Ok Ho Shin

A Complexin/Synaptotagmin 1 Switch Controls Fast Synaptic Vesicle Exocytosis

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.

Three-dimensional structure of the synaptotagmin 1 C2B-domain: Synaptotagmin 1 as a phospholipid binding machine

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.

Synaptotagmins form a hierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca2+‐triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca2+ affinities of synaptotagmin C2‐domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca2+ affinities, with plasma membrane synaptotagmins binding Ca2+ with a 5‐ to 10‐fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca2+ affinities are functionally important, we examined the effects of synaptotagmin C2‐domains on Ca2+‐triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca2+ affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr2+, which was also selectively affected by high‐affinity C2‐domains of synaptotagmins. Together, our results suggest that Ca2+ triggering of exocytosis involves tandem Ca2+ sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high‐affinity Ca2+ sensors involved in slow Ca2+‐dependent exocytosis, whereas vesicular synaptotagmins function as low‐affinity Ca2+ sensors specialized for fast Ca2+‐dependent exocytosis.

Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis

Munc13 is a multidomain protein present in presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here we use biophysical, biochemical and electrophysiological approaches to show that the central C2B domain of Munc13 functions as a Ca2+ regulator of short-term synaptic plasticity. The crystal structure of the C2B domain revealed an unusual Ca2+-binding site with an amphipathic α-helix. This configuration confers onto the C2B domain unique Ca2+-dependent phospholipid-binding properties that favor phosphatidylinositolphosphates. A mutation that inactivated Ca2+-dependent phospholipid binding to the C2B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress release evoked by action-potential trains. In contrast, a mutation that increased Ca2+-dependent phosphatidylinositolbisphosphate binding to the C2B domain enhanced release evoked by isolated action potentials and by action-potential trains. Our data suggest that, during repeated action potentials, Ca2+ and phosphatidylinositolphosphate binding to the Munc13 C2B domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.

Structural Determinants of Synaptobrevin 2 Function in Synaptic Vesicle Fusion

Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca2+-evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca2+-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.

A Gain-of-Function Mutation in Synaptotagmin-1 Reveals a Critical Role of Ca2+-Dependent Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Binding in Synaptic Exocytosis

Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion

Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP‐binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use‐dependent depression, probably by a direct interaction with the SNARE protein SNAP‐25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild‐type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP‐25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP‐25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use‐dependent depression, or has been artificially depleted by deletion of synaptobrevin.

Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca2+. In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine97, and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine97 by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca2+-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

Phosphatidylinositol Phosphates as Co-activators of Ca2+ Binding to C2 Domains of Synaptotagmin 1

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domainβ-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.