The NaV1.7 Sodium Channel: From Molecule to Man*

Voltage-gated sodium channels are essential for electrogenesis in excitable cells. Nine pore-forming α-subunits of such channels (referred to as channels hereinafter), Na_V1.1–Na_V1.9, have been identified in mammals.¹ These isoforms share a common overall structural motif (figure 1). They are each composed of a long poly-peptide (1,700–2,000 amino acids) that folds into four homologous domains (DI–DIV) that are linked by three loops (L1–L3), with each domain having six transmembrane segments (S1–S6).² The recent determination of the crystal structure of the homotetrameric bacterial sodium channel ³ has provided insights into the atomic structure of mammalian sodium channels and the interactions between the voltage-sensing domain (VSD; encompassing S1–S4) and the pore module (PM; S5–S6) within each of the four homologous domains. Genetic, structural and functional studies have shown that Na_V1.7 regulates sensory neuron excitability and is a major contributor to several sensory modalities, and have established the contribution of this sodium channel isoform to human pain disorders (figure 1).

The nine sodium channel isoforms display different kinetics and voltage-dependent properties.¹ Their differential deployment in different types of neurons endows these cells with distinct firing properties. Sodium channels associate with multiple protein partners that regulate channel trafficking and gating,⁴^–⁷ allowing sodium channel properties to be modulated in a cell-type-specific manner (for examples, see refs. 8–10), highlighting the need to study these channels within their native neuronal background whenever practicable. For example, the pathogenic G616R variant of Na_V1.7 displays gating abnormalities within dorsal root ganglion (DRG) neurons that are not seen when these channels are expressed in HEK 293 cells.¹¹ Methods are now available that allow the expression and functional profiling of sodium channels in peripheral neurons, which more closely mimic the in vivo environment of such channels.¹²

In humans, gain-of-function mutations in SCN9A, which encodes Na_V1.7, lead to severe neuropathic pain, whereas loss-of-function mutations in this gene lead to an indifference to pain.¹³ Studies involving animal injury models and functional studies of neuronal excitability following expression of human mutant Na_V1.7 have provided mechanistic insights into the role of this channel in the pathophysiology of pain. Additional studies have linked NA_V1.7 to other sensory modalities, including olfaction,¹⁴^,¹⁵ the afferent limb of the cough reflex ¹⁶ and acid sensing.¹⁷

In this Review, we discuss functional and modeling studies of Na_V1.7 that have yielded new insights into the structure–function relationship of gating mechanisms in this channel and its contribution to neuronal responses under normal and pathological conditions. We also explore strategies for targeting Na_V1.7 in the treatment of pain and, finally, identify unanswered questions regarding the role of Na_V1.7 in pain signaling.

Roles in Pain States

Na_V1.7 in Acquired Pain Conditions

Na_V1.7 has an important role in pain signaling.¹³^,⁵¹ Axotomy of peripheral axons can produce a neuroma in which ectopic impulses arise, causing spontaneous pain.⁵² Application of TTX at concentrations that block only TTX-sensitive channels ameliorates neuropathic pain behavior in a rat axotomy model,⁵³ suggesting that these channels contribute to spontaneous pain. Although the TTX-sensitive sodium channel Na_V1.3 has been implicated in ectopic firing and spontaneous pain,¹³ Na_V1.7 accumulates at nerve endings within neuromas together with activated mitogen-activated protein kinase 1 (MAPK1; also known as ERK2) and MAPK3 (also known as ERK1) in humans ⁵⁴ and in rats.³¹ MAPK1 and MAPK3 phosphorylate Na_V1.7 at four sites within L1, producing a graded hyperpolarizing shift of channel activation. The extent of graded hyperpolarizing shift in Na_V1.7 activation depends on the number of phosphorylated residues.⁵⁵ Together with the finding that MAPK1 and MAPK3 exert a pro-excitatory effect on DRG neurons,⁵⁵ these data suggest that Na_V1.7 can contribute to injury-mediated DRG neuron excitability.

Na_V1.7 expression levels and TTX-sensitive current density are increased in DRG neurons in response to inflammation.⁵⁶ The increase in Na_V1.7 expression levels is more robust than that of Na_V1.3—the other TTX-sensitive channel that is upregulated under these conditions.⁵⁶^,⁵⁷ Na_V1.7 levels in DRG neurons are also increased in diabetic rats,⁵⁸^,⁵⁹ a change that is predicted to contribute to hyperexcitability associated with pain. A direct contribution of Na_V1.7 to pathological DRG neuronal hyperexcitability is further supported by knockdown and knockout studies in rodents. Knockdown of Na_V1.7 attenuates complete Freund’s adjuvant-induced thermal hyperalgesia ⁶⁰ and diabetic pain.⁶¹ Conditional knockout of Na_V1.7 expression in mouse DRG neurons, where Na_V1.8 is expressed, abrogates inflammation-induced and burn injury-induced thermal hyperalgesia, but does not impair mechanical allodynia or hyperalgesia (neuropathic pain).⁴³^,⁶²^,⁶³ However, a recent report⁶² provided evidence suggesting that knocking out Na_V1.7 expression in both DRG and sympathetic neurons abrogated neuropathic pain (box 2).

Box 2
Na_V1.7 in sympathetic neurons and pain signaling

The contribution of Na_V1.7 to electrogenesis in sympathetic neurons and the contribution of these neurons to pain are not well understood. Although Na_V1.7 is normally expressed in sympathetic neurons,¹⁸ individuals with Na_V1.7-related complete insensitivity to pain (CIP) do not report sympathetic deficits,⁶⁶ suggesting that the role of Na_V1.7 in these neurons might be redundant. Gain-of-function mutant Na_V1.7 in patients with severe pain can depolarize the resting membrane potential of dorsal root ganglion (DRG) neurons and sympathetic neurons. The resulting effect renders DRG neurons hyperexcitable and sympathetic neurons hypoexcitable,¹⁰ suggesting that severe pain may still occur even when sympathetic neuron excitability is reduced. However, studies in mice suggest that functional features of both sensory and sympathetic neurons, which are dependent on Na_V1.7, contribute to the manifestation of pain symptoms.⁴³^,⁶² Minett et al.⁶² reported that knocking out Scn9a (the gene encoding Na_V1.7) in DRG neurons alone does not cause a total loss of pain, whereas knocking out the expression of this channel in both sensory and sympathetic neurons recapitulates features of human CIP. Future studies are needed to investigate the role of Na_V1.7 in sympathetic neurons and pain signaling.

Na_V1.7 in Inherited Pain Disorders

The co-segregation of a familial mutation and disease symptoms in more than one generation provides a compelling case for a direct link between a target gene and a disease. Recently, mutations in SCN9A that alter the functional properties of Na_V1.7 in a pro-excitatory manner have been shown to produce familial pain disorders that follow a Mendelian inheritance pattern (inherited sodium channelopathies). These findings provided a causative link in these pain disorders and confirmed that Na_V1.7 has a central role in pain signaling in humans. Dominantly inherited gain-of-function missense mutations in SCN9A are found in individuals with IEM ⁶⁴ and paroxysmal extreme pain disorder (PEPD; previously known as familial rectal pain; OMIM #167400).⁶⁵ By contrast, recessively inherited loss-of-function mutations in SCN9A are linked to complete insensitivity (indifference) to pain (CIP; OMIM #243000).⁶⁶ Functional characterization of these gain-of-function mutations has elucidated the patho-physiological basis for DRG neuron excitability in these disorders, establishing a mechanistic link to pain.

Pain in IEM is localized to the feet and hands, and symptoms of this condition usually appear in early childhood.¹³^,⁵¹ Multiple families with IEM carry mutations in SCN9A that segregate with disease in affected individuals, providing strong genetic evidence for the pathogenicity of these mutations (figure 1). The familial IEM mutations in SCN9A that have been characterized to date all shift the voltage-dependence of Na_V1.7 activation in a hyperpolarized direction (figure 3a), increase ramp current (figure 3b) and slow deactivation. IEM-linked SCN9A mutations can impair slow inactivation (figure 3c), thus enhancing DRG neuron hyperexcitability,⁶⁷ whereas other IEM mutations enhance slow inactivation and therefore attenuate DRG neuron excitability.⁶⁸

Figure 3 Biophysical properties of wild-type and mutant Na_V1.7 channels. (a) Inherited erythromelalgia (IEM)-related *SCN9A* mutations shift the activation of Na_V1.7 in a hyperpolarized direction, allowing the mutant channels to open in response to a weaker depolarization than open wild-type (WT) channels. A comparison of the activation of WT and P1308L mutant Na_V1.7 channels ⁷³ shows that the latter exhibits a hyperpolarizing shift (–9.6 mV) in activation. (b) Activation of Na_V1.7 boosts small, slow depolarizations, producing ramp currents. The ramp currents produced by the IEM I136V mutant Na_V1.7 channel,¹⁴⁰ normalized to maximal peak currents elicited by step depolarizations, are markedly increased compared with the ramp currents for WT Na_V1.7 channels. (c) The slow-inactivated state of Na_V1.7 makes these channels unavailable for further opening after they have been activated by sustained (>10 s) membrane depolarization. Mutations in *SCN9A* that impair slow inactivation (such as N395K and I739V) increase the firing rate of dorsal root ganglion (DRG) neurons.⁶⁷^,¹⁴¹ Error bars represent standard error of the mean. (d) Fast inactivation is a process that transiently makes Na_V1.7 unavailable for further opening after it has been activated by relatively short (100–500 ms) depolarizations. A hallmark of paroxysmal extreme pain disorder (PEPD)-related *SCN9A* mutations is that they cause a depolarizing shift in fast inactivation that results in fewer inactivated channels at any given potential, and resistance of a subpopulation of channels to inactivation. The PEPD G1607R mutant Na_V1.7 channel ⁷⁰ shows a –30 mV depolarizing shift in fast inactivation, and the presence of a subpopulation of channels that resist inactivation (represented by orange shading in the graph). Error bars represent standard error of the mean. (e) Normalized current traces for WT and G1607R Na_V1.7 evoked by a depolarizing pulse to 0 mV show the transient current (I_Na-trans) and that the mutant channels retain a persistent current (I_Na-per) at the end of a 100 ms depolarizing pulse (represented by orange shading in the graph). (f) Resurgent currents (I_Na-res) are triggered by repolarization following a strong depolarization, and support burst firing. Note the increase in resurgent current recorded from DRG neurons expressing the M932L/V991L Na_V1.7 variant from a patient with small-fibre neuropathy.³⁹ Impaired fast- and slow-inactivation and resurgent currents are manifested by PEPD and SFN channel variants, as indicated in the main text, and the panels in this figure should be regarded as examples of these changes. Part a is modified from ref. 73. Part b is modified from ref. 140. Part c is modified, with permission, from ref. 67 © (2007) The Physiological Society. Parts d and e are modified, with permission, from ref. 70 © (2011) Macmillan Publishers Limited. All rights reserved. Part f is modified, with permission, from ref. 39 © (2012) American Neurological Association.

Another distinct set of mutations in SCN9A underlies PEPD, in which severe perirectal pain typically starts in infancy.⁶⁵ The rectal pain is accompanied with skin flushing of the lower or upper body or face and can present in a harlequin pattern,⁶⁹ which can alternate between the left and right sides of the body during different pain episodes.⁷⁰ PEPD-linked SCN9A mutations produce different effects on Na_V1.7 gating compared with IEM-associated mutations.¹³^,⁶⁵ PEPD-linked SCN9A mutations shift the voltage-dependence of steady-state fast inactivation toward a depolarizing direction (figure 3d) and, depending upon the specific mutation, make channel inactivation incomplete, which results in a persistent current (figure 3d,e). PEPD, but not IEM, mutant Na_V1.7 manifests increased resurgent currents³⁸ (figure 3f).

The IEM-linked SCN9A mutations studied to date¹³ lower the threshold for single action potentials (figure 4a–c) and increase the frequency of firing in DRG neurons (figure 4d–f), with many IEM-linked SCN9A mutations causing a depolarizing shift in resting potential.¹³ At the cellular level, PEPD mutant Na_V1.7 lowers the threshold for single action potentials and increases the frequency of firing in DRG neurons, without altering the resting potential.⁷¹^–⁷³ Importantly, these functional profiles have been obtained by recordings from the somas of DRG neurons. It will be important, in the future, to assess the properties of these mutant channels and their effect on excitability near nerve terminals where Na_V1.7 is thought to exert its influence as a threshold channel. A recent study from our group ⁷⁴ has begun to address this point, demonstrating a resting potential for DRG neurites close to −60 mV, a potential at which Na_V1.7 channels are not strongly inactivated and are available for activation in fine diameter axons of DRG neurons. This study also demonstrated that action potential electrogenesis in DRG neurites in culture is driven by the sequential activation of TTX-sensitive and then TTX-resistant sodium currents.

Figure 4 The F1449V mutation in Na_V1.7 makes DRG neurons hyperexcitable. (a,b) Representative traces from small (<30 μm) dorsal root ganglion (DRG) neurons expressing wild-type (WT) Na_V1.7 or Na_V1.7 with the F1449V mutation (the variant linked to inherited erythromelalgia). These traces show that neurons expressing the mutant channel have a lower current threshold for action potential generation. (c) The average current threshold is notably reduced in cells expressing F1449V compared with cells expressing WT channels (*P <0.05). (d,e) A neuron expressing WT Na_V1.7 responds to a 950 ms stimulation of 150 pA with a lower number of action potentials than does the neuron expressing the F1449V mutant (same cells as in panels a and b). (f) There is a sizeable increase in the frequency of firing of action potentials in response to 100 pA and 150 pA stimuli (950 ms) with expression of F1449V versus expression of WT Na_V1.7 (*P <0.05; **P <0.01). Figure is reproduced, with permission, from ref. 94 © (2005) Oxford University Press.

De novo mutations in SCN9A in individuals with IEM, but without a family history of this disorder, produce similar functional changes in mutant Na_V1.7 to those produced by familial mutations and render DRG neurons hyperexcitable, which is consistent with the pathogenicity of these mutant variants.⁷⁵^,⁷⁶ However, the molecular genetic basis of delayed onset of pain in adult-onset IEM is not yet understood. As in IEM, de novo mutations in SCN9A in individuals with PEPD and no family history of this disorder have been identified; the effects of these de novo mutations on Na_V1.7 gating is similar to those in familial PEPD, which is consistent with the pathogenicity of these mutations.⁷⁰

The distinct and focal patterns of pain in IEM and PEPD are remarkable, considering that Na_V1.7 is expressed in most DRG neurons (figure 2) and trigeminal neurons. An individual with a mixed phenotype that included symptoms of IEM and PEPD symptoms was found to carry the SCN9A mutation A1632E, which hyperpolarizes activation and depolarizes steady-state fast inactivation.⁷¹ Thus, Na_V1.7-associated IEM and PEPD might be considered to be part of a clinical and physiological continuum that can produce IEM, PEPD and disorders that have characteristics of both of these conditions.

Recessively inherited SCN9A nonsense or splicing-defective mutations have been linked to Na_V1.7-related CIP.⁶⁶ Heterozygous parent carriers of these mutations are asymptomatic, indicating that the loss of one SCN9A allele does not lead to clinically manifested haploinsufficiency. Truncated Na_V1.7 CIP fragments do not assemble into functional channels⁶⁶^,⁷⁷ and do not act as dominant negative proteins,⁷⁷ which reflects the normal pain experienced in the heterozygous carrier parents of patients with CIP. Although the first cases of Na_V1.7-related CIP were from consanguineous families,⁶⁶ later cases were identified in non-consanguineous marriages,⁴⁴^,⁴⁵ indicating that there is a higher incidence of carriers of non-functional SCN9A alleles in the general population than was predicted from the initial reports. However, neither homozygous nonsense mutations nor compound heterozygous null mutations have been reported in healthy individuals. Patients with Na_V1.7-related CIP do not experience any form of pain. Notably, they do not display motor, cognitive, sympathetic or gastrointestinal deficits, and have intact sensory modalities.⁶⁶^,⁷⁷ An exception to this is that several patients have reported that they have an impaired sense of smell,⁴⁴^,⁴⁵ although a recent study has described several members of a family with a non-sense SCN9A mutation, CIP and normal sense of smell.⁷⁸

Although the expression of wild-type Na_V1.7 at 50% of the normal protein level (that is, there is one functional allele) is sufficient for a normal pain phenotype (that is, there is no haploinsufficiency), the minimum level of functional Na_V1.7 required to maintain the capacity to experience normal pain is not known. Interestingly, an individual with incomplete CIP (the patient retained some pain sensation) was found to carry compound heterozygous mutations in SCN9A, including a missense mutation (C1719R) affecting the S5–S6 extracurricular linker in DIV, and a one base-pair deletion in the 5′ splice donor site of exon 17 of SCN9A.⁷⁹ Impaired splice donor sites, like most splice-site mutations, may cause exclusion of exon 17 and therefore lead to non-functional channels, which is consistent with the phenotype of impaired sensing. The reporting of some pain experience in this individual suggests that successful but inefficient exon 17 inclusion and production of functional Na_V1.7 have occurred, but at levels that do not support full manifestation of pain.

Positive symptoms (pain) or negative symptoms (loss of pain sensing and anosmia) of patients with SCN9A-linked conditions can be explained by the effects of SCN9A gain-of-function and loss-of-function mutations, respectively, on nociceptors. The lack of an effect of SCN9A mutations on other sensory modalities is, however, not well understood. Although Na_V1.7 is expressed in more than 50% of Aβ low-threshold mechanoreceptors,²⁴ individuals with CIP have normal nerve conduction, tactile sense and vibration sense,⁶⁶^,⁷⁷ suggesting that Na_V1.7 function is redundant in these neurons. By contrast, normal proprioception in patients with CIP is consistent with the absence of Na_V1.7 in muscle afferents.²⁴ It is not fully understood why SCN9A gain-of-function mutations do not cause positive symptoms in carriers; for example, causing them to become ‘hyper-smellers’.

Functional Variants as Risk Factors

In agreement with the ‘common disease, common variant’ hypothesis,⁸⁰ the R1150W variant of Na_V1.7 has been associated with enhanced pain sensation.⁸¹^,⁸² Estacion et al.⁸¹ demonstrated that the W1150 minor allele was present in 30% of people in an ethnically matched control population of Caucasian individuals of European descent. The W1150 variant of Na_V1.7 induces hyperexcitability of DRG neurons, suggesting that carriers of this polymorphism might be predisposed to hyperalgesia. Indeed, a genome-wide association study found that the R1150W polymorphism is associated with an increased pain perception in patients with osteoarthritis, phantom limb pain or lumbar root pain, and that the effect is most strongly associated with C-fibre activation.⁸²

About 30% of individuals with idiopathic SFN express functional mutant Na_V1.7 channels arising from gain-of-function SCN9A missense variants,³⁹ which may not be fully penetrant when found in families.⁸³ People carrying these gain-of-function Na_V1.7 variants are hypersensitive to pain, which reflects the expression of this channel in DRG neurons. These individuals also manifest profound autonomic dysfunction, which reflects the expression of Na_V1.7 in sympathetic neurons.¹⁰^,¹⁸ Gain-of-function attributes of Na_V1.7 variants in SFN include depolarized fast inactivation (figure 3d) and/or slow inactivation (figure 3c), or an increase in the fraction of cells that produce resurgent currents (figure 3f). Surprisingly, however, individuals with SCN9A-null mutations do not manifest autonomic system deficits,⁶⁶ suggesting that there is a redundant function for this channel in sympathetic neurons.

Does Na_V1.7 Play a Role in the Dorsal Horn?

Based on studies in HEK 293 cells and DRG neuron somata, and on computer simulations, Na_V1.7 is thought to act as a threshold channel that activates at relatively hyperpolarized potentials, thus amplifying small, slow depolarizations at potentials negative to an action potential threshold.³⁶^,⁸⁴ This role, however, does not explain the total lack of pain sensation in patients with Na_V1.7-related CIP even in response to the most intense stimulation, such as dental work or child-bearing labor. One possible theory is that Na_V1.7 at central termini of primary afferents (figure 2) may play a part in synaptic transmission of pain signals.

Consistent with this hypothesis, Minett et al.⁶² showed that evoked release of substance P into the spinal cord in response to sciatic nerve stimulation, and synaptic potentiation of wide dynamic range neurons receiving input from primary afferents are attenuated in mice that had Na_V1.7 knocked out in DRG neurons. Na_V1.7 may have a role in facilitating the invasion of incoming action potentials from peripheral nociceptors into central pre-terminal exon branches or into terminals within the spinal cord. Alternatively, Na_V1.7 may be involved within the terminals in the process of neurotransmitter release onto second-order dorsal horn neurons. Thus, we speculate that Na_V1.7, deployed near presynaptic terminals in the dorsal horn,³² is important for release of neurotransmitters such as substance P. If this speculation is correct, then Na_V1.7 inhibitors that act on both peripheral and central compartments might be needed for clinical efficacy.

Structural Features of Na_V1.7

Our ability to understand the mechanistic bases of pathogenic SCN9A mutations and to develop rationally designed small-molecule inhibitors for the treatment of hyperexcitability disorders is limited by the lack of a high-resolution crystal structure of a eukaryotic sodium channel. High-resolution crystal structures of ion channels are necessary for a comprehensive understanding of the links between voltage-sensing and channel activation and inactivation, ion selectivity, and drug interactions. Our current understanding of these channel properties was derived from comparative sequence analysis, and from functional assays that measured ion conductance or fluorescence emission of tagged channels.² Atomic structural modeling following the determination of high-resolution crystal structures of potassium channels ⁸⁵^–⁸⁷ and, more recently, a bacterial sodium channel ³ has advanced our understanding of the structure–function relationship of human SCN9A mutations, which is discussed below.

Lessons Learned from Potassium and Bacterial Sodium Channels

Crystallographic studies of potassium channels provided the first direct evidence for the structural basis for ion selectivity, pore gating and coupling of a voltage sensor to the pore components.⁸⁵^–⁸⁷ These studies also yielded valuable insights into kinetics and sequence determinants of different gating mechanisms. Identification of the homotetramer bacterial voltage-gated sodium channel,⁸⁸ with the monomer possessing the six transmembrane segment architecture of the homologous domains in the eukaryotic channels, facilitated the production of sufficient channel protein for crystallization and high-resolution structural studies. Intriguingly, bacterial voltage-gated sodium channels are most similar to DIII of human sodium channels.⁸⁹ The first high-resolution crystal structure (resolved at 2.7 Å) of a pre-open conformation of the voltage-gated sodium channel from the bacterium Arcobacter butzleri (Na_VAb)³ suggested that the S4 segments are in the activated position, but that the activation gate at the cytoplasmic end of the pore domain is closed. This study provided structural evidence for several of the gating steps of sodium channels and demonstrated a possible route for access of small hydrophobic pore-blocking molecules.

Because of the nature of eukaryotic sodium channels as four-domain polypeptides, which are linked by cytoplasmic loops with divergent lengths and sequences in the different members of the sodium channel family, there may be subtle yet important structural differences between these channels and the bacterial homotetrameric channels. Thus, caution is warranted in extrapolating from high-resolution crystal structures of a symmetrical homotetrameric bacterial sodium channel to eukaryotic single polypeptide multi-domain sodium channel isoforms. Moreover, individual channel mutations should optimally be assessed in their native isoform. For example, the S241L mutation within the DI S4–S5 linker of Na_V1.7 produces a marked hyperpolarizing shift in its activation, steady-state fast and slow inactivation, compared with wild-type channels.⁹⁰ By contrast, substitution of the corresponding residue in Na_V1.4, S246L, hyperpolarizes steady-state fast and slow inactivation of the channel but, unlike S241L in Na_V1.7, S246L had no effect on Na_V1.4 activation,⁹¹ thus providing an example of an isoform-specific effect of conserved residues.

Atomic Structural Modeling of the Putative Activation Gate

From a homology model of the Na_V1.7 pore components, based on the crystal structure of the Streptomyces lividans potassium channel KcsA,⁸⁵ we were able to identify a putative activation gate.⁹² This modeling approach identified an aromatic residue within the cytoplasm-proximal portion of each of the pore-lining S6 helices (DI Y405, DII F960, DIII F1449 and DIV F1752) that were predicted to form a hydrophobic ring at the cytoplasmic end of the pore that stabilizes the channel’s pre-open state. These aromatic residues in the four S6 helices form an energetically stable assembly due to extensive van der Waals bonds between their side chains, which is further strengthened by additional edge-face interaction with the adjacent aromatic residues.⁹³ The hydrophobic ring is predicted to raise the energy barrier for the movement of S6, which is necessary to open the channel’s pore, thus stabilizing the closed or pre-open state of the channel. Although the activation gate at the narrow cytoplasmic vestibule of the channel in the Na_VAb crystal structure consists of four methionine 221 residues (one from each monomer),³ modeling of Na_V1.7 based on the Na_VAb crystal structure recapitulates the activation gate that was previously identified based on the KcsA structure (figure 5).

Figure 5 A model of the putative activation gate of Na_V1.7. The folded structure of the two S6 transmembrane segments presented here were based on the crystal structure of a bacterial sodium channel.³ The carboxy-terminal aromatic residue of each S6 is shown in stick representation for wild-type (WT; a) Na_V1.7 and Na_V1.7 with the F1449V mutation (b). The assembly of aromatic residues at the cytoplasmic C terminus of each of the S6 segments forms the putative activation gate of Na_V1.7. The F1449V mutation in the homologous domain III (DIII) disrupts the hydrophobic ring and destabilizes the pre-open state of the channel.

Evidence for the formation of this hydrophobic block is provided by functional studies of the F1449V mutation in Na_V1.7 that is found in patients with IEM. This mutation lowers the threshold for Na_V1.7 activation.⁹⁴ The F1449V substitution is predicted to destabilize interactions with the adjacent aromatic residues, thus reducing the energetic barrier for DIII S6 helix movement and facilitating bending motions associated with pore opening. The increased propensity of the DIII S6 helix to move would be expected to hasten channel activation. Support for this model of activation comes from studies of inwardly rectifying potassium (Kir) channels, which form a similar quadruple phenylalanine hydrophobic ring.⁹⁵ Substitution of F168 in Kir6.2 (which is analogous to F1449 in Na_V1.7) with smaller residues favors the channel’s open-state, whereas substitution of F168 with the aromatic amino acid tryptophan retains wild-type-like function.⁹⁵

There are limitations on the use of the crystal structure of a homotetrameric voltage-gated ion channels (bacterial sodium channel and various potassium channels) for modeling the multi-domain mammalian Na_V1.7, and our functional studies of the effect of F1449V on the activation gate provide an instructive example of such a limitation. Although the model suggested that phenylalanine or tyrosine residues at the carboxyl termini of the S6 segments in Na_V1.7 stabilize the channel’s closed or pre-open state, functional analysis showed that these residues have different effects. Specifically, DII F960V and DIII F1449V markedly hyperpolarize channel activation, whereas DI Y405V and DIV F1752V do not alter channel activation.⁹² This may reflect the functional specialization of the four homologous, yet not identical, domains of eukaryotic sodium channels.

Targeting Na_V1.7 for Pain Treatment

The clear involvement of Na_V1.7 in human pain, and the lack of serious cognitive, cardiac and adverse motor effects with a total loss of Na_V1.7, as demonstrated in individuals with CIP, have fuelled intense efforts to develop Na_V1.7-specific inhibitors or modulators for the treatment of pain. Despite these intensive efforts, progress has been slow.¹⁰⁰ Nonetheless, the occasional reports of patients with IEM who respond to monotherapy using pan-sodium channel blockers,¹⁰¹^,¹⁰² and the responsiveness of patients with PEPD to carbamazepine, suggest that small molecules may be developed to either inhibit or modulate Na_V1.7 in a manner that can reduce excitability of DRG neurons and provide pain relief. Using the IEM Na_V1.7 V400M carbamazepine-responsive mutation¹⁰² as a ‘seed’ for an atomic-level modeling and thermodynamic analysis, Yang et al.¹⁰³ were able to predict carbamazepine-responsiveness of a second IEM mutation, Na_V1.7 S241T, suggesting that, in the future, pharmacogenomic guided therapy may be possible. Alternative strategies may include the development of isoform-specific blockers or modulators of gating states of sodium channels that are differentially altered under pathological conditions; the development of compounds that weakly cross the blood–brain barrier to minimize CNS-related adverse effects; and gene therapy.

Small-Molecule Blockers

Several purportedly selective small-molecule inhibitors of Na_V1.7 have been described and have shown efficacy in animal models of pain.¹⁰⁴^–¹⁰⁷ These reports, however, lack documentation for selectivity against human sodium channel isoforms in a native neuronal environment. Reports detailing the efficacies of these compounds in animal models of pain should therefore be interpreted with caution, as these results could be due to inhibition of any of the neuronal sodium channel isoforms. A small-molecule blocker with robust selectivity for human Na_V1.7 was recently developed.¹⁰⁸ This orally bioavailable compound bound preferentially to the slow-inactivated state of the channel, and showed notable selectivity for Na_V1.7 over other voltage-gated sodium channel isoforms (by 10-fold to 900-fold). The compound also showed 1,000-fold selectivity for Na_V1.7 over potassium and calcium channels. These favorable properties suggest that this small-molecule blocker holds promise for future clinical studies.

State-Dependent Blockers

Local anesthetics, anticonvulsants and tricyclic compounds block sodium channels, mostly in a use-dependent fashion, and are among the first-line treatments that are currently available for neuropathic pain.¹⁰⁹^,¹¹⁰ However, these agents are not isoform-specific and only provide partial pain relief due in part to their limited therapeutic window that results from CNS-related adverse effects, such as dizziness or sedation.¹¹¹^,¹¹² Despite these limitations, lidocaine derivatives and carbamazepine are effective in patients carrying certain SCN9A mutations that render Na_V1.7 pharmacoresponsive,⁶⁵^,¹⁰¹^,¹⁰² suggesting that personalized, genomically based therapeutics for pain is possible.

Patients with PEPD harbouring SCN9A mutations respond favorably to treatment with carbamazepine, which acts to counterbalance impaired fast inactivation of the mutant channel and hence reduces the persistent current caused by these mutations.⁶⁵ Although most patients with Na_V1.7-linked IEM do not respond to pharmacotherapy, a few have reported control of pain symptoms with lidocaine, mexiletine or carbamazepine. Successful lidocaine or mexiletine monotherapy was reported in a patient carrying the Na_V1.7 V872G mutation, possibly resulting from enhanced lidocaine use-dependent block of the mutant channels.¹⁰¹ Three members of a family with IEM, carrying the mutation Na_V1.7 V400M, reported control of their pain symptoms with carbamazepine.¹⁰² Preincubation of V400M channels with clinically relevant concentrations of carbamazepine induced a depolarizing shift in activation, which returned to wild-type voltages.¹⁰² This normalization of activation suggests that carbamazepine acts in an allosteric manner on the mutant Na_V1.7 channel and induces a wild-type-like pre-open state.

Computer simulation studies⁶⁷ and functional characterization of the Na_V1.7 delL955 mutation⁶⁸ suggest that enhancing the slow inactivation of Na_V1.7 may allow an alternative approach to the treatment of pain. Lacosamide, a functionalized amino acid with sodium channel-blocking activity, showed beneficial effects in animal studies and clinical trials of epilepsy, in animal models of acute, inflammatory and neuropathic pain,¹¹³^–¹¹⁶ and in initial clinical trials for diabetic neuropathic pain.¹¹⁷^,¹¹⁸ Lacosamide’s blocking activity is unusual in that it involves selective enhancement of the slow inactivation of voltage-gated sodium channels, including Na_V1.3, Na_V1.7 and Na_V1.8 (ref. 119). Interestingly, lacosamide induces substantially greater inhibition of Na_V1.3, Na_V1.7 and Na_V1.8 when these channels are in an inactivated state.¹¹⁹ This feature of lacosamide might mean it would exhibit a better safety profile and greater tolerability than state-independent voltage-gated sodium channel blockers, as it might preferentially target injured depolarized neurons with hyperactive sodium channels.¹²⁰ Although lacosamide has not been approved for the treatment of human neuropathic pain,¹²¹ targeting of the Na_V1.7 slow-inactivated state might provide a viable drug-development option.

Natural Toxins

Natural peptide toxins might provide a source of isoform-specific inhibitors of sodium channels, because binding of these toxins to channels is regulated by multiple contact points, and minor sequence changes in the channel could have a profound effect on the affinity of the channel–toxin interaction. Venoms of a variety of snails are reservoirs of peptide toxins, and some of these have demonstrated sodium channel isoform selectivity.¹²²^–¹²⁴ However, Na_V1.7 is among the channels that are only weakly blocked by the conotoxins identified to date.¹²²^,¹²³ By contrast, peptide toxins from tarantulas manifest preferential effect on Na_V1.7. For example, ProTx-II is ~50-fold more selective for Na_V1.7 than Na_V1.5 (refs. 125,126). Huwentoxin-I and huwentoxin-IV are potent inhibitors of Na_V1.7 and other neuronal TTX-sensitive channels, but are not effective against Na_V1.4 (refs. 127,128). The exchange of two residues in the DII S3–S4 linker of Na_V1.7 and Na_V1.4 reverses the affinity of huwentoxins to these channels.¹²⁸ Additionally, a charge-conserving substitution in KIIIA, a member of the μ-conotoxin subfamily, enhances the selectivity for Na_V1.7 over Na_V1.2 and Na_V1.4 (ref. 129). It may therefore be possible to engineer peptide toxins with the desirable Na_V1.7 isoform specificity.

Peptide toxins, however, have poor oral bioavailability and it is difficult to deliver them to nerve endings, implying that their use as therapeutic agents remains limited. However, modification of conotoxins by cyclization can enhance their stability in vivo without compromising their biological activity,¹³⁰ and it may be possible to develop cyclized Na_V1.7-specific peptide toxins when such molecules become available.

Gene Therapy

Advances in virus-mediated gene therapy have led to the initiation of Phase I trials for pain involving a herpes simplex virus (HSV) platform to transfer human preproencephalin (PENK) to DRG neurons.¹³¹ Local delivery of a gene product within the projection zone of an injured or diseased nerve associated with a focal pain syndrome (as in post-herpetic neuralgia or peripheral nerve injury) could be used to treat pain in a topologically defined manner, reducing systemic adverse effects. Animal studies have provided the proof-of-principle for this approach, showing that anti-Na_V1.7 antisense constructs, delivered by a HSV virion, can attenuate pain behavior in mice following peripheral inflammation⁶⁰ and in diabetic rats.⁶¹ We have recently succeeded in targeting another sodium channel, Na_V1.3, in DRG neurons using RNA interference molecules (shRNA for gene knockdown) delivered using the non-virulent AAV platform,¹³² which is less immunogenic than other viral delivery platforms, suggesting that a similar strategy for targeting Na_V1.7 using AAV-mediated delivery of shRNA may be successful.

Notes

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The Na_V1.7 Sodium Channel: From Molecule to Man *

Cellular and Subcellular Distribution

Box 1
Na_V1.7 contributes most of the sodium current in OSNs

Biophysical Properties

Roles in Multiple Sensory Modalities

Pain

Olfaction

Cough Reflex

Acid Sensing

Putative Role in Epilepsy

Roles in Pain States

Na_V1.7 in Acquired Pain Conditions

Box 2
Na_V1.7 in sympathetic neurons and pain signaling

Na_V1.7 in Inherited Pain Disorders

Functional Variants as Risk Factors

Does Na_V1.7 Play a Role in the Dorsal Horn?

Structural Features of Na_V1.7

Lessons Learned from Potassium and Bacterial Sodium Channels

Atomic Structural Modeling of the Putative Activation Gate

Dependence on Neuronal Background

Targeting Na_V1.7 for Pain Treatment

Small-Molecule Blockers

State-Dependent Blockers

Natural Toxins

Gene Therapy

Summary and Future Directions

Acknowledgments

About the Authors

Competing Interests Statement

Databases

Further Information

Glossary

Notes