Synaptic vesicle tethering and the CaV2.2 distal C-terminal

Abstract

Evidence that synaptic vesicles (SVs) can be gated by a single voltage sensitive calcium channel (CaV2.2) predict a molecular linking mechanism or “tether” (Stanley, 1993). Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel (Kaeser et al., 2011; Wong et al., 2013) while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site (Kaeser et al., 2011). Using a novel in vitro SV pull down binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137–2357; Wong et al., 2013). Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137–2299). To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349–2357) and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or “grabbed,” from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be “locked” close to the channel by a second attachment mechanism in preparation for single channel domain gating.

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…Second, although they are able to bind to syntaxin 1A in vitro, Ca V 2.3 calcium channels do not have a synprint-like motif. Finally, work from several groups has identified postsynaptic density protein PSD95, Drosophila disc large tumor suppressor Dlg1, and Zona occludens-1 protein-containing proteins such as Rab3-interacting molecule and MINT-1 as critical anchors between Ca V 2 channels and synaptic vesicles (Maximov and Bezprozvanny, 2002;Han et al, 2011Han et al, , 2015Kaeser et al, 2011;Wong et al, 2013Wong et al, , 2014, with the interactions being critically dependent on the C-terminal region of the channel.…”

A system for studying mechanisms of neuromuscular junction development and maintenance

“…The following antibodies were used: mouse anti-βIII tubulin (R&D systems, MAB1195, clone #TuJ1, 1/400), rabbit anti-ryanodine receptor (Braubach et al, 2014) (Millipore, AB9078, 1/200), goat anti-VaChT (Atasoy et al, 2014) (Millipore, ABN100, 1/100), goat anti-ChAT (Sümbül et al, 2014) (Millipore, AB144, 1/100), chicken anti-neurofilament H (NFH) (Wainger et al, 2015) (Millipore, AB5539, 1/400), mouse anti-dihydropyridine receptor (DHPR) (Bradley et al, 2014) (Abcam, Ab2864, 1/400), mouse anti-synaptotagmin (Wong et al, 2014) (Abcam, ab13259 clone ASV30, 1/100), mouse anti-α-actinin (Falcone et al, 2014) (Sigma-Aldrich, A5044 clone BM-75.2, 1/500), mouse anti-sodium channel pan (Bailey et al, 2003) (Sigma-Aldrich, clone K58/35, S8809, 1/100), mouse anti-ankyrin (Bailey et al, 2003) (Thermo Scientific, 33-8800, clone 4G3F8, 1/100), mouse antibassoon (Jing et al, 2013) (Abcam, ab82958, 1/100), rabbit anti-glial fibrillary acidic protein (GFAP) (Achtstätter et al, 1986) (Dako, Z0334, 1/100), mouse anti-oligodendrocytic marker O4 (Paintlia et al, 2004) (Sigma-Aldrich, O7139, clone O4, 1/400), rabbit anti-MuSK (serum T194, gift from Markus Ruegg, Biozentrum, University of Basel, Switzerland, 1/500), mouse anti-rapsyn [Abcam, ab11423 (1234), 1/200], mouse antiSyne1 (clone 8c3, gift from Glenn Morris, Keele University, UK, 1/200).…”

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

“…Early platinum-shadowed 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). Recently 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). At least two main types have been identified: a single, ‘long tether’ link (∼>45 nm) and multiple ‘short tethers’ (Fernández-Busnadiego et al, 2013; Wong et al, 2014; Cole et al, 2016).…”

“…Recently 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). At least two main types have been identified: a single, ‘long tether’ link (∼>45 nm) and multiple ‘short tethers’ (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 then ‘locked’ in place and within range of the Ca 2+ influx of CaVs by the shorter links (Fernández-Busnadiego et al, 2013; Wong et al, 2014; Cole et al, 2016).…”

“…While such a connection could conceivably contribute to the short tether type because of maximum extension of the II–III loop backbone (∼60 nm max), they are unlikely to account for the long tethers filaments, which may be up to 190 nm in length (Wong et al, 2014). …”