Search

The sodium channels Na v 1.7, Na v 1.8 and Na v 1.9 are critical for pain perception in peripheral nociceptors. Loss of function of Na v 1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of K v 4.2, restores nociception in Na v 1.7 knockout (Na v 1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Na v 1.7 and activates Na v 1.9 but does not affect Na v 1.8. This toxin produces pain in wild-type (WT) and Na v 1.7-KO mice, and attenuates nociception in Na v 1.9-KO mice, but has no effect in Na v 1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Na v 1.9 activation and offers pharmacological insight into the relationship of the three Na v channels in pain signalling.

Acute pruritus occurs in various disorders. Despite severe repercussions on quality of life treatment options remain limited. Voltage-gated sodium channels (Na V ) are indispensable for transformation and propagation of sensory signals implicating them as drug targets. Here, Na V 1.7, 1.8 and 1.9 were compared for their contribution to itch by analysing Na V -specific knockout mice. Acute pruritus was induced by a comprehensive panel of pruritogens (C48/80, endothelin, 5-HT, chloroquine, histamine, lysophosphatidic acid, trypsin, SLIGRL, β-alanine, BAM8-22), and scratching was assessed using a magnet-based recording technology. We report an unexpected stimulus-dependent diversity in Na V channel-mediated itch signalling. Na V 1.7 -/- showed substantial scratch reduction mainly towards strong pruritogens. Na V 1.8 -/- impaired histamine and 5-HT-induced scratching while Na V 1.9 was involved in itch signalling towards 5-HT, C48/80 and SLIGRL. Furthermore, similar microfluorimetric calcium responses of sensory neurons and expression of itch-related TRP channels suggest no change in sensory transduction but in action potential transformation and conduction. The cumulative sum of scratching over all pruritogens confirmed a leading role of Na V 1.7 and indicated an overall contribution of Na V 1.9. Beside the proposed general role of Na V 1.7 and 1.9 in itch signalling, scrutiny of time courses suggested Na V 1.8 to sustain prolonged itching. Therefore, Na V 1.7 and 1.9 may represent targets in pruritus therapy.

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems ( e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aa Nav1.7 , which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies., Competing Interests: The authors have declared that no competing interests exist.