Capsaicin-Induced Endocytosis of Endogenous Presynaptic Ca(V)2.2 in DRG-Spinal Cord Co-Cultures Inhibits Presynaptic Function

The N-type calcium channel, Ca(V)2.2 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(V)2.2_HA knock-in mice, because the exofacial epitope tag in Ca(V)2.2_HA enables accurate detection and localization of endogenous Ca(V)2.2. Ca(V)2.2_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(V)2.2_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(V)2.2_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(V)2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in Ca(V)2.2_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 Ca(2+) influx. The mechanism of this down-regulation of Ca(V)2.2_HA involves a Rab11a-dependent trafficking process, since dominant-negative Rab11a (S25N) occludes the effect of capsaicin on presynaptic Ca(V)2.2_HA expression, and also prevents the effect of capsaicin on action potential-induced Ca(2+) influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface Ca(V)2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway.