Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy.

Sodium channel Nav1.9 is expressed in peripheral nociceptive neurons, as well as visceral afferents, and has been shown to act as a threshold channel. Painful peripheral neuropathy represents a significant public health challenge and may involve gain-of-function variants in sodium channels that are preferentially expressed in peripheral sensory neurons. Although gain-of-function variants of peripheral sodium channels Nav1.7 and Nav1.8 have recently been found in painful small fibre neuropathy, the aetiology of peripheral neuropathy in many cases remains unknown. We evaluated 459 patients who were referred for possible painful peripheral neuropathy, and confirmed the diagnosis of small fibre neuropathy in a cohort of 393 patients (369 patients with pure small fibre neuropathy, and small fibre neuropathy together with large fibre involvement in an additional 24 patients). From this cohort of 393 patients with peripheral neuropathy, we sequenced SCN11A in 345 patients without mutations in SCN9A and SCN10A, and found eight variants in 12 patients. Functional profiling by electrophysiological recordings showed that these Nav1.9 mutations confer gain-of-function attributes to the channel, depolarize resting membrane potential of dorsal root ganglion neurons, enhance spontaneous firing, and increase evoked firing of these neurons. Our data show, for the first time, missense mutations of Nav1.9 in individuals with painful peripheral neuropathy. These genetic and functional observations identify missense mutations of Nav1.9 as a cause of painful peripheral neuropathy.

A new Nav1.7 mutation in an erythromelalgia patient.

Gain-of-function missense mutations of SCN9A gene, which encodes voltage-gated sodium channel Nav1.7, alter channel's biophysical properties causing painful disorders which are refractory to pharmacotherapy in the vast majority of patients. Here we report a novel SCN9A mutation (ca.T3947C) in exon 20 in a 9 year old patient, not present in 200 ethnically-matched control alleles; the mutation substitutes the invariant valine 1316 residue within DIII/S5 by alanine (V1316A). Voltage-clamp studies show that Nav1.7 V1316A mutation hyperpolarizes activation (-9 mV), and enhances response to ramp stimuli (3-fold), changes that are predicted to cause hyperexcitability of DRG neurons. V1316A also hyperpolarizes steady-state slow-inactivation (-9.9 mV), which is predicted to attenuate the effect of this mutation on DRG neuron firing. These changes are consistent with previously characterized Erytheromelalgia associated mutations of Nav1.7.

Structural modelling and mutant cycle analysis predict pharmacoresponsiveness of a Na(V)1.7 mutant channel.

Sodium channel Na(V)1.7 is critical for human pain signalling. Gain-of-function mutations produce pain syndromes including inherited erythromelalgia, which is usually resistant to pharmacotherapy, but carbamazepine normalizes activation of Na(V)1.7-V400M mutant channels from a family with carbamazepine-responsive inherited erythromelalgia. Here we show that structural modelling and thermodynamic analysis predict pharmacoresponsiveness of another mutant channel (S241T) that is located 159 amino acids distant from V400M. Structural modelling reveals that Na(v)1.7-S241T is ~2.4 Å apart from V400M in the folded channel, and thermodynamic analysis demonstrates energetic coupling of V400M and S241T during activation. Atomic proximity and energetic coupling are paralleled by pharmacological coupling, as carbamazepine (30 µM) depolarizes S214T activation, as previously reported for V400M. Pharmacoresponsiveness of S241T to carbamazepine was further evident at a cellular level, where carbamazepine normalized the hyperexcitability of dorsal root ganglion neurons expressing S241T. We suggest that this approach might identify variants that confer enhanced pharmacoresponsiveness on a variety of channels.

A channelopathy contributes to cerebellar dysfunction in a model of multiple sclerosis.

OBJECTIVE: Cerebellar dysfunction in multiple sclerosis (MS) contributes significantly to disability, is relatively refractory to symptomatic therapy, and often progresses despite treatment with disease-modifying agents. We previously observed that sodium channel Nav1.8, whose expression is normally restricted to the peripheral nervous system, is present in cerebellar Purkinje neurons in a mouse model of MS (experimental autoimmune encephalomyelitis [EAE]) and in humans with MS. Here, we tested the hypothesis that upregulation of Nav1.8 in cerebellum in MS and EAE has functional consequences contributing to symptom burden. METHODS: Electrophysiology and behavioral assessment were performed in a new transgenic mouse model overexpressing Nav1.8 in Purkinje neurons. We also measured EAE symptom progression in mice lacking Nav1.8 compared to wild-type littermates. Finally, we administered the Nav1.8-selective blocker A803467 in the context of previously established EAE to determine reversibility of MS-like deficits. RESULTS: We report that, in the context of an otherwise healthy nervous system, ectopic expression of Nav1.8 in Purkinje neurons alters their electrophysiological properties, and disrupts coordinated motor behaviors. Additionally, we show that Nav1.8 expression contributes to symptom development in EAE. Finally, we demonstrate that abnormal patterns of Purkinje neuron firing and MS-like deficits in EAE can be partially reversed by pharmacotherapy using a Nav1.8-selective blocker. INTERPRETATION: Our results add to the evidence that a channelopathy contributes to cerebellar dysfunction in MS. Our data suggest that Nav1.8-specific blockers, when available for humans, merit study in MS.

Deletion mutation of sodium channel Na(V)1.7 in inherited erythromelalgia: enhanced slow inactivation modulates dorsal root ganglion neuron hyperexcitability.

Gain-of-function missense mutations of voltage-gated sodium channel Na(V)1.7 have been linked to the painful disorder inherited erythromelalgia. These mutations hyperpolarize activation, slow deactivation and enhance currents evoked by slow ramp stimuli (ramp currents). A correlation has recently been suggested between the age of onset of inherited erythromelalgia and the extent of hyperpolarizing shifts in mutant Na(V)1.7 channel activation; mutations causing large activation shifts have been linked to early age of onset inherited erythromelalgia, while mutations causing small activation shifts have been linked to age of onset within the second decade of life. Here, we report a family with inherited erythromelalgia with an in-frame deletion of a single residue--leucine 955 (Del-L955) in DII/S6. The proband did not show symptoms until the age of 15 years, and her affected mother only experienced mild symptoms during adolescence, which disappeared at the age of 38 years. Del-L955 shows no effect on Na(V)1.7 current density and fast inactivation, but causes an approximately -24 mV shift in activation, together with increases in amplitude of persistent currents and ramp currents. The mutation also produces an approximately -40 mV shift in slow inactivation, which reduces channel availability. Comparison of the effects of the Del-L955 mutation on dorsal root ganglion neuron hyperexcitability with those produced by another inherited erythromelalgia mutation (L858F) that does not enhance slow inactivation suggests that a delayed age of onset and milder symptoms in association with a large shift of channel activation, enhanced persistent and enhanced ramp currents may be related to the approximately -40 mV shift in slow inactivation for Del-L955, the largest shift thus far demonstrated in mutant Na(V)1.7 channels. Our results suggest that despite the pivotal role of activation shift in inherited erythromelalgia development, slow inactivation may regulate clinical phenotype by altering channel availability.

Nav1.7 is the predominant sodium channel in rodent olfactory sensory neurons.

BACKGROUND: Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons). METHODS: We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs. RESULTS: We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system. CONCLUSIONS: Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.

Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK.

Sodium channel Na(v)1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Na(v)1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Na(v)1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser(553)-Pro motif in its intracellular loop L1 reduces Na(v)1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser(553)-Pro motif is a putative binding site to Nedd4 ubiquitin ligases, and we hypothesized that Nedd4-like ubiquitin ligases may contribute to channel ubiquitination and internalization. We report here that p38 activation in hippocampal neurons from wild-type mice, but not from Scn8a(medtg) mice that lack Na(v)1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Na(v)1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Na(v)1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Na(v)1.6 via a Pro-Ser-Tyr(1945) motif in the C terminus of the channel and reduces Na(v)1.6 current density, and we show that this regulation requires both the Pro-Gly-Ser-Pro motif in L1 and the Pro-Ser-Tyr motif in the C terminus. Similarly, both motifs are necessary for p38-mediated reduction of Na(v)1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Na(v)1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Na(v)1.6 is necessary for stress-induced current modulation, with positive or negative regulation depending upon the availability of the C-terminal Pro-Ser-Tyr motif to bind Nedd4-2.

Alternative splicing may contribute to time-dependent manifestation of inherited erythromelalgia.

The Na(v)1.7 sodium channel is preferentially expressed in nocioceptive dorsal root ganglion and sympathetic ganglion neurons. Gain-of-function mutations in Na(v)1.7 produce the nocioceptor hyperexcitability underlying inherited erythromelalgia, characterized in most kindreds by early-age onset of severe pain. Here we describe a mutation (Na(v)1.7-G616R) in a pedigree with adult-onset of pain in some family members. The mutation shifts the voltage-dependence of channel fast-inactivation in a depolarizing direction in the adult-long, but not in the neonatal-short splicing isoform of Na(v)1.7 in dorsal root ganglion neurons. Altered inactivation does not depend on the age of the dorsal root ganglion neurons in which the mutant is expressed. Expression of the mutant adult-long, but not the mutant neonatal-short, isoform of Na(v)1.7 renders dorsal root ganglion neurons hyperexcitable, reducing the current threshold for generation of action potentials, increasing spontaneous activity and increasing the frequency of firing in response to graded suprathreshold stimuli. This study shows that a change in relative expression of splice isoforms can contribute to time-dependent manifestation of the functional phenotype of a sodium channelopathy.

Mutations at opposite ends of the DIII/S4-S5 linker of sodium channel Na V 1.7 produce distinct pain disorders.

BACKGROUND: Two groups of gain-of-function mutations in sodium channel NaV1.7, which are expressed in dorsal root ganglion (DRG) neurons, produce two clinically-distinct pain syndromes - inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). IEM is characterized by intermittent burning pain and skin redness in the feet or hands, triggered by warmth or mild exercise, while PEPD is characterized by episodes of rectal, ocular and mandibular pain accompanied with skin flushing, triggered by bowel movement and perianal stimulation. Most of the IEM mutations are located within channel domains I and II, while most of the PEPD mutations are located within domains III and IV. The structural dichotomy parallels the biophysical effects of the two types of mutations, with IEM mutations shifting voltage-dependence of NaV1.7 activation in a hyperpolarized direction, and PEPD mutations shifting fast-inactivation of NaV1.7 in a depolarized direction. While four IEM and four PEPD mutations are located within cytoplasmic linkers joining segments 4 and 5 (S4-S5 linkers) in the different domains (IEM: domains I and II; PEPD: domains III and IV), no S4-S5 linker has been reported to house both IEM and PEPD mutations thus far. RESULTS: We have identified a new IEM mutation P1308L within the C-terminus of the DIII/S4-S5 linker of NaV1.7, ten amino acids from a known PEPD mutation V1298F which is located within the N-terminus of this linker. We used voltage-clamp to compare the biophysical properties of the two mutant channels and current-clamp to study their effects on DRG neuron excitability. We confirm that P1308L and V1298F behave as prototypical IEM and PEPD mutations, respectively. We also show that DRG neurons expressing either P1308L or V1298F become hyperexcitable, compared to DRG neurons expressing wild-type channels. CONCLUSIONS: Our results provide evidence for differential roles of the DIII/S4-S5 linker N- and C-termini in channel inactivation and activation, and demonstrate the cellular basis for pain in patients carrying these mutations.

A new Nav1.7 sodium channel mutation I234T in a child with severe pain.

Dominant gain-of-function mutations that hyperpolarize activation of the Na(v)1.7 sodium channel have been linked to inherited erythromelalgia (IEM), a disorder characterized by severe pain and redness in the feet and hands in response to mild warmth. Pharmacotherapy remains largely ineffective for IEM patients with cooling and avoidance of triggers being the most reliable methods to relieve pain. We now report a 5 year old patient with pain precipitated by warmth, together with redness in her hands and feet. Her pain episodes were first reported at 12 months, and by the age of 15-16 months were triggered by sitting as well as heat. Pain has been severe, inducing self-mutilation, with limited relief from drug treatment. Our analysis of the patient's genomic DNA identified a novel Na(v)1.7 mutation which replaces isoleucine 234 by threonine (I234T) within domain I/S4-S5 linker. Whole-cell voltage-clamp analysis shows a I234T-induced shift of -18 mV in the voltage-dependence of activation, accelerated time-to-peak, slowed deactivation and enhanced responses to slow ramp depolarizations, together with a -21 mV shift in the voltage-dependence of slow-inactivation. Our data show that I234T induces the largest activation shift for Na(v)1.7 mutations reported thus far. Although enhanced slow-inactivation may attenuate the gain-of-function of the I234T mutation, the shift in activation appears to be dominant, and is consistent with the severe pain symptoms reported in this patient.

ERK1/2 mitogen-activated protein kinase phosphorylates sodium channel Na(v)1.7 and alters its gating properties.

Na(v)1.7 sodium channels can amplify weak stimuli in neurons and act as threshold channels for firing action potentials. Neurotrophic factors and pro-nociceptive cytokines that are released during development and under pathological conditions activate mitogen-activated protein kinases (MAPKs). Previous studies have shown that MAPKs can transduce developmental or pathological signals by regulating transcription factors that initiate a gene expression response, a long-term effect, and directly modulate neuronal ion channels including sodium channels, thus acutely regulating dorsal root ganglion (DRG) neuron excitability. For example, neurotrophic growth factor activates (phosphorylates) ERK1/2 MAPK (pERK1/2) in DRG neurons, an effect that has been implicated in injury-induced hyperalgesia. However, the acute effects of pERK1/2 on sodium channels are not known. We have shown previously that activated p38 MAPK (pp38) directly phosphorylates Na(v)1.6 and Na(v)1.8 sodium channels and regulates their current densities without altering their gating properties. We now report that acute inhibition of pERK1/2 regulates resting membrane potential and firing properties of DRG neurons. We also show that pERK1 phosphorylates specific residues within L1 of Na(v)1.7, inhibition of pERK1/2 causes a depolarizing shift of activation and fast inactivation of Na(v)1.7 without altering current density, and mutation of these L1 phosphoacceptor sites abrogates the effect of pERK1/2 on this channel. Together, these data are consistent with direct phosphorylation and modulation of Na(v)1.7 by pERK1/2, which unlike the modulation of Na(v)1.6 and Na(v)1.8 by pp38, regulates gating properties of this channel but not its current density and contributes to the effects of MAPKs on DRG neuron excitability.

A sodium channel gene SCN9A polymorphism that increases nociceptor excitability.

Sodium channel Na(V)1.7, encoded by the SCN9A gene, is preferentially expressed in nociceptive primary sensory neurons, where it amplifies small depolarizations. In studies on a family with inherited erythromelalgia associated with Na(V)1.7 gain-of-function mutation A863P, we identified a nonsynonymous single-nucleotide polymorphism within SCN9A in the affected proband and several unaffected family members; this polymorphism (c. 3448C&T, Single Nucleotide Polymorphisms database rs6746030, which produces the amino acid substitution R1150W in human Na(V)1.7 [hNa(V)1.7]) is present in 1.1 to 12.7% of control chromosomes, depending on ethnicity. In this study, we examined the effect of the R1150W substitution on function of the hNa(V)1.7 channel, and on the firing of dorsal root ganglion (DRG) neurons in which this channel is normally expressed. We show that this polymorphism depolarizes activation (7.9-11mV in different assays). Current-clamp analysis shows that the 1150W allele depolarizes (6mV) resting membrane potential and increases ( approximately 2-fold) the firing frequency in response to depolarization in DRG neurons in which it is present. Our results suggest that polymorphisms in the Na(V)1.7 channel may influence susceptibility to pain.

Erythromelalgia mutation L823R shifts activation and inactivation of threshold sodium channel Nav1.7 to hyperpolarized potentials.

Erythromelalgia (also termed erythermalgia) is a neuropathic pain syndrome, characterized by severe burning pain combined with redness in the extremities, triggered by mild warmth. The inherited form of erythromelalgia (IEM) has recently been linked to mutations in voltage-gated sodium channel Nav1.7, which is expressed in peripheral nociceptors. Here, we used whole-cell voltage-clamp recordings in HEK293 cells to characterize the IEM mutation L823R, which introduces an additional positive charge into the S4 voltage sensor of domain II. The L823R mutation produces an approximately 15mV hyperpolarizing shift in the midpoint of activation and also affects the activation slope factor. Closing of the channel from the open state (deactivation) is slowed, increasing the likelihood of the channel remaining in the open state. The L823R mutation induces a approximately 10mV hyperpolarizing shift in fast-inactivation. L823R is the only naturally-occurring IEM mutation studied thus far to shift fast-inactivation to more negative potentials. We conclude that introduction of an additional charge into the S4 segment of domain II of Nav1.7 leads to a pronounced hyperpolarizing shift of activation, a change that is expected to increase nociceptor excitability despite the hyperpolarizing shift in fast-inactivation, which is unique among the IEM mutations.

Transfection of rat or mouse neurons by biolistics or electroporation.

Properties of ion channels are affected by the background of the cells in which they are expressed. Thus, it is important for investigators interested in neuronal function to study these proteins in post-mitotic neurons. However, post-mitotic neurons, and many cell lines, are difficult to transfect by standard methods. Here we provide detailed protocols for two different procedures, biolistic and electroporation, which have been used to transfect peripheral sensory neurons from mice or rats with expression constructs of voltage-gated sodium channels. Neurons can be prepared, transfected and currents recorded within 48 h. Using these methods, primary sensory neurons can be transfected with an efficiency of 5-20%, which has permitted studying biophysical properties of sodium channels and their naturally occurring mutants in a native neuronal cell background. Although we have used sodium channels for the examples that we show here, these methods can also be used to study other types of molecules.

Early- and late-onset inherited erythromelalgia: genotype-phenotype correlation.

Inherited erythromelalgia (IEM), an autosomal dominant disorder characterized by severe burning pain in response to mild warmth, has been shown to be caused by gain-of-function mutations of sodium channel Na(v)1.7 which is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Almost all physiologically characterized cases of IEM have been associated with onset in early childhood. Here, we report the voltage-clamp and current-clamp analysis of a new Na(v)1.7 mutation, Q10R, in a patient with clinical onset of erythromelalgia in the second decade. We show that the mutation in this patient hyperpolarizes activation by only -5.3 mV, a smaller shift than seen with early-onset erythromelalgia mutations, but similar to that of I136V, another mutation that is linked to delayed-onset IEM. Using current-clamp, we show that the expression of Q10R induces hyperexcitability in DRG neurons, but produces an increase in excitability that is smaller than the change produced by I848T, an early-onset erythromelalgia mutation. Our analysis suggests a genotype-phenotype relationship at three levels (clinical, cellular and molecular/ion channel), with mutations that produce smaller effects on sodium channel activation being associated with a smaller degree of DRG neuron excitability and later onset of clinical signs.

Mexiletine-responsive erythromelalgia due to a new Na(v)1.7 mutation showing use-dependent current fall-off.

Inherited erythromelalgia (IEM), characterized by episodic burning pain and erythema of the extremities, is produced by gain-of-function mutations in sodium channel Na(v)1.7, which is preferentially expressed in nociceptive and sympathetic neurons. Most patients do not respond to pharmacotherapy, although occasional reports document patients as showing partial relief with lidocaine or mexiletine. A 7-year-old girl, with a two-year history of symmetric burning pain and erythema in her hands and feet, was diagnosed with erythromelalgia. Treatment with mexiletine reduced the number and severity of pain episodes. We report here a new IEM Na(v)1.7 mutation in this patient, and its response to mexiletine. SCN9A exons from the proband were amplified and sequenced. We identified a single nucleotide substitution (T2616G) in exon 15, not present in 200 ethnically-matched control alleles, which substitutes valine 872 by glycine (V872G) within DII/S5. Whole-cell patch-clamp analysis of wild-type and mutant Na(v)1.7 channels in mammalian cells show that V872G shifts activation by -10 mV, slows deactivation, and generates larger ramp currents. We observed a stronger use-dependent fall-off in current following exposure to mexiletine for V872G compared to wild-type channels. These observations suggest that some patients with IEM may show a favorable response to mexiletine due to a use-dependent effect on mutant Na(v)1.7 channels. Continued relief from pain, even after mexiletine was discontinued in this patient, might suggest that early treatment may slow the progression of the disease.

Paroxysmal extreme pain disorder M1627K mutation in human Nav1.7 renders DRG neurons hyperexcitable.

BACKGROUND: Paroxysmal extreme pain disorder (PEPD) is an autosomal dominant painful neuropathy with many, but not all, cases linked to gain-of-function mutations in SCN9A which encodes voltage-gated sodium channel Nav1.7. Severe pain episodes and skin flushing start in infancy and are induced by perianal probing or bowl movement, and pain progresses to ocular and mandibular areas with age. Carbamazepine has been effective in relieving symptoms, while other drugs including other anti-epileptics are less effective. RESULTS: Sequencing of SCN9A coding exons from an English patient, diagnosed with PEPD, has identified a methionine 1627 to lysine (M1627K) substitution in the linker joining segments S4 and S5 in domain IV. We confirm that M1627K depolarizes the voltage-dependence of fast-inactivation without substantially altering activation or slow-inactivation, and inactivates from the open state with slower kinetics. We show here that M1627K does not alter development of closed-state inactivation, and that M1627K channels recover from fast-inactivation faster than wild type channels, and produce larger currents in response to a slow ramp stimulus. Using current-clamp recordings, we also show that the M1627K mutant channel reduces the threshold for single action potentials in DRG neurons and increases the number of action potentials in response to graded stimuli. CONCLUSION: M1627K mutation was previously identified in a sporadic case of PEPD from France, and we now report it in an English family. We confirm the initial characterization of mutant M1627K effect on fast-inactivation of Nav1.7 and extend the analysis to other gating properties of the channel. We also show that M1627K mutant channels render DRG neurons hyperexcitable. Our new data provide a link between altered channel biophysics and pain in PEPD patients.

A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Nav1.7 channel is disrupted by the erythromelalgia-associated F1449V mutation.

Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.

Phosphorylation of sodium channel Na(v)1.8 by p38 mitogen-activated protein kinase increases current density in dorsal root ganglion neurons.

The sensory neuron-specific sodium channel Na(v)1.8 and p38 mitogen-activated protein kinase are potential therapeutic targets within nociceptive dorsal root ganglion (DRG) neurons in inflammatory, and possibly neuropathic, pain. Na(v)1.8 channels within nociceptive DRG neurons contribute most of the inward current underlying the depolarizing phase of action potentials. Nerve injury and inflammation of peripheral tissues cause p38 activation in DRG neurons, a process that may contribute to nociceptive neuron hyperexcitability, which is associated with pain. However, how substrates of activated p38 contribute to DRG neuron hyperexcitability is currently not well understood. We report here, for the first time, that Na(v)1.8 and p38 are colocalized in DRG neurons, that Na(v)1.8 within DRG neurons is a substrate for p38, and that direct phosphorylation of the Na(v)1.8 channel by p38 regulates its function in these neurons. We show that direct phosphorylation of Na(v)1.8 at two p38 phospho-acceptor serine residues on the L1 loop (S551 and S556) causes an increase in Na(v)1.8 current density that is not accompanied by changes in gating properties of the channel. Our study suggests a mechanism by which activated p38 contributes to inflammatory, and possibly neuropathic, pain through a p38-mediated increase of Na(v)1.8 current density.

Mutation I136V alters electrophysiological properties of the Na(v)1.7 channel in a family with onset of erythromelalgia in the second decade.

BACKGROUND: Primary erythromelalgia is an autosomal dominant pain disorder characterized by burning pain and skin redness in the extremities, with onset of symptoms during the first decade in the families whose mutations have been physiologically studied to date. Several mutations of voltage-gated Na+ channel NaV1.7 have been linked with primary erythromelalgia. Recently, a new substitution Na(v)1.7/I136V has been reported in a Taiwanese family, in which pain appeared at later ages (9-22 years, with onset at 17 years of age or later in 5 of 7 family members), with relatively slow progression (8-10 years) to involvement of the hands. The proband reported onset of symptoms first in his feet at the age of 11, which then progressed to his hands at the age of 19. The new mutation is located in transmembrane segment 1 (S1) of domain I (DI) in contrast to all Na(v)1.7 mutations reported to date, which have been localized in the voltage sensor S4, the linker joining segments S4 and S5 or pore-lining segments S5 and S6 in DI, II and III. RESULTS: In this study, we characterized the gating and kinetic properties of I136V mutant channels in HEK293 cells using whole-cell patch clamp. I136V shifts the voltage-dependence of activation by -5.7 mV, a smaller shift in activation than the other erythromelalgia mutations that have been characterized. I136V also decreases the deactivation rate, and generates larger ramp currents. CONCLUSION: The I136V substitution in Na(v)1.7 alters channel gating and kinetic properties. Each of these changes may contribute to increased excitability of nociceptive dorsal root ganglion neurons, which underlies pain in erythromelalgia. The smaller shift in voltage-dependence of activation of Na(v)1.7, compared to the other reported cases of inherited erythromelalgia, may contribute to the later age of onset and slower progression of the symptoms reported in association with this mutation.