ROP, the Drosophila Sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner

Noncanonical secretagogues such as hypertonicity or α-latrotoxin have been extremely informative in studying neurotransmission. Lanthanum and lanthanides can also trigger neurotransmitter release through an unknown mechanism. Here, we studied the effect of lanthanides on neurotransmission in hippocampal cultures. Application of 2 mM La3+ caused rapid and robust neurotransmitter release within seconds. In addition, transient application of La3+ uncovered a sustained facilitation of miniature neurotransmission. The response to La3+ was detectable at 2 μM and increased in a concentration-dependent manner ≤2 mM. Rapid effect of La3+ was independent of extracellular and intracellular Ca2+ and did not require La3+ entry into cells or activation of phospholipaseCβ. Synapses deficient in synaptobrevin-2, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in the brain, did not display any rapid release in response to La3+, whereas the slow facilitation of release detected after La3+ removal remained intact. In contrast, preincubation with intracellular Ca2+ chelators selectively attenuated the delayed release triggered by La3+. Moreover, synapses deficient in synaptotagmin-1 maintained a rapid response to La3+, suggesting that La3+-triggered neurotransmitter release does not require synaptotagmin-1 as a sensor. Therefore La3+ has two separate effects on synaptic transmission. For its rapid action, La3+ interacts with a target on the surface membrane, and unlike other forms of release, it triggers strictly synaptobrevin-2–dependent fusion, implying that in central synapses synaptobrevin-2 function is secretagogue specific. For the delayed action, La3+ may act intracellularly after its entry or through intracellular Ca2+ via a mechanism that does not require synaptobrevin-2.

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Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle fusion and replenishment in a dosage-dependent manner

10.1101/2020.01.30.926246 ◽

2020 ◽

Author(s):

Zhuo Guan ◽

Mónica C. Quiñones-Frías ◽

Yulia Akbergenova ◽

J. Troy Littleton

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Active Zone ◽

Neurotransmitter Release ◽

Dependent Manner ◽

Short Term ◽

Sensor Model ◽

Synaptotagmin 1 ◽

Asynchronous Release ◽

Synaptic Vesicle Fusion

AbstractSynchronous neurotransmitter release is triggered by Ca2+ binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). We generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca2+ sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and robust facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at release sites. These findings indicate the two Ca2+ sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.

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Neto-α controls synapse organization and homeostasis at the Drosophila neuromuscular junction

10.1101/812040 ◽

2019 ◽

Author(s):

Tae Hee Han ◽

Rosario Vicidomini ◽

Cathy Isaura Ramos ◽

Qi Wang ◽

Peter Nguyen ◽

...

Keyword(s):

Neuromuscular Junction ◽

Motor Neurons ◽

Neurotransmitter Release ◽

Cell Biology ◽

Expression Patterns ◽

Dependent Manner ◽

Independent Function ◽

Postsynaptic Receptors ◽

Auxiliary Protein ◽

And Function

SummaryGlutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and post-synaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology and electrophysiology we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptors field. In motor neurons, Neto-α controls neurotransmitter release in a KAR-dependent manner. Furthermore, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensured NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

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CB1 Cannabinoid Receptor Inhibits Synaptic Release of Glutamate in Rat Dorsolateral Striatum

Journal of Neurophysiology ◽

10.1152/jn.2001.85.1.468 ◽

2001 ◽

Vol 85 (1) ◽

pp. 468-471 ◽

Cited By ~ 277

Author(s):

Gregory Gerdeman ◽

David M. Lovinger

Keyword(s):

Basal Ganglia ◽

Synaptic Transmission ◽

Neurotransmitter Release ◽

Cannabinoid Receptors ◽

Glutamate Release ◽

Brain Slices ◽

Cb1 Receptor ◽

Motor Deficits ◽

Dependent Manner ◽

Dorsolateral Striatum

CB1 cannabinoid receptors in the neostriatum mediate profound motor deficits induced when cannabinoid drugs are administered to rodents. Because the CB1 receptor has been shown to inhibit neurotransmitter release in various brain areas, we investigated the effects of CB1 activation on glutamatergic synaptic transmission in the dorsolateral striatum of the rat where the CB1 receptor is highly expressed. We performed whole cell voltage-clamp experiments in striatal brain slices and applied the CB1 agonists HU-210 or WIN 55,212–2 during measurement of synaptic transmission. Excitatory postsynaptic currents (EPSCs), evoked by electrical stimulation of afferent fibers, were significantly reduced in a dose-dependent manner by CB1 agonist application. EPSC inhibition was accompanied by an increase in two separate indices of presynaptic release, the paired-pulse response ratio and the coefficient of variation, suggesting a decrease in neurotransmitter release. These effects were prevented by application of the CB1 antagonist SR141716A. When Sr2+ was substituted for Ca2+ in the extracellular solution, application of HU-210 (1 μM) significantly reduced the frequency, but not amplitude, of evoked, asynchronous quantal release events. Spontaneous release events were similarly decreased in frequency with no change in amplitude. These findings further support the interpretation that CB1 activation leads to a decrease of glutamate release from afferent terminals in the striatum. These results reveal a novel potential role for cannabinoids in regulating striatal function and thus basal ganglia output and may suggest CB1-targeted drugs as potential therapeutic agents in the treatment of Parkinson's disease and other basal ganglia disorders.

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Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III

Biochemical Journal ◽

10.1042/bj3230421 ◽

1997 ◽

Vol 323 (2) ◽

pp. 421-425 ◽

Cited By ~ 30

Author(s):

Mitsunori FUKUDA ◽

Toshio KOJIMA ◽

Katsuhiko MIKOSHIBA

Keyword(s):

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Binding Proteins ◽

Binding Activity ◽

Binding Property ◽

Dependent Manner ◽

Phospholipid Binding ◽

Bivalent Cations ◽

Dependent Interaction ◽

C2a Domain

Synaptotagmins are Ca2+-and phospholipid-binding proteins of synaptic vesicles that might function as Ca2+ receptors for neurotransmitter release via their first C2 (C2A) domain. Here we describe the effect of Mg2+ on phospholipid binding to the C2A domains of multiple synaptotagmins (II–VI), and demonstrate that only synaptotagmin III can bind negatively charged phospholipids [phosphatidylserine (PS) and phosphatidylinositol] in a Mg2+-dependent manner. The Mg2+-dependent interaction with PS was found to have an EC50 of approx. 30 μM Mg2+, which is comparable to that of Sr2+ and Ba2+ (EC50 values of approx. 10 μM). This binding property of the C2A domain is specific to synaptotagmin III, because none of the C2A domains of other proteins, such as rabphilin 3A, Doc2α, Doc2β or Gap1m, showed phospholipid binding activity in the presence of 1 mM Mg2+. Our results suggest that synaptotagmin III is involved in presynaptic functions different from those of synaptotagmins I and II.

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Neuronal calcium sensor-1: a multifunctional regulator of secretion

Biochemical Society Transactions ◽

10.1042/bst0310828 ◽

2003 ◽

Vol 31 (4) ◽

pp. 828-832 ◽

Cited By ~ 38

Author(s):

S. Hilfiker

Keyword(s):

Guanylate Cyclase ◽

Neurotransmitter Release ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Current Knowledge ◽

Photoreceptor Cells ◽

Cell Types ◽

Dependent Manner ◽

Calcium Sensor ◽

Neuronal Calcium Sensor

Ca2+ ions play a crucial role not only as the trigger for neurotransmitter release, but also in other aspects of brain function, such as short-term and long-term modulation of synaptic efficacy, which may underlie certain forms of learning and memory. The actions of Ca2+ are mediated by Ca2+-binding proteins, including a group of proteins known as neuronal calcium sensor (NCS) proteins. The NCS family includes NCS-1, visinin-like proteins, recoverins, guanylate cyclase-activating proteins and potassium channel-interacting proteins. Some members of this family, such as recoverin and guanylate cyclase-activating protein, are only expressed in photoreceptor cells and have been implicated in the control of visual transduction pathways, while the functional roles of the other members are largely unknown. NCS-1 was originally identified in Drosophila in a screen for neuronal hyperexcitability mutants. NCS-1 is an N-terminally myristoylated protein that contains four EF-hand motifs, three of which are able to bind Ca2+ in the submicromolar range. Overexpression of NCS-1 has been shown to enhance evoked neurotransmitter release, paired-pulse facilitation and exocytosis in several neuronal and neuroendocrine cell types. Recent experiments suggest that NCS-1 interacts directly with phosphatidylinositol 4-hydroxykinase in yeast as well as mammalian cells, suggesting that it may enhance neuronal secretion by modulating cellular trafficking steps in a phosphoinositide-dependent manner. In contrast, an involvement of NCS-1 in the expression and regulation of voltage-gated Ca2+ channels and K+ channels has also been proposed, which may be attributed, at least in part, to the effects of NCS-1 on vesicular trafficking pathways. The present review describes current knowledge about the cellular functions and molecular mechanisms by which NCS-1 may regulate neurotransmitter release.

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Intracellular Mg2+ Enhances the Function of Bk-Type Ca2+-Activated K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.118.5.589 ◽

2001 ◽

Vol 118 (5) ◽

pp. 589-606 ◽

Cited By ~ 84

Author(s):

Jingyi Shi ◽

Jianmin Cui

Keyword(s):

Binding Sites ◽

Neurotransmitter Release ◽

Single Channel ◽

Bk Channels ◽

Bk Channel ◽

Dependent Manner ◽

Physiological Conditions ◽

Channel Function ◽

Open Conformation ◽

Voltage Dependent

BK channels modulate neurotransmitter release due to their activation by voltage and Ca2+. Intracellular Mg2+ also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg2+ blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg2+ activates mslo1 BK channels independently of Ca2+ and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca2+ binding sites in the tail, is not activated by Mg2+. However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg2+ sensitivity similar to mslo1 channels, indicating that Mg2+ sites differ from Ca2+ sites. We discovered that Mg2+ also binds to Ca2+ sites and competitively inhibits Ca2+-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg2+ under physiological conditions is to enhance BK channel function.

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Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420312112 ◽

2015 ◽

Vol 112 (12) ◽

pp. 3793-3798 ◽

Cited By ~ 19

Author(s):

Jihye Lee ◽

J. Troy Littleton

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Temporal Dynamics ◽

Transgenic Animals ◽

Vesicle Fusion ◽

Full Length ◽

Dependent Manner ◽

Membrane Phospholipids ◽

C2 Domains

Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca2+-dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1’s ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.

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Peptide modulation of ACh receptor desensitization controls neurotransmitter release from chicken sympathetic neurons

Journal of Neurophysiology ◽

10.1152/jn.1993.69.3.928 ◽

1993 ◽

Vol 69 (3) ◽

pp. 928-942 ◽

Cited By ~ 33

Author(s):

D. C. Valenta ◽

J. E. Downing ◽

L. W. Role

Keyword(s):

Time Constant ◽

Neurotransmitter Release ◽

Time Course ◽

Sympathetic Neurons ◽

Nerve Fibers ◽

Dependent Manner ◽

Transmitter Secretion ◽

Whole Cell ◽

Rate Of Decay

1. The distribution and release of substance P (SP) in embryonic chicken lumbar sympathetic ganglia was examined with the use of immunohistochemistry and radioimmunoassay, respectively. SP immunoreactivity was detected in nerve fibers surrounding individual sympathetic neurons and was released by ganglionic depolarization. 2. Effects of SP on nicotinic acetylcholine receptor (AChR) function was assayed in embryonic sympathetic neurons in vitro by whole-cell patch clamp. SP (0.1–20 microM) accelerated the rate of decay (desensitization) of ACh-induced currents. The AChR desensitization time course is biphasic and described by the sum of two exponential functions dependent on agonist concentration (time constant of the faster component, tau f = 1-2 s, and the slower time constant, tau s = 10-25 s). SP selectively decreased tau s and the contribution of the slow component to the overall rate of current decay. The effects of SP on desensitization were concentration dependent and reversible. SP slowed recovery from desensitization by 2.5-fold. 3. SP shifted the dose-response curve for ACh-induced desensitization, reducing the concentration of ACh required to produce half-maximal desensitization by approximately twofold. 4. Preapplication of SP was equivalent to SP applied together with ACh in accelerating AChR desensitization. SP did not alter the time course of currents elicited by nondesensitizing concentrations of ACh, carbamylcholine (CARB), or dimethylphenylpiperazinium (DMPP). These data suggest that AChR activation is neither necessary nor sufficient for the peptide to modulate receptor function. A kinetic model of the effects of SP on specific steps in AChR desensitization is presented. 5. SP enhanced the rate of decay of synaptic currents in sympathetic neurons innervated in vitro, decreasing the synaptic current duration by up to 80%. 6. Effects of SP on neurotransmitter release from sympathetic neurons were evaluated by measuring the release of [3H]-norepinephrine (NE). ACh and CARB stimulated NE release in a concentration- and calcium-dependent manner. SP alone had no effect on NE secretion, but the peptide inhibited NE release induced by ACh or CARB by 40–50%. 7. Although agonists specific for either nicotinic or muscarinic receptors stimulated release of NE, SP selectively inhibited the nicotinic component of transmitter secretion. Thus SP suppressed NE release induced by DMPP by up to 80% but had no effect on muscarine or depolarization-induced NE secretion. 8. Parallel studies of the modulatory effects of SP on whole-cell currents and NE secretion revealed that SP inhibition of transmitter release from sympathetic neurons is directly proportional to the extent of potentiation of AChR desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

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Inhibitory role of Src family tyrosine kinases on Ca2+-dependent insulin release

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00103.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. E845-E852 ◽

Cited By ~ 12

Author(s):

Haiying Cheng ◽

Susanne G. Straub ◽

Geoffrey W. G. Sharp

Keyword(s):

Insulin Secretion ◽

Tyrosine Kinases ◽

Neurotransmitter Release ◽

Src Family Kinases ◽

Dependent Manner ◽

Inhibitory Role ◽

Intracellular Ca ◽

Src Family Tyrosine Kinases ◽

Sites Of Action

Both neurotransmitter release and insulin secretion occur via regulated exocytosis and share a variety of similar regulatory mechanisms. It has been suggested that Src family tyrosine kinases inhibit neurotransmitter release from neuronal cells (H. Ohnishi, S. Yamamori, K. Ono, K. Aoyagi, S. Kondo, and M. Takahashi. Proc Natl Acad Sci USA 98: 10930–10935, 2001). Thus the potential role of Src family kinases in the regulation of insulin secretion was investigated in this study. Two structurally different inhibitors of Src family kinases, SU-6656 and PP2, but not the inactive compound, PP3, enhanced Ca2+-induced insulin secretion in both rat pancreatic islets and INS-1 cells in a concentration-dependent and time-dependent manner. Furthermore, Src family kinase-mediated insulin secretion appears to be dependent on elevated intracellular Ca2+ and independent of glucose metabolism, the ATP-dependent K+ channel, adenylyl cyclase, classical PKC isoforms, extracellular signal-regulated kinase 1/2, and insulin synthesis. The sites of action for Src family kinases seem to be distal to the elevation of intracellular Ca2+ level. These results indicate that one or more Src family tyrosine kinases exert a tonic inhibitory role on Ca2+-dependent insulin secretion.

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