Small-diameter vesicular glutamate transporter 3-lineage (Vglut3^lineage) 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^lineage neurons. Whole-cell recordings showed that small-diameter Vglut3^lineage 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^lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3^lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (Na_V) currents in menthol-sensitive Vglut3^lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive Na_V transcripts between TRPM8-positive and -negative Vglut3^lineage neurons; however, Na_V1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of Na_V subunits, with Na_V1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3^lineage neurons rely primarily on TTX-resistant Na_V channels. Additionally, when Na_V1.1 channels were blocked, the remaining Na_V current readily entered into slow inactivation in menthol-sensitive Vglut3^lineage neurons. Thus, these data demonstrate that TTX-sensitive Na_Vs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT 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 Na_V1.1 as a therapeutic target for sensory disorders.

TRAAK, TREK-1, and TREK-2 are mechanosensitive two-pore domain K+ (K2P) channels that contribute to action potential propagation, sensory transduction, and muscle contraction. While structural and functional studies have led to models that explain their mechanosensitivity, we lack a quantitative understanding of channel activation by membrane tension. Here, we define the tension response of mechanosensitive K2Ps using patch-clamp recording and imaging. All are low-threshold mechanosensitive channels (T10%/50% 0.6-2.7 / 4.4-6.4 mN/m) with distinct response profiles. TRAAK is most sensitive, TREK-1 intermediate, and TREK-2 least sensitive. TRAAK and TREK-1 are activated broadly over a range encompassing nearly all physiologically relevant tensions. TREK-2, in contrast, activates over a narrower range like mechanosensitive channels Piezo1, MscS, and MscL. We further show that low-frequency, low-intensity focused ultrasound increases membrane tension to activate TRAAK and MscS. This work provides insight into tension gating of mechanosensitive K2Ps relevant to understanding their physiological roles and potential applications for ultrasonic neuromodulation.