Capsaicin-Induced Endocytosis of Endogenous Presynaptic CaV2.2 in DRG-Spinal Cord Co-Cultures Inhibits Presynaptic Function.

The N-type calcium channel, CaV2.2 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 CaV2.2_HA knock-in mice, because the exofacial epitope tag in CaV2.2_HA enables accurate detection and localization of endogenous CaV2.2. CaV2.2_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. CaV2.2_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 CaV2.2_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 CaV2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in CaV2.2_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 CaV2.2_HA involves a Rab11a-dependent trafficking process, since dominant-negative Rab11a (S25N) occludes the effect of capsaicin on presynaptic CaV2.2_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 CaV2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway.

Involvement of CaV 2.2 channels and α2 δ-1 in homeostatic synaptic plasticity in cultured hippocampal neurons.

In the mammalian brain, presynaptic CaV 2 channels play a pivotal role in synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone of presynaptic terminals. However, the distribution and modulation of CaV 2.2 channels at plastic hippocampal synapses remains to be elucidated. Here, we assess CaV 2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. We show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV 2.2 channels at the presynaptic site. This work focused further on the role of α2 δ-1 subunits, important regulators of synaptic transmission and CaV 2.2 channel abundance at the presynaptic membrane. We found that α2 δ-1 overexpression reduces the contribution of CaV 2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Levels of endogenous α2 δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2 δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV 2.2 channels and novel roles for α2 δ-1 during synaptic plastic adaptation. KEY POINTS: The roles of CaV 2.2 channels and α2 δ-1 in homeostatic synaptic plasticity in hippocampal neurons in culture were examined. Chronic silencing of neuronal activity resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV 2.2 channels at presynaptic sites. The level of endogenous α2 δ-1 decreased during homeostatic synaptic plasticity, whereas overexpression of α2 δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV 2.2 channels and novel roles for α2 δ-1 during synaptic plastic adaptation.