Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

Genetic, electrophysiological, and pathological studies on patients with SCN9A-related pain disorders.

BACKGROUND AND AIMS: Voltage-gated sodium channel Nav1.7, encoded by the SCN9A gene, has been linked to diverse painful peripheral neuropathies, represented by the inherited erythromelalgia (EM) and paroxysmal extreme pain disorder (PEPD). The aim of this study was to determine the genetic etiology of patients experiencing neuropathic pain, and shed light on the underlying pathogenesis. METHODS: We enrolled eight patients presenting with early-onset painful peripheral neuropathies, consisting of six cases exhibiting EM/EM-like disorders and two cases clinically diagnosed with PEPD. We conducted a gene-panel sequencing targeting 18 genes associated with hereditary sensory and/or autonomic neuropathy. We introduced novel SCN9A mutation (F1624S) into a GFP-2A-Nav1.7rNS plasmid, and the constructs were then transiently transfected into HEK293 cells. We characterized both wild-type and F1624S Nav1.7 channels using an automated high-throughput patch-clamp system. RESULTS: From two patients displaying EM-like/EM phenotypes, we identified two SCN9A mutations, I136V and P1308L. Among two patients diagnosed with PEPD, we found two additional mutations in SCN9A, F1624S (novel) and A1632E. Patch-clamp analysis of Nav1.7-F1624S revealed depolarizing shifts in both steady-state fast inactivation (17.4 mV, p < .001) and slow inactivation (5.5 mV, p < .001), but no effect on channel activation was observed. INTERPRETATION: Clinical features observed in our patients broaden the phenotypic spectrum of SCN9A-related pain disorders, and the electrophysiological analysis enriches the understanding of genotype-phenotype association caused by Nav1.7 gain-of-function mutations.

Structural basis for severe pain caused by mutations in the S4-S5 linkers of voltage-gated sodium channel NaV1.7.

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: NaV1.7/I234T, NaV1.7/I848T, and NaV1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel NaVAb recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the NaV1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of NaV1.7 and lead to severe pain in this debilitating disease.

A case of sporadic erythromelalgia presenting with small fibre neuropathy.

Erythromelalgia (EM) is a rare disorder of small nerve fibres that leads to painful flushing and burning paresthesisas of the distal extremities and is typically associated with heat or physical activity; relief is found using cooling measures. Its effects are often debilitating in the general population, but this patient had an excellent response to specific treatment options and continues to maintain employment, something many individuals suffering from EM are unable to do. His presentation was also unique in that he had isolated, proximal involvement as his condition progressed whereas typically only the distal extremities are affected. Routine electromyography and nerve conduction studies were normal, whereas nerve biopsy demonstrated findings of small fibre neuropathy. Ultimately, his condition was managed with carbamazepine and his symptoms have almost entirely resolved to date.

[Erythromelalgia: skin redness and pain]. / Erythromelalgie: rote Haut und Schmerz.

Erythromelalgia is a rare disease that is associated with hemato-oncological diseases or after taking certain drugs and toxins, but it can also occur as an independent clinical picture, for example, due to mutations in the sodium channel NaV1.7. Clinically, there is a characteristic triad of attack-like burning pain and skin redness in the area of the distal extremities, which can be alleviated by excessive cooling. The attacks are triggered by heat, exertion, and stress. The diagnosis is primarily made clinically and can be confirmed by genetic testing if a sodium channel NaV1.7 mutation is present. Important differential diagnoses are complex regional pain syndrome, the non-freezing cold injury, and small fiber neuropathies. Therapy is multidisciplinary and has to be planned individually and include physical therapy and psychotherapy as well as drug therapy as integral components.

Bone scan findings in erythromelalgia.

The use of hyaluronic acid nanoshells has been proposed to encapsulate prodrugs and exploit the mechanisms of interactions between living cells, like endocytes or cancer cells and hyaluronic acid, which is a natural component of the extracellular matrix. In this review we describe the potential and the limits of this promising research trend and discuss the theoretical advantages of such an engineering approach. Is it a possible scalability to increase the efficacy and biodegradability of molecules like contrast media and radiotracers especially for neuroradiology and nuclear medicine studies.

A Novel SCN9A Mutation (F826Y) in Primary Erythromelalgia Alters the Excitability of Nav1.7.

BACKGROUND: Primary erythromelalgia (PE) is a dominant inherited disorder characterized by recurrent pain, redness, and warmth of the extremities that is caused by gain-of-function mutations in Nav1.7 encoding gene SCN9A. Most of the PE-causing mutations of Nav1.7 have been shown to be able to render Nav1.7-expressing cells hyperexcitable, however in most PE cases the symptoms are refractory to treatment with sodium channel blockers and the mechanism underlying the intractability has not been clearly clarified. OBJECTIVE: To identify the mutation of SCN9A in a Chinese Han family with typical symptoms of PE and study the electrophysiological effect of the identified mutation. METHODS: A Chinese Han family with typical symptoms of PE was collected and the proband's response to treatment was recorded. All the exons and flanking intronic sequences of SCN9A were amplified with PCR and sequenced. Several online programs were used to predict the damaging effect of variants. The functional effect of variants was studied by voltage-clamp analysis in CHO-K1 cells. RESULTS: The PE symptoms of the proband are refractory to all kinds of reported medications. Sequence analysis of SCN9A showed that a novel c.2477T>A (p. F826Y) mutation co-segregated with the disease phenotype. Several online programs predicted that the F826Y mutation has a deleterious effect on the gene product. Voltage-clamp analysis showed that while compared with the wild-type channel, activation of the F826Y mutant channel was shifted by 7.7 mV in a hyperpolarizing direction, whereas steadystate inactivation was shifted by 4.3 mV in a depolarizing direction. CONCLUSION: A novel disease-causing SCN9A Mutation (F826Y) was identified in a Chinese family with typical PE symptoms refractory to treatment. F826Y of Nav1.7 could render DRG neurons hyperexcitable, contributing to the pathogenesis of PE.

Burning pain: axonal dysfunction in erythromelalgia.

Erythromelalgia (EM) is a rare neurovascular disorder characterized by intermittent severe burning pain, erythema, and warmth in the extremities on heat stimuli. To investigate the underlying pathophysiology, peripheral axonal excitability studies were performed and changes with heating and therapy explored. Multiple excitability indices (stimulus-response curve, strength-duration time constant (SDTC), threshold electrotonus, and recovery cycle) were investigated in 23 (9 EMSCN9A+ and 14 EMSCN9A-) genetically characterized patients with EM stimulating median motor and sensory axons at the wrist. At rest, patients with EM showed a higher threshold and rheobase (P < 0.001) compared with controls. Threshold electrotonus and current-voltage relationships demonstrated greater changes of thresholds in both depolarizing and hyperpolarizing preconditioning electrotonus in both EM cohorts compared with controls in sensory axons (P < 0.005). When average temperature was raised from 31.5°C to 36.3°C in EMSCN9A+ patients, excitability changes showed depolarization, specifically SDTC significantly increased, in contrast to the effects of temperature previously established in healthy subjects (P < 0.05). With treatment, 4 EMSCN9A+ patients (4/9) reported improvement with mexiletine, associated with reduction in SDTC in motor and sensory axons. This is the first study of primary EM using threshold tracking techniques to demonstrate alterations in peripheral axonal membrane function. Taken together, these changes may be attributed to systemic neurovascular abnormalities in EM, with chronic postischaemic resting membrane potential hyperpolarization due to Na/K pump overactivity. With heating, a trigger of acute symptoms, axonal depolarization developed, corresponding to acute axonal ischaemia. This study has provided novel insights into EM pathophysiology.

Nav1.7-A1632G Mutation from a Family with Inherited Erythromelalgia: Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli.

UNLABELLED: Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. SIGNIFICANCE STATEMENT: Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients.

Erythromelalgia as a manifestation of autonomic nervous system involvement in multiple sclerosis.

Erythromelalgia is a rare condition characterized by burning pain, erythema and increased temperature of the hands or the feet. Its etiology is not completely understood but it is believed that the underlying cause is a peripheral vascular dysfunction that leads to simultaneous tissue hypoxia and hyperemia. We present a rare co-occurrence of erythromelalgia and multiple sclerosis in a patient with autonomic nervous system dysfunction and propose a causative interconnection.

A case of secondary erythromelalgia with perivascular and intramural mucin.

We present a 49-year-old man with type I erythromelalgia, demonstrating a newly reported histological feature of striking perivascular mucin. There is a single previously reported case in the literature describing these histological features. This patient had a comorbid history of primary myelofibrosis diagnosed 2 years prior to his presentation.

The Role and Importance of Small Fiber Neuropathy in Fibromyalgia Pain.

Serious investigators of fibromyalgia (FM) realize the profound implications of finding features of small fiber neuropathy (SFN) in this disorder. For the first time, an easily reproducible and generally agreed upon, peripheral tissue lesion has been reported from multiple investigative centers. Understanding how this discovery relates to other features of FM, and how one might utilize it to better comprehend, and care for, afflicted patients' painful complaints remains a challenge, however. In this article we review how the SFN seen in FM may be placed in context, and suggest how such a tissue abnormality might be used to better understand the pathophysiology of FM, and plan for its effective treatment. We also suggest how finding SFN in FM implies the need for continued focused research within the area of neuropathic disease in FM.

Corneal Confocal Microscopy Detects Small Fibre Neuropathy in Patients with Upper Gastrointestinal Cancer and Nerve Regeneration in Chemotherapy Induced Peripheral Neuropathy.

There are multiple neurological complications of cancer and its treatment. This study assessed the utility of the novel non-invasive ophthalmic technique of corneal confocal microscopy in identifying neuropathy in patients with upper gastrointestinal cancer before and after platinum based chemotherapy. In this study, 21 subjects with upper gastrointestinal (oesophageal or gastric) cancer and 21 healthy control subjects underwent assessment of neuropathy using the neuropathy disability score, quantitative sensory testing for vibration perception threshold, warm and cold sensation thresholds, cold and heat induced pain thresholds, nerve conduction studies and corneal confocal microscopy. Patients with gastro-oesophageal cancer had higher heat induced pain (P = 0.04) and warm sensation (P = 0.03) thresholds with a significantly reduced sural sensory (P<0.01) and peroneal motor (P<0.01) nerve conduction velocity, corneal nerve fibre density (CNFD), nerve branch density (CNBD) and nerve fibre length (CNFL) (P<0.0001). Furthermore, CNFD correlated significantly with the time from presentation with symptoms to commencing chemotherapy (r = -0.54, P = 0.02), and CNFL (r = -0.8, P<0.0001) and CNBD (r = 0.63, P = 0.003) were related to the severity of lymph node involvement. After the 3rd cycle of chemotherapy, there was no change in any measure of neuropathy, except for a significant increase in CNFL (P = 0.003). Corneal confocal microscopy detects a small fibre neuropathy in this cohort of patients with upper gastrointestinal cancer, which was related to disease severity. Furthermore, the increase in CNFL after the chemotherapy may indicate nerve regeneration.

Epidermal innervation morphometry by immunofluorescence and bright-field microscopy.

We investigated the agreement between simple indirect immunofluorescence (IF) and bright-field immunohistochemistry (BFI) on free-floating sections for intraepidermal nerve fiber density (IENFD) quantification. Fifty-five healthy subjects and 63 patients with probable small fiber neuropathy (SFN) underwent two adjacent skin biopsies at the distal leg processed by IF and BFI technique. Agreement between IENFD pairs obtained by each method was assessed by Bland-Altman testing. The area under the curve of the receiving operating characteristics (ROC) curves was used to compare the discrimination ability. The diagnostic judgment was based on sex and age-adjusted normative values. IF and BFI showed good correlation (r = 0.81), with a ratio of about 2:1 and a mean difference of 5.5 ± 3.0 IENF per millimeter between paired measures, as demonstrated by linear regression and Bland-Altman test analyses. The square root transformation confirmed a Poisson distribution of the data and a fixed bias between IF and BFI measurements. The ROC curves analysis demonstrated a striking overlap between IF and BFI (0.83 and 0.82; p = 0.72). The diagnosis of SFN disagreed in only 6.7% of cases when the judgment was based on a difference of >1 IENF from 5% cut-off value. IF and BFI showed comparable diagnostic efficiency when referred to appropriate normative reference values.

Small fiber neuropathy in women with fibromyalgia. An in vivo assessment using corneal confocal bio-microscopy.

OBJECTIVE: A consistent line of investigation suggests that fibromyalgia is a neuropathic pain syndrome. This outlook has been recently reinforced by several controlled studies that describe decreased small nerve fiber density in skin biopsies of patients with fibromyalgia. The cornea receives the densest small fiber innervation of the body. Corneal confocal bio-microscopy is a new noninvasive method to evaluate small nerve fiber morphology. Our objective was to assess corneal small nerve fiber morphology in patients with fibromyalgia, and to associate corneal nerve microscopic features with neuropathic pain descriptors and other fibromyalgia symptoms. METHODS: We studied 17 female patients with fibromyalgia and 17 age-matched healthy control subjects. All the participants completed different questionnaires regarding the symptoms of fibromyalgia, including a neuropathic pain survey. A central corneal thickness scan was obtained with a confocal microscope. Nerve measurements were made by a single ophthalmologist without knowledge of the clinical diagnosis. Stromal nerve thickness was defined as the mean value between the widest and the narrowest portion of each analyzed stromal nerve. Corneal sub-basal plexus nerve density was also assessed. RESULTS: Patients with fibromyalgia had stromal nerve thickness of 5.0 ± 1.0 µm (mean ± standard deviation) significantly different from that of control's values (6.1 ± 1.3) p = 0.01. Patients also had decreased sub-basal plexus nerve density per square millimeter (85 ± 29) vs. 107 ± 26 of controls p = 0.02. When controls and patients were grouped together, there was an association between stromal nerve slenderness and neuropathic pain descriptors (Fisher's exact test p = 0.007). CONCLUSION: Women suffering from fibromyalgia have thinner corneal stromal nerves and diminished sub-basal plexus nerve density when compared to healthy controls. Nerve scarcity is associated with neuropathic pain descriptors. Small fiber neuropathy may play a role in the pathogenesis of fibromyalgia pain. Corneal confocal microscopy could become a useful test in the study of patients with fibromyalgia.

Specific changes in conduction velocity recovery cycles of single nociceptors in a patient with erythromelalgia with the I848T gain-of-function mutation of Nav1.7.

Seven patients diagnosed with erythromelalgia (EM) were investigated by microneurography to record from unmyelinated nerve fibers in the peroneal nerve. Two patients had characterized variants of sodium channel Nav1.7 (I848T, I228M), whereas no mutations of coding regions of Navs were found in 5 patients with EM. Irrespective of Nav1.7 mutations, more than 50% of the silent nociceptors in the patients with EM showed spontaneous activity. In the patient with mutation I848T, all nociceptors, but not sympathetic efferents, displayed enhanced early subnormal conduction in the velocity recovery cycles and the expected late subnormality was reversed to supranormal conduction. The larger hyperpolarizing shift of activation might explain the difference to the I228M mutation. Sympathetic fibers that lack Nav1.8 did not show supranormal conduction in the patient carrying the I848T mutation, confirming in human subjects that the presence of Nav1.8 crucially modulates conduction in cells expressing EM mutant channels. The characteristic pattern of changes in conduction velocity observed in the patient with the I848T gain-of function mutation in Nav1.7 could be explained by axonal depolarization and concomitant inactivation of Nav1.7. If this were true, activity-dependent hyperpolarization would reverse inactivation of Nav1.7 and account for the supranormal CV. This mechanism might explain normal pain thresholds under resting conditions.

Corneal Confocal Microscopy Identifies Small-Fiber Neuropathy in Subjects With Impaired Glucose Tolerance Who Develop Type 2 Diabetes.

OBJECTIVE: Impaired glucose tolerance (IGT) through to type 2 diabetes is thought to confer a continuum of risk for neuropathy. Identification of subjects at high risk of developing type 2 diabetes and, hence, worsening neuropathy would allow identification and risk stratification for more aggressive management. RESEARCH DESIGN AND METHODS: Thirty subjects with IGT and 17 age-matched control subjects underwent an oral glucose tolerance test, assessment of neuropathic symptoms and deficits, quantitative sensory testing, neurophysiology, skin biopsy, and corneal confocal microscopy (CCM) to quantify corneal nerve fiber density (CNFD), branch density (CNBD), and fiber length (CNFL) at baseline and annually for 3 years. RESULTS: Ten subjects who developed type 2 diabetes had a significantly lower CNFD (P = 0.003), CNBD (P = 0.04), and CNFL (P = 0.04) compared with control subjects at baseline and a further reduction in CNFL (P = 0.006), intraepidermal nerve fiber density (IENFD) (P = 0.02), and mean dendritic length (MDL) (P = 0.02) over 3 years. Fifteen subjects who remained IGT and 5 subjects who returned to normal glucose tolerance had no significant baseline abnormality on CCM or IENFD but had a lower MDL (P < 0.0001) compared with control subjects. The IGT subjects showed a significant decrease in IENFD (P = 0.02) but no change in MDL or CCM over 3 years. Those who returned to NGT showed an increase in CNFD (P = 0.05), CNBD (P = 0.04), and CNFL (P = 0.05), but a decrease in IENFD (P = 0.02), over 3 years. CONCLUSIONS: CCM and skin biopsy detect a small-fiber neuropathy in subjects with IGT who develop type 2 diabetes and also show a dynamic worsening or improvement in corneal and intraepidermal nerve morphology in relation to change in glucose tolerance status.

Progress in the treatment of small fiber peripheral neuropathy.

Small fiber neuropathy is a syndrome of diverse disease etiology because of multiple pathophysiologic mechanisms with major presentations of neuropathic pain and autonomic symptoms. Over the past decade, there has been substantial progress in the treatments for neuropathic pain, dysautonomia and disease-modifying strategy. In particular, anticonvulsants and antidepressants alleviate neuropathic pain based on randomized clinical trials.

Erythromelalgia mutation Q875E Stabilizes the activated state of sodium channel Nav1.7.

The human voltage-gated sodium channel Nav1.7 plays a crucial role in transmission of noxious stimuli. The inherited pain disorder erythromelalgia (IEM) has been linked to Nav1.7 gain-of-function mutations. Here we show that the IEM-associated Q875E mutation located on the pore module of Nav1.7 produces a large hyperpolarizing shift (-18 mV) in the voltage dependence of activation. Three-dimensional homology modeling indicates that the side chains of Gln-875 and the gating charge Arg-214 of the domain I voltage sensor are spatially close in the activated conformation of the channel. We verified this proximity by using an engineered disulfide bridge approach. The Q875E mutation introduces a negative charge that may modify the local electrical field experienced by the voltage sensor and, upon activation, interact directly via a salt bridge with the Arg-214 gating charge residue. Together these processes could promote transition to, and stabilization of, the domain I voltage sensor in the activated conformation and thus produce the observed gain of function. In support of this hypothesis, an increase in the extracellular concentration of Ca(2+) or Mg(2+) reverted the voltage dependence of activation of the IEM mutant to near WT values, suggesting a cation-mediated electrostatic screening of the proposed interaction between Q875E and Arg-214.