Background: Extracellular acidosis mediates pain and inflammation by activating sensory afferent neurons. Results: Protons activate and sensitize human TRPA1 in a strongly species-specific manner encoded by transmembrane domains 5 and 6. Conclusion: Our data identify TRPA1 as an ion channel likely to mediate acid-induced pain in humans. Significance: Protons are the first known endogenous agonists of TRPA1 with species-specificity for human TRPA1.

SummaryHuman pluripotent stem cells (hPSCs) offer the opportunity to generate neuronal cells, including nociceptors. Using a chemical-based approach, we generated nociceptive sensory neurons from HUES6 embryonic stem cells and retrovirally reprogrammed induced hPSCs derived from fibroblasts. The nociceptive neurons expressed respective markers and showed tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) voltage-gated sodium currents in patch-clamp experiments. In contrast to their counterparts from rodent dorsal root ganglia, TTXr currents of hPSC-derived nociceptors unexpectedly displayed a significantly more hyperpolarized voltage dependence of activation and fast inactivation. This apparent discrepancy is most likely due to a substantial expression of the developmentally important sodium channel NAV1.5. In view of the obstacles to recapitulate neuropathic pain in animal models, our data advance hPSC-derived nociceptors as a better model to study developmental and pathogenetic processes in human nociceptive neurons and to develop more specific small molecules to attenuate pain.

BackgroundGain-of-function mutations of the nociceptive voltage-gated sodium channel Nav1.7 lead to inherited pain syndromes, such as paroxysmal extreme pain disorder (PEPD). One characteristic of these mutations is slowed fast-inactivation kinetics, which may give rise to resurgent sodium currents. It is long known that toxins from Anemonia sulcata, such as ATX-II, slow fast inactivation and skin contact for example during diving leads to various symptoms such as pain and itch. Here, we investigated if ATX-II induces resurgent currents in sensory neurons of the dorsal root ganglion (DRGs) and how this may translate into human sensations.ResultsIn large A-fiber related DRGs ATX-II (5 nM) enhances persistent and resurgent sodium currents, but failed to do so in small C-fiber linked DRGs when investigated using the whole-cell patch-clamp technique. Resurgent currents are thought to depend on the presence of the sodium channel β4-subunit. Using RT-qPCR experiments, we show that small DRGs express significantly less β4 mRNA than large sensory neurons. With the β4-C-terminus peptide in the pipette solution, it was possible to evoke resurgent currents in small DRGs and in Nav1.7 or Nav1.6 expressing HEK293/N1E115 cells, which were enhanced by the presence of extracellular ATX-II. When injected into the skin of healthy volunteers, ATX-II induces painful and itch-like sensations which were abolished by mechanical nerve block. Increase in superficial blood flow of the skin, measured by Laser doppler imaging is limited to the injection site, so no axon reflex erythema as a correlate for C-fiber activation was detected.ConclusionATX-II enhances persistent and resurgent sodium currents in large diameter DRGs, whereas small DRGs depend on the addition of β4-peptide to the pipette recording solution for ATX-II to affect resurgent currents. Mechanical A-fiber blockade abolishes all ATX-II effects in human skin (e.g. painful and itch-like paraesthesias), suggesting that it mediates its effects mainly via activation of A-fibers.

Background: Mutations in the sodium channel Nav1.7 cause the inherited pain syndromes IEM and PEPD. Results:The new IEM mutation A1632T impairs channel inactivation, whereas an IEM/PEPD crossover mutation (A1632E) at the same position additionally increases resurgent sodium currents. Conclusion: Reduced inactivation without increased resurgent currents induces symptoms of IEM. Significance: Resurgent currents are likely to determine whether a mutation leads to IEM or PEPD.

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation.