Ca²⁺ sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca²⁺ and a number of Ca²⁺ sensors including synaptotagmins (Syts) and Double C₂ domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C₂ domain architecture, but are functionally diverse in their subcellular location, Ca²⁺-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca²⁺-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca²⁺ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca²⁺ sensors that may be implicated in the spontaneous release of glutamate.