Tetrodotoxin-Sensitive Sodium Channels Mediate Action Potential Firing and Excitability in Menthol-Sensitive Vglut3-Lineage Sensory Neurons.

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3 neurons. Whole-cell recordings showed that small-diameter Vglut3 DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3 neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3 neurons. A biophysical analysis revealed voltage-gated sodium channel (Na) currents in menthol-sensitive Vglut3 neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive Na transcripts between TRPM8-positive and -negative Vglut3 neurons; however, Na1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of Na subunits, with Na1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3 neurons rely primarily on TTX-resistant Na channels. Additionally, when Na1.1 channels were blocked, the remaining Na current readily entered into slow inactivation in menthol-sensitive Vglut3 neurons. Thus, these data demonstrate that TTX-sensitive Nas drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability. Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of Na1.1 as a therapeutic target for sensory disorders.