The Calcium Channel C-Terminal and Synaptic Vesicle Tethering: Analysis by Immuno-Nanogold Localization

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.

Vesicle sub‐pool organization at inner hair cell ribbon synapses

“…Still, neither protein can fully account for tether formation at the membrane. A recent study could show in chicken synaptosomal preparations that the C‐term of Ca V 2.1 and 2.2 Ca 2+ channels might directly tether SVs to the membrane . However, whether the Ca V 1.2 Ca 2+ channel could also fulfill this function has not been tested so far.…”

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal

“…Software predictions suggest that the C-terminal exhibits very little predicted secondary structure and its tip could, at least in theory, range far into the surrounding cytoplasm (Wong et al, 2014 ). We recently used nanogold immunocytochemistry to localize the different regions of the CaV2.2 C-terminal within cytoplasm-vacated, but tethered SV-retaining, nerve terminals (Chen et al, 2017 ). We found that the tip of the C-terminal, in the region corresponding to our first identified (C3) SV-binding motif, contacts SVs up to and over 100 nm from the active zone, at least in nerve terminals vacated of cytoplasmic constituents.…”

“…These peptides were made with a terminal cysteine to permit conjugation to a carrier protein, KLH (Biomatik, Cambridge, Canada). Immunization of rabbits were done as previously described (Chen et al, 2017 ). All antibodies used in the present study are listed in Table 1 .…”

“…Electron micrographic analysis of untethered-SV-vacated synaptosomes identified a small population of luminal SVs that were observed to be attached as far as ~200 nm from the surface membrane via faint fibrous processes (Wong et al, 2014 ). Further, nanogold immunolabeling suggests that these tethers include the unraveled channel C-terminal (Chen et al, 2017 ). Such long tethers could contribute to SV loading into the docking site near the channel but shorter links might be predicted to account for the short, ~25 nm distance, requirement for single domain gating (Stanley, 1993 , 2015 ; Matveev et al, 2011 ; Dittrich and Meriney, 2016 ).…”

Cellular and Structural Studies of Eukaryotic Cells by Cryo-Electron Tomography

“…A common way to tag a protein of interest for EM analysis is immuno-gold labeling [108]. Several studies have impressively shown how antibodies coupled to colloidal gold could be used to identify specific proteins by electron microscopy, e.g., to determine the localization of individual components of NPCs or to investigate the co-localization of neurotransmitters in neurons [89,109,110,111,112,113]. In a proof-of-concept study, native immuno-gold labeling was applied to living cells to identify hRSV assembly sites in mammalian cells and study virus–host interactions [114].…”