Two forms of the membrane-bound state of the first C2 domain (C2A) of synaptotagminⅠand calcium-triggered membrane insertion

Neurotransmitter release from presynaptic neurons into the synaptic cleft is an essential step in neurotrans-mission. The release is triggered by Ca2+ and completed by the fusion of neurotransmitters containing synaptic vesicles with the presynaptic membrane. Over the last decades, there has been a virtual explosion in identifica-tion of proteins that play critical roles in the release proc-ess. The leading candidates proposed as the Ca2+-sensors that regulate fusion are members of the synapt…     

Unfolding of C2A Domain of Synaptotagmin I in the Presence of Guanidine Hydrochloride

The C2 domain originally referred to the second of four constant structural motifs in protein kinase C (PKC). Now this domain represents a large structural family sharing a homologous dimensional structure in many proteins that play important roles in many organisms. The C2A domain is one of the two C2 domains of synaptotagmin I involved in the Ca^2 regulation of exocytosis. This domain is mostly composed of β-sheet except for a small fraction of α-helix, and therefore provides an ideal model for a protein folding study. In this report, the unfolding equilibrium of the C2A domain in guanidine hydrochloride (GdnHCI) containing solutions has been studied using ultraviolet (UV) difference spectrum, fluorescence spectrum, size exclusion chromatography (SEC), and circular dichroism (CD) spectrum. The results suggest that unfolding of the C2A domain occurs as a two-state process during GdnHCI titration. By examining the changes of both tertiary structure and secondary structure, no intermediates could be detected during this unfolding study. However, it has been found that the native state of the C2A domain has a large hydrophobic surface. This result suggests that as a fragment of a protein, the C2A domain itself may exist in a state with large hydrophobic surface. This hydrophobic surface may be the molecular basis for interaction between domains in the whole protein.Furthermore, the hydrophobic behavior may play a role during the oligomerization of svnaptotagmin.     

Characterization of docking and fusion of synaptic-like microvesicles in PC12 cells using TIRFM

Neurotransmitters are released by the fusion of synaptic vesicles with presynaptic membrane,which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of "kiss and run" and "kiss and stay" which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation,docking and priming along the exo-cytotic pathway play important roles in neurotransmitter release and its regulation. In the present report,we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope(TIRFM) to monitor the docking and fusion of synaptic-like microvesicles(SLMVs) in PC12 cells stimulated by high K . Our results show that "kiss and run" is a dominative fu-sion mode in PC12 cells under high K -challenge,and the dwell time of SLMVs is prolonged by the high K stimulation that suggests an enhancement of vesicle priming.     

C2 domain of synaptotagmin I associates with lipid rafts of plasma membrane

In this paper we report that the C2 domain of synaptotagmin I （syt I） could associate with lipid rafts of plasma membrane. We demonstrate that phosphatidylinositol 4,5-bisphosphate （PIP2） in the target membrane and Ca^2＋ are the key factors to enhance the raft association of the C2 domain. We also found that the raft association of the C2 domain could be fulfilled by either C2A or C2B alone, suggesting that their raft association might be complementary. Finally, we indicate that destroying lipid rafts or blocking syt I-raft association could significantly reduce the Ca^2＋-driven release of glutamates. Our data indicate that the raft association of the C2 domain might play an important role in the regulated exocytosis.     

Development of the model of pancreatic β-cell membrane chromatography and its chromatographic characteristics

A new model of pancreatic β-cell membrane chromatography has been established by using a β-cell membrane stationary phase （β-CMSP） prepared by immobilizing the β-cell membrane onto the surface of silica carrier. The protein level and K^＋, Na^＋-ATP enzymatic activity of the β-CMSP were detected respectively. The surface characteristics of the β-CMSP were tested by using the scanning electron microscope and surface energy spectrometer. In this model, the column （10 mm × 2 mm, I.D.） packed with β-CMSP, 25 mmol/ammonium sulfate buffer solution （pH 7.4） as mobile phase with the flow rate of 0.2 mL/min at 37 ± 0.5℃ were used in the following studies. The retention characteristics of the sulfonylureas （gliquidone, glibenclamide, gliclazide and glipizide） were investigated under the chromatographic conditions above. The affinities of the sulfonylureas on β-cell membrane and receptor will be expressed by using the logk＇ values （the logarithm of capacity factor of a solute） in the model. The correlation of the affinity with the pharmacological effect of the sulfonylureas was analyzed also.     

Interaction of SynaptotagminⅠ with Phospholipid Membrane: A Monolayer Study

IntroductionSynaptotagmin is a family of vesicletransmembrane proteins present in synapticvesicles and large secretary granules of neuronsand endocrine cells[1 ] .It is a major constituent ofsynaptic vesicle membranes,comprising7% 8%of the total vesicle protein,characterized by ashort intravesiclar N-terminus,a singletransmembrane region,and a long plasmicdomain.　The best-charaterized form of synaptotagmin,syt ,is found abundantly in rostrol brain.Syt was first described in 1 981 [2 ] ,and i…     

Effects of heavy metal on dielectric properties of E. coli revealed by dielectric spectroscopy

Dielectric spectroscopy of E. coli cell before and after exposure to heavy metals Cd^2＋ , Cu^2＋ , Zn^2＋ and Ca^2＋ was investigated. The results indicate that changes in dielectric spectra reflect effects of heavy metal on the structure and function of E. coli cells. Heavy metal can change membrane capacitance as well as pennittivity and conductivity of the cytoplasm. Changes in volume fraction suggested that dielectric measurement could monitor the growth of E. coli cells. These results demonstrated that dielectric spectroscopy was a potential effective technique for studying electric properties of biological cells.     

Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek''s pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek's pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek's pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek's pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

The C(2)B Ca(2+)-binding motif of synaptotagmin is required for synaptic transmission in vivo

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission.     

Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.     

Spontaneous Neurotransmitter Release Depends on Intracellular Rather than ER Calcium Stores in Cultured Xenopus NMJ

Introduction During development, many cell types exhibit sponta-neous neurotransmitter release, with synaptic transmis-sions crucial for normal nervous system activity. Syn-aptic transmissions are initiated when an action poten-tial triggers the neurotran…     

Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion 'willingness' in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (approximately 4) is gradually reduced below 3 microM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.     

α-LTX and α-LTXN4C induce [Ca2+]i elevation through different mechanisms in pancreatic β cells

α-latrotoxin (α-LTX) is the only neurotoxin from black-widow spider which has secretagogue effects in the vertebrates. It causes massive neurotransmitter and hormone release via two instinct mechanisms after binding with its high-affinity membrane recep…     

RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone.

Neurotransmitters are released by synaptic vesicle fusion at the active zone. The active zone of a synapse mediates Ca2+-triggered neurotransmitter release, and integrates presynaptic signals in regulating this release. Much is known about the structure of active zones and synaptic vesicles, but the functional relation between their components is poorly understood. Here we show that RIM1alpha, an active zone protein that was identified as a putative effector for the synaptic vesicle protein Rab3A, interacts with several active zone molecules, including Munc13-1 (ref. 6) and alpha-liprins, to form a protein scaffold in the presynaptic nerve terminal. Abolishing the expression of RIM1alpha in mice shows that RIM1alpha is essential for maintaining normal probability of neurotransmitter release, and for regulating release during short-term synaptic plasticity. These data indicate that RIM1alpha has a central function in integrating active zone proteins and synaptic vesicles into a molecular scaffold that controls neurotransmitter release.     

Synaptotagmins I and IV promote transmitter release independently of Ca(2+) binding in the C(2)A domain

At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+) (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.     

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.