Molecular Dissection of Active Zone Functions in Neurotransmitter Release

Summary Within a nerve terminal, neurotransmitter-filled vesicles fuse at the active zone. The active zone consists of protein scaffolds that are anchored to the plasma membrane and form sites for vesicle fusion, and active zone proteins mediate both the release of synaptic and neuromodulatory transmitters. Interactions between active zone proteins and voltage-gated Ca2+ channels (CaVs) have long been of central interest. Ca2+ influx through channels of the CaV2 family triggers secretion, and CaV2 positioning determines the sub-millisecond timing, magnitude and plasticity of release. In previous work, we distinguished between two competing models of CaV2s. In the “assembly model”, CaV2s are structural scaffolds with functions in active zone assembly and vesicle docking. In the “anchored model”, active zone proteins recruit CaV2s to anchor them to release sites, and presynaptic assembly is CaV2-independent. We found that active zone assembly and vesicle docking are unaffected by genetic ablation of CaV2s. This argues strongly against the assembly model. The alternate anchored model remains difficult to establish. It is uncertain how CaV2s are targeted to active zones and whether this model applies broadly to synaptic and neuromodulatory transmission. One line of work has found that disrupting binding between CaV2 channels and active zone proteins results in impaired CaV2 recruitment. However, no specific CaV2 binding activity is required, and it remains unknown which CaV2 sequences suffice to mediate CaV2 active zone delivery. We hypothesize that the CaV2 C-terminus contains multiple interaction motifs that together provide a module for CaV delivery to release sites in most synaptic and neuromodulatory transmitter systems. We build on two lines of preliminary data. First, we find that multiple sequence elements in the CaV2 C-terminus together mediate presynaptic delivery, and that they can serve to target non-active zone CaVs to release sites. Second, our experiments suggest that release of two volume transmitters, serotonin and acetylcholine, fully depends on CaV2s. In aim 1, we define CaV2 sequences that mediate CaV active zone targeting at hippocampal synapses. In aim 2, we determine the CaV2-dependence of neuromodulatory transmitter release in the brain, focusing on serotonin, acetylcholine and norepinephrine. Our work will establish mechanisms of CaV2 targeting to the active zone and will determine whether these mechanisms can fundamentally operate in both synaptic and neuromodulatory transmission through assessing their CaV2 dependence. Altogether, the proposed experiments will likely lead to broad support for the anchored model across transmitter systems. The understanding of CaV2s that we develop in this line of work is important for determining how neurotransmission operates and how it breaks down in disease.