Mark Estacion

Gain of function NaV1.7 mutations in idiopathic small fiber neuropathy

Small nerve fiber neuropathy (SFN) often occurs without apparent cause, but no systematic genetic studies have been performed in patients with idiopathic SFN (I‐SFN). We sought to identify a genetic basis for I‐SFN by screening patients with biopsy‐confirmed idiopathic SFN for mutations in the SCN9A gene, encoding voltage‐gated sodium channel NaV1.7, which is preferentially expressed in small diameter peripheral axons.

NaV1.7 Gain-of-Function Mutations as a Continuum: A1632E Displays Physiological Changes Associated with Erythromelalgia and Paroxysmal Extreme Pain Disorder Mutations and Produces Symptoms of Both Disorders

Gain-of-function mutations of NaV1.7 have been shown to produce two distinct disorders: NaV1.7 mutations that enhance activation produce inherited erythromelalgia (IEM), characterized by burning pain in the extremities; NaV1.7 mutations that impair inactivation produce a different, nonoverlapping syndrome, paroxysmal extreme pain disorder (PEPD), characterized by rectal, periocular, and perimandibular pain. Here we report a novel NaV1.7 mutation associated with a mixed clinical phenotype with characteristics of IEM and PEPD, with an alanine 1632 substitution by glutamate (A1632E) in domain IV S4–S5 linker. Patch-clamp analysis shows that A1632E produces changes in channel function seen in both IEM and PEPD mutations: A1632E hyperpolarizes (−7 mV) the voltage dependence of activation, slows deactivation, and enhances ramp responses, as observed in NaV1.7 mutations that produce IEM. A1632E depolarizes (+17mV) the voltage dependence of fast inactivation, slows fast inactivation, and prevents full inactivation, resulting in persistent inward currents similar to PEPD mutations. Using current clamp, we show that A1632E renders dorsal root ganglion (DRG) and trigeminal ganglion neurons hyperexcitable. These results demonstrate a NaV1.7 mutant with biophysical characteristics common to PEPD (impaired fast inactivation) and IEM (hyperpolarized activation, slow deactivation, and enhanced ramp currents) associated with a clinical phenotype with characteristics of both IEM and PEPD and show that this mutation renders DRG and trigeminal ganglion neurons hyperexcitable. These observations indicate that IEM and PEPD mutants are part of a physiological continuum that can produce a continuum of clinical phenotypes.

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 novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia†

Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain‐of‐function mutations of Nav1.7. We now report a novel mutation (V400M) in a three‐generation Canadian family in which pain is relieved by carbamazepine (CBZ).

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

BackgroundParoxysmal 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.ResultsSequencing 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.ConclusionM1627K 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 sodium channel gene SCN9A polymorphism that increases nociceptor excitability.

Sodium channel NaV1.7, 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 NaV1.7 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 NaV1.7 [hNaV1.7]) 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 hNaV1.7 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 (∼2‐fold) the firing frequency in response to depolarization in DRG neurons in which it is present. Our results suggest that polymorphisms in the NaV1.7 channel may influence susceptibility to pain. Ann Neurol 2009;66:862–866

A sodium channel mutation linked to epilepsy increases ramp and persistent current of Nav1.3 and induces hyperexcitability in hippocampal neurons

Voltage-gated sodium channelopathies underlie many excitability disorders. Genes SCN1A, SCN2A and SCN9A, which encode pore-forming alpha-subunits Na(V)1.1, Na(V)1.2 and Na(V)1.7, are clustered on human chromosome 2, and mutations in these genes have been shown to underlie epilepsy, migraine, and somatic pain disorders. SCN3A, the gene which encodes Na(V)1.3, is part of this cluster, but until recently was not associated with any mutation. A charge-neutralizing mutation, K345Q, in the Na(V)1.3 DI/S5-6 linker has recently been identified in a patient with cryptogenic partial epilepsy. Pathogenicity of the Na(V)1.3/K354Q mutation has been inferred from the conservation of this residue in all sodium channels and its absence from control alleles, but functional analysis has been limited to the corresponding substitution in the cardiac muscle sodium channel Na(V)1.5. Since identical mutations may produce different effects within different sodium channel isoforms, we assessed the K354Q mutation within its native Na(V)1.3 channel and studied the effect of the mutant Na(V)1.3/K354Q channels on hippocampal neuron excitability. We show here that the K354Q mutation enhances the persistent and ramp currents of Na(V)1.3, reduces current threshold and produces spontaneous firing and paroxysmal depolarizing shift-like complexes in hippocampal neurons. Our data provide a pathophysiological basis for the pathogenicity of the first epilepsy-linked mutation within Na(V)1.3 channels and hippocampal neurons.

Intra- and interfamily phenotypic diversity in pain syndromes associated with a gain-of-function variant of NaV1.7

BackgroundSodium channel NaV1.7 is preferentially expressed within dorsal root ganglia (DRG), trigeminal ganglia and sympathetic ganglion neurons and their fine-diamter axons, where it acts as a threshold channel, amplifying stimuli such as generator potentials in nociceptors. Gain-of-function mutations and variants (single amino acid substitutions) of NaV1.7 have been linked to three pain syndromes: Inherited Erythromelalgia (IEM), Paroxysmal Extreme Pain Disorder (PEPD), and Small Fiber Neuropathy (SFN). IEM is characterized clinically by burning pain and redness that is usually focused on the distal extremities, precipitated by mild warmth and relieved by cooling, and is caused by mutations that hyperpolarize activation, slow deactivation, and enhance the channel ramp response. PEPD is characterized by perirectal, periocular or perimandibular pain, often triggered by defecation or lower body stimulation, and is caused by mutations that severely impair fast-inactivation. SFN presents a clinical picture dominated by neuropathic pain and autonomic symptoms; gain-of-function variants have been reported to be present in approximately 30% of patients with biopsy-confirmed idiopathic SFN, and functional testing has shown altered fast-inactivation, slow-inactivation or resurgent current. In this paper we describe three patients who house the NaV1.7/I228M variant.MethodsWe have used clinical assessment of patients, quantitative sensory testing and skin biopsy to study these patients, including two siblings in one family, in whom genomic screening demonstrated the I228M NaV1.7 variant. Electrophysiology (voltage-clamp and current-clamp) was used to test functional effects of the variant channel.ResultsWe report three different clinical presentations of the I228M NaV1.7 variant: presentation with severe facial pain, presentation with distal (feet, hands) pain, and presentation with scalp discomfort in three patients housing this NaV1.7 variant, two of which are from a single family. We also demonstrate that the NaV1.7/I228M variant impairs slow-inactivation, and produces hyperexcitability in both trigeminal ganglion and DRG neurons.ConclusionOur results demonstrate intra- and interfamily phenotypic diversity in pain syndromes produced by a gain-of-function variant of NaV1.7.

Voltage-clamp and current-clamp recordings from mammalian DRG neurons

We provide here detailed electrophysiological protocols to study voltage-gated sodium channels and to investigate how wild-type and mutant channels influence firing properties of transfected mammalian dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings permit us to analyze kinetic and voltage-dependence properties of ion channels and to determine the effect and mode of action of pharmaceuticals on specific channel isoforms. They also permit us to analyze the role of individual sodium channels and their mutant derivatives in regulating firing of DRG neurons. Five to ten cells can be recorded daily, depending on the extent of analysis that is required. Because of different internal solutions that are used in voltage-clamp and current-clamp recordings, only limited information can be obtained from recording the same neuron in both modes. These electrophysiological studies help to elucidate the role of specific channels in setting threshold and suprathreshold responses of neurons, under normal and pathological conditions.

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.