Sensor Synaptotagmin Research Articles

THE SYNAPTIC VESICLE CYCLE

Neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. To support rapid and repeated rounds of release, synaptic vesicles undergo a trafficking cycle. The focal point of the vesicle cycle is Ca2+-triggered exocytosis that is followed by different routes of endocytosis and recycling. Recycling then leads to the docking and priming of the vesicles for another round of exo- and endocytosis. Recent studies have led to a better definition than previously available of how Ca2+ triggers exocytosis and how vesicles recycle. In particular, insight into how Munc18-1 collaborates with SNARE proteins in fusion, how the vesicular Ca2+ sensor synaptotagmin 1 triggers fast release, and how the vesicular Rab3 protein regulates release by binding to the active zone proteins RIM1 alpha and RIM2 alpha has advanced our understanding of neurotransmitter release. The present review attempts to relate these molecular data with physiological results in an emerging view of nerve terminals as macromolecular machines.

Identification and characterization of Snapin as a ubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells.

Members of the SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) superfamily [syntaxins, VAMPs (vesicle-associated membrane proteins) and SNAP25 (synaptosome-associated protein-25)-related proteins] are required for intracellular membrane-fusion events in eukaryotes. In neurons, assembly of SNARE core complexes comprising the presynaptic membrane-associated SNAREs syntaxin 1 and SNAP25, and the vesicle-associated SNARE VAMP2, is necessary for synaptic vesicle exocytosis. Several accessory factors have been described that associate with the synaptic SNAREs and modulate core complex assembly or mediate Ca2+ regulation. One such factor, Snapin, has been reported to be a brain-specific protein that interacts with SNAP25, and regulates association of the putative Ca2+-sensor synaptotagmin with the synaptic SNARE complex [Ilardi, Mochida and Sheng (1999) Nat. Neurosci. 2, 119-124]. Here we demonstrate that Snapin is expressed ubiquitously in neuronal and non-neuronal cells. Furthermore, using protein-protein-interaction assays we show that Snapin interacts with SNAP23, the widely expressed homologue of SNAP25, and that the predicted C-terminal helical domain of Snapin contains the SNAP23-binding site. Subcellular localization experiments revealed that Snapin is a soluble protein that exists in both cytosolic and peripheral membrane-bound pools in adipocytes. Moreover, association of Snapin with the plasma membrane was detected in cells overexpressing a Snapin-green fluorescent protein fusion protein. Finally, we show that Snapin is able to form a ternary complex with SNAP23 and syntaxin 4, suggesting that it is a component of non-neuronal SNARE complexes. An important implication of our results is that Snapin is likely to perform a general role in SNARE-mediated vesicle fusion events in non-neuronal cells in addition to its participation in Ca2+-regulated neurosecretion.

Facile detection of protein-protein interactions by one-dimensional NMR spectroscopy.

Two methods for detecting protein-protein interactions in solution using one-dimensional (1D) NMR spectroscopy are described. Both methods rely on measurement of the intensity of the strongest methyl resonance (SMR), which for most proteins is observed at 0.8-0.9 ppm. The severe resonance overlap in this region facilitates detection of the SMR at low micromolar and even sub-micromolar protein concentrations. A decreased SMR intensity in the 1H NMR spectrum of a protein mixture compared to the added SMR intensities of the isolated proteins reports that the proteins interact (SMR method). Decreased SMR intensities in 1D 13C-edited 1H NMR spectra of 13C-labeled proteins upon addition of unlabeled proteins or macromolecules also demonstrate binding (SMRC method). Analysis of the interaction between XIAP and Smac, two proteins involved in apoptosis, illustrates both methods. A study showing that phospholipids compete with the neuronal core complex for Ca2+-dependent binding to the presynaptic Ca2+-sensor synaptotagmin 1 illustrates the usefulness of the SMRC method in studying multicomponent systems.

SNAREs in Mammalian Sperm: Possible Implications for Fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm–egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm–egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.

Serotonin, via 5-HT 2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release

Previously, serotonin (5-HT) was found to induce a marked increase in glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) in apical dendrites of layer V pyramidal cells of prefrontal cortex; this effect was mediated by 5-HT 2A receptors, a proposed site of action of hallucinogenic and atypical antipsychotic drugs. Unexpectedly, although the effect of 5-HT was Ca 2+-dependent and tetrodotoxin-sensitive, it did not appear to involve the activation of excitatory afferent impulse flow. This paradox prompted us to investigate (in rat brain slices) whether 5-HT was acting through an atypical mode of excitatory transmitter release. We found that the frequency of 5-HT-induced spontaneous EPSCs was fully supported by Sr 2+ in the absence of added Ca 2+, implicating the mechanism of asynchronous transmitter release which has been linked to the high-affinity Ca 2+-sensor synaptotagmin III. Although the early, synchronous component of electrically evoked EPSCs was reduced while 5-HT was being applied, late, nonsynchronous components were enhanced during 5-HT washout and also by the 5-HT 2 partial agonist 1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI); the effect of DOI was blocked by a selective 5-HT 2A antagonist (MDL 100,907). This late, nonsynchronous component was distinct from conventional polysynaptic EPSCs evoked in the presence of the GABA A antagonist bicuculline, but resembled asynchronous glutamatergic excitatory postsynaptic potentials (EPSPs) evoked in the presence of Sr 2+. An enhancement of asynchronous EPSCs by a specific neurotransmitter receptor has not been reported previously. The possible role of excessive asynchronous transmission in the cerebral cortex in mediating the hallucinogenic effects of 5-HT 2A agonists such as DOI is discussed.

Are mechanisms of exocytosis neurotransmitter specific?

In their review, Langley and Grant (1997) investigate the question whether mechanisms of exocytosis are neurotransmitter specific. There is now much evidence that the mechanisms governing the exocytosis of the two principal storage organelles—granules (large dense core vesicles) and electron-lucent vesicles—differ. But much less is known concerning potential differences in the release mechanisms of electron-lucent vesicles that store different types of fast neurotransmitters or of granules in different types of neurons. It is an open question whether there is a unifying control mechanism for the exocytosis of, for example, a peptide-containing granule of a glutamatergic neuron, a chromaffin granule, a noradrenergic granule or a granule from a neurosecretory neuron in the pituitary. The small electron-lucent synaptic vesicles of various kind apparently share common molecular components of regulated release. They carry the calcium sensor synaptotagmin, small GTP-binding proteins of the rab3 group or the v-SNARE synaptobrevin. Nevertheless, there may be differences in the regulatory mechanisms. This concerns the type of calcium channel involved or the absence of some of the presynaptic molecules such as rab3a, synapsin I or the t-SNAREs SNAP-25 or syntaxin from distinct types of neurons or sensory cells.

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A 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]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A 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 degrees 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.

Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses.

Neurotransmitter release requires the specific docking of synaptic vesicles to the presynaptic plasma membrane followed by a calcium-triggered fusion event. Herein we report a previously unsuspected interaction of the synaptic vesicle protein and likely calcium sensor synaptotagmin with the plasma membrane t-SNARE SNAP-25. This interaction appears to resolve the apparent paradox that synaptic vesicles are capable of docking even when VAMP (vesicle-associated membrane protein) or syntaxin is cleaved or deleted and suggests that two species of v-SNAREs (VAMP and synaptotagmin) and two species of t-SNAREs (SNAP-25 and syntaxin) interact to functionally dock synaptic vesicles.