Synaptic vesicle capture by CaV2.2 calcium channels

Abstract

The fusion of synaptic vesicles (SVs) at the presynaptic transmitter release face is gated by Ca2+ influx from nearby voltage-gated calcium channels (CaVs). Functional studies favor a direct molecular “tethering” attachment and recent studies have proposed a direct link to the channel C-terminal. To test for direct CaV–SV attachment we developed an in vitro assay, termed SV pull-down (SV-PD), to test for capture of purified, intact SVs. Antibody-immobilized presynaptic or expressed CaV2.2 channels but not plain beads, IgG or pre-blocked antibody successfully captured SVs, as assessed byWestern blot for a variety of protein markers. SV-PD was also observed with terminal fusion proteins of the distal half of the C-terminal, supporting involvement of this CaV region in tethering. Thus our results support a model in which the SV tethers directly to the CaV. Since the tip of the C-terminal could extend as far as 200 nm into the cytoplasm, we hypothesize that this link may serve as the initial SV capture mechanism by the release site. Further studies will be necessary to evaluate the molecular basis of C-terminal tethering and whether the SV binds to the channel by additional, shorter-range attachments.

Variable priming of a docked synaptic vesicle

“…Because the complexes link the VM to the PM as do the proximal rib segments/pegs and pins, their apparent length [∼14 nm (48)] is similar to the average length of the proximal rib segments/pegs (∼17 nm) and the pins (∼13 nm), and there are no discernible structures in the vicinity of the VM-PM contact site other than proximal rib segments/pegs and pins, it is reasonable to suggest that the SNARE core complex and its auxiliary proteins are components of these structures. Accordingly, whereas the SNARE core complex and its auxiliary proteins would be contained in each pin, the PM's syntaxin and SNAP25 would reach the proximal rib segments to form the core complex with the SV's synaptobrevin via the proximal pegs alongside the cytosolic portion of Ca 2+ channels, which are also thought to occupy the pegs and extend into the ribs (2,11,49). Biochemical findings indicate that the SNARE core complex and its auxiliary proteins can be linked to N-type Ca 2+ channels such as those at active zones of the frog's NMJ (50).…”

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.…”

The structure and function of ‘active zone material’ at synapses

“…the rib segment between the vesicle membrane and the peg proximal to it, while synaptotagmin is also likely to be a component of the proximal segment of the ribs. The frequency and distribution of the peg-presynaptic membrane connection sites is similar to that of the macromolecules in the presynaptic membrane seen in freeze-fracture replicas [17,18], indicating that the pegs are connected to the presynaptic membrane macromolecules and that they probably include the cytosolic portions of the presynaptic membrane's Ca 2þ -channels [38] and Ca 2þ -activated K þ -channels.…”