Evolutionary diversity of the dual Ca sensor system for neurotransmitter release.

Several proteins containing C2 domains have been identified as Ca sensors for neurotransmitter release. In several cases, multiple C2 domain containing proteins function together to sustain evoked synchronous and asynchronous release as well as Ca-dependent forms of spontaneous release. Most recent publication by Li and colleagues have identified a novel Ca sensor at the C.

Presynaptic store-operated Ca entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress.

Store-operated calcium entry (SOCE) is activated by depletion of Ca from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca depletion increases presynaptic Ca levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function.

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies.

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders.

Dysregulation of Norepinephrine Release in the Absence of Functional Synaptotagmin 7.

Synaptotagmin 7 (Syt7) is expressed in cardiac sympathetic nerve terminals where norepinephrine (NE) is released in both Ca(2+)-dependent exocytosis and Ca(2+)-independent norepinephrine transporter (NET)-mediated overflow. The role of Syt7 in the regulation of NE release from cardiac sympathetic nerve terminals is tested by employing a Syt7 knock-in mouse line that expresses a non-functional mutant form of Syt7. In cardiac sympathetic nerve terminals prepared from these Syt7 knock-in mice, the Ca(2+)-dependent component of NE release was diminished.

Exocytosis and synaptic vesicle function.

Synaptic vesicles release their vesicular contents to the extracellular space by Ca(2+)-triggered exocytosis. The Ca(2+)-triggered exocytotic process is regulated by synaptotagmin (Syt), a vesicular Ca(2+)-binding C2 domain protein. Synaptotagmin 1 (Syt1), the most studied major isoform among 16 Syt isoforms, mediates Ca(2+)-triggered synaptic vesicle exocytosis by interacting with the target membranes and SNARE/complexin complex.

Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron.

Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons.

Interactions among the SNARE proteins and complexin analyzed by a yeast four-hybrid assay.

Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25 kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex.

Structural and mutational analysis of functional differentiation between synaptotagmins-1 and -7.

Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7).

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 C(2)B domain of Munc13 functions as a Ca(2+) regulator of short-term synaptic plasticity. The crystal structure of the C(2)B domain revealed an unusual Ca(2+)-binding site with an amphipathic alpha-helix.

Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C2 domains mediate neurotransmitter release.

Neurotransmitter release is triggered by cooperative Ca2+-binding to the Ca2+-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca2+ with similar properties and affinities. However, Ca2+ binding to the C2A domain is not required for release, whereas Ca2+ binding to the C2B domain is essential for release.

Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release.

Spontaneous 'mini' release occurs at all synapses, but its nature remains enigmatic. We found that >95% of spontaneous release in murine cortical neurons was induced by Ca2+-binding to synaptotagmin-1 (Syt1), the Ca2+ sensor for fast synchronous neurotransmitter release. Thus, spontaneous and evoked release used the same Ca2+-dependent release mechanism.

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 Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes.

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.

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.

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.

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.

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 domain beta-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.

Assays and properties of arfaptin 2 binding to Rac1 and ADP-ribosylation factors (Arfs).

Arfaptin 1 and 2 were identified as targets for GTP bound ADP-ribosylation factors (Arfs). Arfaptin 1 had no significant effects on guanine nucleotide binding to Arfs, nor enzymatic activities of guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) acting on Arfs. However, arfaptin 1 inhibited Arf activation of cholera toxin and phospholipase D (PLD) in a dose-dependent manner.

Evolutionarily conserved multiple C2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties.

C2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C2 domains. In both classes of proteins, C2 domains usually regulate the respective protein's function by forming Ca(2+)-dependent or Ca(2+)-independent phospholipid complexes. We now describe MCTPs (multiple C2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C2 domain proteins with unusual Ca(2+)-dependent properties.

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4.

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently.

Unexpected Ca2+-binding properties of synaptotagmin 9.

Synaptotagmin 1 (Syt 1) functions as an essential Ca2+ sensor for the fast but not slow component of Ca2+-triggered exocytosis. One hypothesis to account for this selective function, based on the close homology of Syt 1 with synaptotagmin 9 (Syt 9), is that these Syts are redundant for the slow but not the fast component of release. We now show, however, that Syt 9 has unique properties that set it apart from Syt 1.

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.

Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1.

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain.

Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells.

Ca(2+)-triggered dense-core vesicle exocytosis in PC12 cells does not require vesicular synaptotagmins 1 and 2, but may use plasma membrane synaptotagmins 3 and 7 as Ca(2+) sensors. In support of this hypothesis, C(2) domains from the plasma membrane but not vesicular synaptotagmins inhibit PC12 cell exocytosis. Ca(2+) induces binding of both plasma membrane and vesicular synaptotagmins to phospholipids and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors), although with distinct apparent Ca(2+) affinities.

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins.