Capsaicin-Induced Endocytosis of Endogenous Presynaptic Ca_V2.2 in DRG-Spinal Cord Co-Cultures Inhibits Presynaptic...

The N-type calcium channel, Ca_V2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their postsynaptic targets in the spinal cord. In this study, we have utilized Ca_V2.2_HA knock-in mice, because the exofacial epitope tag in Ca_V2.2_HA enables accurate detection and localization of endogenous Ca_V2.2. Ca_V2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. Ca_V2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in Ca_V2.2_HA expression and increased association with the postsynaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface Ca_V2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in Ca_V2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca²⁺ influx. The mechanism of this down-regulation of Ca_V2.2_HA involves a Rab11a-dependent trafficking process, since dominant-negative Rab11a (S25N) occludes the effect of capsaicin on presynaptic Ca_V2.2_HA expression, and also prevents the effect of capsaicin on action potential-induced Ca²⁺ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface Ca_V2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway.