to an effective mono-therapy with non-selecitve Na_V blockers. This is shown by the satisfying effectiveness of carbamazepine (CBZ) in patients with PEPD. CBZ reduces the persistent current of Na_V1.7 mutations and shifts the voltage dependence of fast inactivation in the hyperpolarizing direction. Additionally, some reports describe a sufficient effectiveness of mexiletine or CBZ in some but not all patients with PE.

The putative essential and non-redundant requirement of Na_V1.7 for nociception in humans has triggered an extensive search for selective Na_V blockers, of which only few substances have found their way into clinical studies. The most promising candidates at present are semi-selective and activity-dependent Na_V blockers (Zakrzewska et al., 2013), peptide derivatives of the spider toxin protoxin-II, that stabilize the closed state of the channel (Flinspach et al., 2017) as well as small subtype-selective molecules that stabilize the domain IV voltage-sensor in its activated conformation, thus effectively reducing the activity of Na_V1.7 (Ahuja et al., 2015). It is unclear yet, whether or not one of these substances will reach the market.