Michael Seagar

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the α1A subunit of the P/Q-type calcium channel

Synaptotagmins are synaptic vesicle proteins containing two calcium‐binding C2 domains which are involved in coupling calcium influx through voltage‐gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q‐type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co‐immunoprecipitated [125I]ω‐conotoxin MVIIC‐labelled calcium channels. Direct interactions were studied between in vitro‐translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the α1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780–969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium‐independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4°C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore‐forming subunit of the P/Q‐type channel may optimally locate the calcium‐binding sites that initiate exocytosis within a zone of voltage‐gated calcium entry.

Calmodulin and lipid binding to synaptobrevin regulates calcium‐dependent exocytosis

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP‐25. Calcium influx is thought to control this process via Ca2+‐binding proteins that associate with components of the SNARE complex. Ca2+/calmodulin or phospholipids bind in a mutually exclusive fashion to a C‐terminal domain of VAMP (VAMP77–90), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild‐type VAMP77–90, but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre‐incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca2+‐dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT‐resistant (Q76V, F77W) VAMP, but additional targeted mutations in VAMP77–90 abolished its ability to rescue release. The calmodulin‐ and phospholipid‐binding domain of VAMP 2 is thus required for Ca2+‐dependent exocytosis, possibly to regulate SNARE complex assembly.

A fast, single-vesicle fusion assay mimics physiological SNARE requirements

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5–10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5–11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.

INTERACTION OF CYSTEINE STRING PROTEINS WITH THE ALPHA 1A SUBUNIT OF THE P/Q-TYPE CALCIUM CHANNEL

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with125I-ω-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparentK D = 700 nm at pH 7.4 and 4 °C) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the α1A calcium channel subunit (BI isoform, residues 780–969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.

Synaptotagmins in membrane traffic: Which vesicles do the tagmins tag?

The aim of this review is to give a broad picture of what is actually known about the synaptotagmin family. Synaptotagmin I is an abundant synaptic vesicle and secretory granule protein in neurons and endocrine cells which plays a key role in Ca(2+)-induced exocytosis. It belongs to the large family of C2 domain-proteins as it contains two internal repeats that have homology to the C2 domain of protein kinase C. Eleven synaptotagmin genes have been described in rat and mouse. Except for synaptotagmin I, and by analogy synaptotagmin II, the functions of these proteins are unknown. In this review we focus on data obtained on the various isoforms without exhaustively discussing the role of synaptotagmin I in neurotransmission. Numerous in vitro interactions of synaptotagmin I with key components of the exocytosis-endocytosis machinery have been reported. Additional data concerning the other synaptotagmins are now becoming available and are reviewed here. Only interactions which have been described for several synaptotagmins, are mentioned. It is unlikely that a single isoform displays all of these potential interactions in vivo and probably the subcellular distribution of the protein will favor some of them and preclude others. Therefore, to discuss the putative role of the various synaptotagmins we have examined in detail published data concerning their localization.

V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release.

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels

Nerve terminal protein complexes implicated in exocytosis were examined by immuno‐isolation from rat brain synaptosomes. Immunoprecipitation with anti‐syntaxin or anti‐VAMP antibodies revealed a syntaxin‐SNAP25‐VAMP‐synaptotagmin complex. Anti‐VAMP antibodies also trapped a distinct VAMP‐synaptophysin complex. A similar fraction (about 70%) of N‐type calcium channels ([125I]ω conotoxin GVIA receptors), was immunoprecipitated by either anti‐syntaxin or anti‐VAMP antibodies, but not by anti‐synaptophysin antibodies (< 4%). The majority of N‐ but not L‐type calcium channels ([3H]PN200‐110 receptors), appear to be associated with a synaptic vesicle prefusion complex.

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized ω conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.

Synaptotagmin I and IV define distinct populations of neuronal transport vesicles

Mammalian synaptotagmins constitute a multigene family of at least 11 membrane proteins. We have characterized synaptotagmin IV using antibodies directed against the C2A domain of the protein. Antibodies reacted specifically with a protein band that migrated as a 41–44 kDa doublet. Synaptotagmin IV expression was regulated throughout development. A strong decrease in the amount detected by Western blotting occurred between postnatal day 5 and adulthood, in agreement with studies on the expression of synaptotagmin IV transcripts. In subcellular fractionation, synaptotagmin IV was not detected in the synaptic vesicle‐enriched fraction. Immunofluorescence microscopy was concordant with this finding. In 6‐day‐old rat cerebellum and cultured hippocampal neurons the subcellular distribution of synaptotagmin IV was clearly different from that of synaptotagmin I. Synaptotagmin IV displayed a punctate non‐polarized distribution on neuronal extensions, whereas synaptotagmin I staining was essentially synaptic. Synaptotagmin IV staining was also observed in the soma in strong perinuclear fluorescent puncta superimposed on that of Golgi/TGN markers. Furthermore, synaptotagmin IV was seen in the proximal part of the growth cone domain and not in the microfilament‐rich region which includes filopodia. Co‐localizations with the adhesion molecules vinculin and zyxin at the proximal part of growth cones were observed. Synaptotagmin IV may thus be involved in the regulation of specific membrane‐trafficking pathways during brain development.

A role for V-ATPase subunits in synaptic vesicle fusion?

J. Neurochem. (2011) 117, 603–612.