Ca_v2.2 Channels Sustain Vesicle Recruitment at a Mature Glutamatergic Synapse

Abstract

The composition of voltage-gated Ca²⁺ channel (Ca_v) subtypes that gate action potential (AP)-evoked release changes during the development of mammalian CNS synapses. Ca_v2.2 and Ca_v2.3 lose their function in gating-evoked release during postnatal synapse maturation. In mature boutons, Ca_v2.1 currents provide the almost exclusive trigger for evoked release, and Ca_v2.3 currents are required for the induction of presynaptic long-term potentiation. However, the functional significance of Ca_v2.2 remained elusive in mature boutons, although they remain present at active zones and continue contributing significantly to presynaptic Ca²⁺ influx. Here, we addressed the functional significance of Ca_v2.2 and Ca_v2.3 at mature parallel-fiber (PF) to Purkinje neuron synapses of mice of either sex. These synapses are known to exhibit the corresponding developmental Ca_v subtype changes in gating release. We addressed two hypotheses, namely that Ca_v2.2 and Ca_v2.3 are involved in triggering spontaneous glutamate release and that they are engaged in vesicle recruitment during repetitive evoked release. We found that spontaneous miniature release is Ca²⁺ dependent. However, experiments with Ca_v subtype-specific blockers excluded the spontaneous opening of Ca_vs as the Ca²⁺ source for spontaneous glutamate release. Thus, neither Ca_v2.2 nor Ca_v2.3 controls spontaneous release from PF boutons. Furthermore, vesicle recruitment during brief bursts of APs was also independent of Ca²⁺ influx through Ca_v2.2 and Ca_v2.3. However, Ca_v2.2, but not Ca_v2.3, currents significantly boosted vesicle recruitment during sustained high-frequency synaptic transmission. Thus, in mature PF boutons Ca_v2.2 channels are specifically required to sustain synaptic transmission during prolonged neuronal activity.

SIGNIFICANCE STATEMENT At young CNS synapses, action potential-evoked release is gated via three subtypes of voltage-gated Ca²⁺ channels: Ca_v2.1, Ca_v2.2, and Ca_v2.3. During postnatal maturation, Ca_v2.2 and Ca_v2.3 lose their function in gating evoked release, such that at mature synapses Ca_v2.1 provides the almost exclusive source for triggering evoked release. Ca_v2.3 currents are required for the induction of presynaptic long-term potentiation. However, the function of the still abundant Ca_v2.2 in mature boutons remained largely elusive. Here, we studied mature cerebellar parallel-fiber synapses and found that Ca_v2.2 does not control spontaneous release. However, Ca²⁺ influx through Ca_v2.2 significantly boosted vesicle recruitment during trains of action potentials. Thus, Ca_v2.2 in mature parallel-fiber boutons participate in sustaining synaptic transmission during prolonged activity.