Abstract
At chemical synapses the incoming action potential triggers the influx of Ca2+ through voltage-sensitive calcium channels (CaVs, typically CaV2.1 and 2.2) and the ions binds to sensors associated with docked, transmitter filled synaptic vesicles (SVs), triggering their fusion and discharge. The CaVs and docked SVs are located within the active zone (AZ) region of the synapse which faces a corresponding neurotransmitter receptor-rich region on the post-synaptic cell. Evidence that the fusion of a SV can be gated by Ca2+ influx through a single CaV suggests that the channel and docked vesicle are linked by one or more molecular tethers (Stanley, 1993). Short and long fibrous SV-AZ linkers have been identified in presynaptic terminals by electron microscopy and we recently imaged these in cytosol-vacated synaptosome ‘ghosts.’ Using CaV fusion proteins combined with blocking peptides we previously identified a SV binding site near the tip of the CaV2.2 C-terminal suggesting that this intracellular channel domain participates in SV tethering. In this study, we combined the synaptosome ghost imaging method with immunogold labeling to localize CaV intracellular domains. L45, raised against the C-terminal tip, tagged tethered SVs often as far as 100 nm from the AZ membrane whereas NmidC2, raised against a C-terminal mid-region peptide, and C2Nt, raised against a peptide nearer the C-terminal origin, resulted in gold particles that were proportionally closer to the AZ. Interestingly, the observation of gold-tagged SVs with NmidC2 suggests a novel SV binding site in the C-terminal mid region. Our results implicate the CaV C-terminal in SV tethering at the AZ with two possible functions: first, capturing SVs from the nearby cytoplasm and second, contributing to the localization of the SV close to the channel to permit single domain gating.
Highlights
The fusion of synaptic vesicles (SVs) at the active zone (AZ) of classical fast-transmitting presynaptic terminals is gated by the influx of calcium ions (Ca2+) through voltage-gated calcium channels (CaVs) at the presynaptic membrane (Katz and Miledi, 1965; Llinás et al, 1992)
To test the hypothesis that the channel C-terminal contributes to long cytoplasmic SV tethers, we addressed two main questions: whether the tip of the presynaptic calcium channel C-terminal contacts tethered SVs, and whether the C-terminal mid-region is located in the cytoplasmic gap between the surface membrane and the tethered SV
When we measured the distances of gold particle clusters along tethers to the surface membrane, we found that distribution of gold clusters for each of the C2Nt, NmidC2, and L45 antibodies (Figure 9B) relatively corresponded to the respective regions that the antibodies were targeted against (Figure 1)
Summary
The fusion of synaptic vesicles (SVs) at the active zone (AZ) of classical fast-transmitting presynaptic terminals is gated by the influx of calcium ions (Ca2+) through voltage-gated calcium channels (CaVs) at the presynaptic membrane (Katz and Miledi, 1965; Llinás et al, 1992). Platinumshadowed freeze-etched images of various presynaptic terminals revealed a network of cytoplasmic filaments that linked SVs to each other and to the AZ surface membrane (Landis et al, 1988; Hirokawa et al, 1989) including long, >100 nm, AZ surface membrane to SV fibrous structures (Hirokawa et al, 1989) Such SV-AZ fibers have been imaged in more detail by means of a variety of electron microscopy-based methods (Harlow et al, 2001; Siksou et al, 2007; Fernández-Busnadiego et al, 2013; Wong et al, 2014; Cole et al, 2016). A simple hypothesis to account for these types of tethers is that the SV is initially snared, or ‘grabbed’ by the long tether which serves to guide the SV to the surface membrane where it is ‘locked’ in place and within range of the Ca2+ influx of CaVs by the shorter links (Fernández-Busnadiego et al, 2013; Wong et al, 2014; Cole et al, 2016)
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