Tetrodotoxin-sensitive sodium channels mediate action potential firing and excitability in menthol-sensitive Vglut3-lineage sensory neurons

Abstract

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) 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 Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage 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 Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.