AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPARlike. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process.

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The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel NaV1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased NaV1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of NaV1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live-imaging approach that allows visualization of NaV1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration- and time-dependent effects of PTX on vesicular trafficking and membrane localization of NaV1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous NaV1.7 mRNA and current density in DRG neurons. However, the current produced by transfection of DRG neurons with Halo-tag NaV1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-NaV1.7 that we observed by live imaging in tranfected DRG neurons after treatment with PTX might be independent of an increased pool of NaV1.7 channels. After exposure to inflammatory mediators (IM) to mimic the inflammatory condition seen during chemotherapy, both NaV1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous NaV1.7 channels in DRG neurons and enhances trafficking and surface distribution of NaV1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

Diabetes mellitus is a global challenge with many diverse health sequelae, of which diabetic peripheral neuropathy is one of the most common. A substantial number of patients with diabetic peripheral neuropathy develop chronic pain, but the genetic and epigenetic factors that predispose diabetic peripheral neuropathy patients to develop neuropathic pain are poorly understood. Recent targeted genetic studies have identified mutations in a-subunits of voltage-gated sodium channels (Na v s) in patients with painful diabetic peripheral neuropathy. Mutations in proteins that regulate trafficking or functional properties of Na v s could expand the spectrum of patients with Na v-related peripheral neuropathies. The auxiliary sodium channel b-subunits (b1-4) have been reported to increase current density, alter inactivation kinetics, and modulate subcellular localization of Na v. Mutations in b-subunits have been associated with several diseases, including epilepsy, cancer, and diseases of the cardiac conducting system. However, mutations in b-subunits have never been shown previously to contribute to neuropathic pain. We report here a patient with painful diabetic peripheral neuropathy and negative genetic screening for mutations in SCN9A, SCN10A, and SCN11A-genes encoding sodium channel a-subunit that have been previously linked to the development of neuropathic pain. Genetic analysis revealed an aspartic acid to asparagine mutation, D109N, in the b2-subunit. Functional analysis using current-clamp revealed that the b2-D109N rendered dorsal root ganglion neurons hyperexcitable, especially in response to repetitive stimulation. Underlying the hyperexcitability induced by the b2-subunit mutation, as evidenced by voltage-clamp analysis, we found a depolarizing shift in the voltage dependence of Na v 1.7 fast inactivation and reduced use-dependent inhibition of the Na v 1.7 channel.

Neuronal excitability relies on coordinated action of functionally distinction channels. Voltage-gated sodium (Na V ) and potassium (K V ) channels have distinct but complementary roles in firing action potentials: Na V channels provide depolarizing current while K V channels provide hyperpolarizing current. Mutations and dysfunction of multiple Na V and K V channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance imaging to investigate trafficking of Na V and K V channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to Na V 1.7 channels, Na V 1.8 and K V 7.2 channels are transported in Rab6a-positive vesicles, and that each of the Na V channel isoforms expressed in healthy, mature sensory neurons (Na V 1.6, Na V 1.7, Na V 1.8, and Na V 1.9) is cotransported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions (Na V 1.7, K V 7.2, and TNFR1) are cotransported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein (NCX2) is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.

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Recapitulating human disease pathophysiology using genetic animal models is a powerful approach to enable mechanistic understanding of genotype-phenotype relationships for drug development. Na V 1.7 is a sodium channel expressed in the peripheral nervous system with strong human genetic validation as a pain target. Efforts to identify novel analgesics that are nonaddictive resulted in industry exploration of a class of sulfonamide compounds that bind to the fourth voltage-sensor domain of Na V 1.7. Due to sequence differences in this region, sulfonamide blockers generally are potent on human but not rat Na V 1.7 channels. To test sulfonamide-based chemical matter in rat models of pain, we generated a humanized Na V 1.7 rat expressing a chimeric Na V 1.7 protein containing the sulfonamide-binding site of the human gene sequence as a replacement for the equivalent rat sequence. Unexpectedly, upon transcription, the human insert was spliced out, resulting in a premature stop codon. Using a validated antibody, Na V 1.7 protein was confirmed to be lost in the brainstem, dorsal root ganglia, sciatic nerve, and gastrointestinal tissue but not in nasal turbinates or olfactory bulb in rats homozygous for the knock-in allele (HOM-KI). HOM-KI rats exhibited normal intraepidermal nerve fiber density with reduced tetrodotoxin-sensitive current density and action potential firing in small diameter dorsal root ganglia neurons. HOM-KI rats did not exhibit nociceptive pain responses in hot plate or capsaicin-induced flinching assays and did not exhibit neuropathic pain responses following spinal nerve ligation. Consistent with expression of chimeric Na V 1.7 in olfactory tissue, HOM-KI rats retained olfactory function. This new genetic model highlights the necessity of Na V 1.7 for pain behavior in rats and indicates that sufficient inhibition of Na V 1.7 in humans may reduce pain in neuropathic conditions. Due to preserved olfactory function, this rat model represents an alternative to global Na V 1.7 knockout mice that require time-intensive hand feeding during early postnatal development.

The inhibition of voltage-gated sodium (NaV) channels in somatosensory neurons presents a promising novel modality for the treatment of pain. However, the precise contribution of these channels to neuronal excitability, the cellular correlate of pain, is unknown; previous studies using genetic knockout models or pharmacologic block of NaV channels have identified general roles for distinct sodium channel isoforms, but have never quantified their exact contributions to these processes. To address this deficit, we have utilized dynamic clamp electrophysiology to precisely tune in varying levels of NaV1.8 and NaV1.9 currents into induced pluripotent stem cell-derived sensory neurons (iPSC-SNs), allowing us to quantify how graded changes in these currents affect different parameters of neuronal excitability and electrogenesis. We quantify and report direct relationships between NaV1.8 current density and action potential half-width, overshoot, and repetitive firing. We additionally quantify the effect varying NaV1.9 current densities have on neuronal membrane potential and rheobase. Furthermore, we examined the simultaneous interplay between NaV1.8 and NaV1.9 on neuronal excitability. Finally, we show that minor biophysical changes in the gating of NaV1.8 can render human iPSC-SNs hyperexcitable, in a first-of-its-kind investigation of a gain-of-function NaV1.8 mutation in a human neuronal background.