Building sensory axons: Delivery and distribution of NaV1.7 channels and effects of inflammatory mediators.

Sodium channel NaV1.7 controls firing of nociceptors, and its role in human pain has been validated by genetic and functional studies. However, little is known about NaV1.7 trafficking or membrane distribution along sensory axons, which can be a meter or more in length. We show here with single-molecule resolution the first live visualization of NaV1.7 channels in dorsal root ganglia neurons, including long-distance microtubule-dependent vesicular transport in Rab6A-containing vesicles. We demonstrate nanoclusters that contain a median of 12.5 channels at the plasma membrane on axon termini. We also demonstrate that inflammatory mediators trigger an increase in the number of NaV1.7-carrying vesicles per axon, a threefold increase in the median number of NaV1.7 channels per vesicle and a ~50% increase in forward velocity. This remarkable enhancement of NaV1.7 vesicular trafficking and surface delivery under conditions that mimic a disease state provides new insights into the contribution of NaV1.7 to inflammatory pain.

Resilience to Pain: A Peripheral Component Identified Using Induced Pluripotent Stem Cells and Dynamic Clamp.

Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing NaV1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, KCNQ in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain.SIGNIFICANCE STATEMENT Individual-to-individual differences in pain are well documented, but not well understood. In this study, we show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing and dynamic clamp, we show that it is possible to pinpoint a specific gene variant that modulates pain signaling and contributes to interindividual differences in pain.

A novel gain-of-function Nav1.7 mutation in a carbamazepine-responsive patient with adult-onset painful peripheral neuropathy.

Voltage-gated sodium channel Nav1.7 is a threshold channel in peripheral dorsal root ganglion (DRG), trigeminal ganglion, and sympathetic ganglion neurons. Gain-of-function mutations in Nav1.7 have been shown to increase excitability in DRG neurons and have been linked to rare Mendelian and more common pain disorders. Discovery of Nav1.7 variants in patients with pain disorders may expand the spectrum of painful peripheral neuropathies associated with a well-defined molecular target, thereby providing a basis for more targeted approaches for treatment. We screened the genome of a patient with adult-onset painful peripheral neuropathy characterized by severe burning pain and report here the new Nav1.7-V810M variant. Voltage-clamp recordings were used to assess the effects of the mutation on biophysical properties of Nav1.7 and the response of the mutant channel to treatment with carbamazepine (CBZ), and multi-electrode array (MEA) recordings were used to assess the effects of the mutation on the excitability of neonatal rat pup DRG neurons. The V810M variant increases current density, shifts activation in a hyperpolarizing direction, and slows kinetics of deactivation, all gain-of-function attributes. We also show that DRG neurons that express the V810M variant become hyperexcitable. The patient responded to treatment with CBZ. Although CBZ did not depolarize activation of the mutant channel, it enhanced use-dependent inhibition. Our results demonstrate the presence of a novel gain-of-function variant of Nav1.7 in a patient with adult-onset painful peripheral neuropathy and the responsiveness of that patient to treatment with CBZ, which is likely due to the classical mechanism of use-dependent inhibition.

Atypical changes in DRG neuron excitability and complex pain phenotype associated with a Nav1.7 mutation that massively hyperpolarizes activation.

Sodium channel Nav1.7 plays a central role in pain-signaling: gain-of-function Nav1.7 mutations usually cause severe pain and loss-of-function mutations produce insensitivity to pain. The Nav1.7 I234T gain-of-function mutation, however, is linked to a dual clinical presentation of episodic pain, together with absence of pain following fractures, and corneal anesthesia. How a Nav1.7 mutation that produces gain-of-function at the channel level causes clinical loss-of-function has remained enigmatic. We show by current-clamp that expression of I234T in dorsal root ganglion (DRG) neurons produces a range of membrane depolarizations including a massive shift to >-40 mV that reduces excitability in a small number of neurons. Dynamic-clamp permitted us to mimic the heterozygous condition via replacement of 50% endogenous wild-type Nav1.7 channels by I234T, and confirmed that the I234T conductance could drastically depolarize DRG neurons, resulting in loss of excitability. We conclude that attenuation of pain sensation by I234T is caused by massively depolarized membrane potential of some DRG neurons which is partly due to enhanced overlap between activation and fast-inactivation, impairing their ability to fire. Our results demonstrate how a Nav1.7 mutation that produces channel gain-of-function can contribute to a dual clinical presentation that includes loss of pain sensation at the clinical level.

Reverse pharmacogenomics: carbamazepine normalizes activation and attenuates thermal hyperexcitability of sensory neurons due to Nav 1.7 mutation I234T.

BACKGROUND AND PURPOSE: Pharmacotherapy for pain currently involves trial and error. A previous study on inherited erythromelalgia (a genetic model of neuropathic pain due to mutations in the sodium channel, Nav 1.7) used genomics, structural modelling and biophysical and pharmacological analyses to guide pharmacotherapy and showed that carbamazepine normalizes voltage dependence of activation of the Nav 1.7-S241T mutant channel, reducing pain in patients carrying this mutation. However, whether this approach is applicable to other Nav channel mutants is still unknown. EXPERIMENTAL APPROACH: We used structural modelling, patch clamp and multi-electrode array (MEA) recording to assess the effects of carbamazepine on Nav 1.7-I234T mutant channels and on the firing of dorsal root ganglion (DRG) sensory neurons expressing these mutant channels. KEY RESULTS: In a reverse engineering approach, structural modelling showed that the I234T mutation is located in atomic proximity to the carbamazepine-responsive S241T mutation and that activation of Nav 1.7-I234T mutant channels, from patients who are known to respond to carbamazepine, is partly normalized with a clinically relevant concentration (30 µM) of carbamazepine. There was significantly higher firing in intact sensory neurons expressing Nav 1.7-I234T channels, compared with neurons expressing the normal channels (Nav 1.7-WT). Pre-incubation with 30 µM carbamazepine also significantly reduced the firing of intact DRG sensory neurons expressing Nav 1.7-I234T channels. Although the expected use-dependent inhibition of Nav 1.7-WT channels by carbamazepine was confirmed, carbamazepine did not enhance use-dependent inhibition of Nav 1.7-I234T mutant channels. CONCLUSION AND IMPLICATIONS: These results support the utility of a pharmacogenomic approach to treatment of pain in patients carrying sodium channel variants. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.