Surface mobility of postsynaptic AMPARs tunes synaptic transmission

AMPA glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission. Upon fast consecutive synaptic stimulation, transmission can be depressed. Recuperation from fast synaptic depression has been attributed solely to recovery of transmitter release and/or AMPAR desensitization. We show that AMPAR lateral diffusion, observed in both intact hippocampi and cultured neurons, allows fast exchange of desensitized receptors with na?ve functional ones within or near the postsynaptic density. Recovery from depression in the tens of millisecond time range can be explained in part by this fast receptor exchange. Preventing AMPAR surface movements through cross-linking, endogenous clustering, or calcium rise all slow recovery from depression. Physiological regulation of postsynaptic receptor mobility affects the fidelity of synaptic transmission by shaping the frequency dependence of synaptic responses.     

Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs

We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca2+-binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca2+-triggered exocytosis.     

Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.     

A G protein directly regulates mammalian cardiac calcium channels

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.     

Vesicle endocytosis requires dynamin-dependent GTP hydrolysis at a fast CNS synapse

Molecular dependence of vesicular endocytosis was investigated with capacitance measurements at the calyx of Held terminal in brainstem slices. Intraterminal loading of botulinum toxin E revealed that the rapid capacitance transient implicated as "kiss-and-run" was unrelated to transmitter release. The release-related capacitance change decayed with an endocytotic time constant of 10 to 25 seconds, depending on the magnitude of exocytosis. Presynaptic loading of the nonhydrolyzable guanosine 5'-triphosphate (GTP) analog GTPgS or dynamin-1 proline-rich domain peptide abolished endocytosis. These compounds had no immediate effect on exocytosis, but caused a use-dependent rundown of exocytosis. Thus, the guanosine triphosphatase dynamin-1 is indispensable for vesicle endocytosis at this fast central nervous system (CNS) synapse.     

N- to C-terminal SNARE complex assembly promotes rapid membrane fusion

Assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) syntaxin 1, SNAP-25, and synaptobrevin 2 is thought to be the driving force for the exocytosis of synaptic vesicles. However, whereas exocytosis is triggered at a millisecond time scale, the SNARE-mediated fusion of liposomes requires hours for completion, which challenges the idea of a key role for SNAREs in the final steps of exocytosis. We found that liposome fusion was dramatically accelerated when a stabilized syntaxin/SNAP-25 acceptor complex was used. Thus, SNAREs do have the capacity to execute fusion at a speed required for neuronal secretion, demonstrating that the maintenance of acceptor complexes is a critical step in biological fusion reactions.     

Neurexin and neuroligin mediate retrograde synaptic inhibition in C. elegans

The synaptic adhesion molecules neurexin and neuroligin alter the development and function of synapses and are linked to autism in humans. Here, we found that Caenorhabditis elegans neurexin (NRX-1) and neuroligin (NLG-1) mediated a retrograde synaptic signal that inhibited neurotransmitter release at neuromuscular junctions. Retrograde signaling was induced in mutants lacking a muscle microRNA (miR-1) and was blocked in mutants lacking NLG-1 or NRX-1. Release was rapid and abbreviated when the retrograde signal was on, whereas release was slow and prolonged when retrograde signaling was blocked. The retrograde signal adjusted release kinetics by inhibiting exocytosis of synaptic vesicles (SVs) that are distal to the site of calcium entry. Inhibition of release was mediated by increased presynaptic levels of tomosyn, an inhibitor of SV fusion.     

A 32-kD GTP-binding protein associated with the CD4-p56lck and CD8-p56lck T cell receptor complexes

The guanosine triphosphate (GTP)-binding proteins include signal-transducing heterotrimeric G proteins (for example, Gs, Gi), smaller GTP-binding proteins that function in protein sorting, and the oncogenic protein p21ras. The T cell receptor complexes CD4-p56lck and CD8-p56lck were found to include a 32- to 33-kilodalton phosphoprotein (p32) that was recognized by an antiserum to a consensus GTP-binding region in G proteins. Immunoprecipitated CD4 and CD8 complexes bound GTP and hydrolyzed it to guanosine diphosphate (GDP). The p32 protein was covalently linked to [alpha-32P]GTP by ultraviolet photoaffinity labeling. These results demonstrate an interaction between T cell receptor complexes and an intracellular GTP-binding protein.     

Proportional release of norepinephrine and dopamine- -hydroxylase from sympathetic nerves

Dopamine-beta-hydroxylase(DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, is localized in the vesicles containing catecholamine in sympathetic nerves. This enzyme is released with norepinephrine when the nerves to the guinea pig vas deferens are stimulated in vitro, and the amount of enzyme discharged increases as the length of stimulation periods increases. The amount of DBH released is proportional to the amount of norepinephrine released, and the ratio of norepinephrine to DBH discharged into the incubation medium is similar to that in the soluble portion of the contents of the synaptic vesicles from the vas deferens. These data are compatible with the release of the neurotransmitter norepinephrine and DBH from symnpathetic nerves by a process of exocytosis.     

Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein

The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with the Golgi apparatus in an energy, GTP, and fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, the 110-kilodalton cytosolic coat protein beta-COP. Addition of beta gamma subunits of G proteins inhibited the association of both ARF and beta-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Thus, heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane.     

植物GTP结合蛋白的研究进展

GTP结合蛋白(G蛋白)广泛存在于生物界,在细胞信息传导中起着极为重要的开关作用,它可以调控真核生物中高度保守的跨膜信号传导通路。G蛋白可以划分为异源三聚体G蛋白和低分子量G蛋白(Ran)两种类型。Ran是一种大量分布于细胞核内的小分子的GTP酶,在核质运输以及微管蛋白成核过程中具有十分重要的作用。本文主要对G蛋白以及低分子量G蛋白Ran的研究进展做一综述。     

Pentobarbital: selective depression of excitatory postsynaptic potentials

The effects of pentobarbital (Nembutal) on synaptic transmission and postsynaptic potentials were studied by the use of several invertebrate preparations. Pentobarbital selectively and reversibly depressed both excitatory postsynaptic potentials and sodium-dependent postsynaptic responses to putative excitatory transmitters without affecting either inhibitory postsynaptic potentials or chloride- and potassium-dependent postsynaptic responses to putative transmitters. A selective depression of postsynaptic excitatory events was also observed with other central nervous system depressants (ethanol, chloroform, chloralose, diphenylhydantoin, and urethane). The results suggest that central and peripheral depression observed during general anesthesia is due to a selective depression of excitatory synaptic events.     

Astrocytic purinergic signaling coordinates synaptic networks

To investigate the role of astrocytes in regulating synaptic transmission, we generated inducible transgenic mice that express a dominant-negative SNARE domain selectively in astrocytes to block the release of transmitters from these glial cells. By releasing adenosine triphosphate, which accumulates as adenosine, astrocytes tonically suppressed synaptic transmission, thereby enhancing the dynamic range for long-term potentiation and mediated activity-dependent, heterosynaptic depression. These results indicate that astrocytes are intricately linked in the regulation of synaptic strength and plasticity and provide a pathway for synaptic cross-talk.     

RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release

The molecular machinery mediating the fusion of synaptic vesicles (SVs) at presynaptic active zone (AZ) membranes has been studied in detail, and several essential components have been identified. AZ-associated protein scaffolds are viewed as only modulatory for transmission. We discovered that Drosophila Rab3-interacting molecule (RIM)-binding protein (DRBP) is essential not only for the integrity of the AZ scaffold but also for exocytotic neurotransmitter release. Two-color stimulated emission depletion microscopy showed that DRBP surrounds the central Ca(2+) channel field. In drbp mutants, Ca(2+) channel clustering and Ca(2+) influx were impaired, and synaptic release probability was drastically reduced. Our data identify RBP family proteins as prime effectors of the AZ scaffold that are essential for the coupling of SVs, Ca(2+) channels, and the SV fusion machinery.     

A pertussis toxin-sensitive G protein in hippocampal long-term potentiation

High-frequency (tetanic) stimulation of presynaptic nerve tracts in the hippocampal region of the brain can lead to long-term synaptic potentiation (LTP). Pertussis toxin prevented the development of tetanus-induced LTP in the stratum radiatum-CA1 synaptic system of rat hippocampal slices, indicating that a guanosine triphosphate-binding protein (G protein) may be required for the initiation of LTP. This G protein may be located at a site distinct from the postsynaptic neuron (that is, in presynaptic terminals or glial cells) since maximal activation of CA1 neuronal G proteins by intracellular injection of guanosine-5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of guanosine 5'-triphosphate, did not occlude LTP.     

Emerging roles of presynaptic proteins in Ca++-triggered exocytosis

The twinning of techniques from biophysics and molecular biology has led to remarkable progress in understanding the molecular mechanisms of synaptic transmission. Here we review the current picture of Ca++-triggered exocytosis, which has emerged from studies of a simple cellular model, the adrenal chromaffin cell. We discuss the molecular players that have been assigned a specific role in a particular step of this process and give a brief outlook on what these insights might tell us about mechanisms of short-term plasticity at classical synapses.     

Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter

Previous work has identified two families of proteins that transport classical neurotransmitters into synaptic vesicles, but the protein responsible for vesicular transport of the principal excitatory transmitter glutamate has remained unknown. We demonstrate that a protein that is unrelated to any known neurotransmitter transporters and that was previously suggested to mediate the Na(+)-dependent uptake of inorganic phosphate across the plasma membrane transports glutamate into synaptic vesicles. In addition, we show that this vesicular glutamate transporter, VGLUT1, exhibits a conductance for chloride that is blocked by glutamate.     

Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) dissociate into guanosine triphosphate (GTP)-bound alpha subunits and a complex of beta and gamma subunits after interaction with receptors. The GTP-alpha subunit complex activates appropriate effectors, such as adenylyl cyclase, retinal phosphodiesterase, phospholipase C, and ion channels. G protein beta gamma subunits have been found to have regulatory effects on certain types of adenylyl cyclase. In the presence of Gs alpha, the alpha subunit of the G protein that activates adenylyl cyclase, one form of adenylyl cyclase was inhibited by beta gamma, some forms were activated by beta gamma, and some forms were not affected by beta gamma. These interactions suggest mechanisms for communication between distinct signal-transducing pathways.     

Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein

The function of the guanosine triphosphate (GTP)-binding protein Ypt1 in regulating vesicular traffic was studied in a cell-free system that reconstitutes transport from the endoplasmic reticulum to the Golgi. Blocking the Ypt1 protein activity resulted in accumulation of vesicles that act as an intermediate passing between the two compartments. The Ypt1 protein was found on the outer side of these vesicles. The transport process is completed by fusion of these vesicles with the acceptor compartment, and Ypt1 protein activity was needed for this step. Thus, a specific GTP-binding protein is required for either attachment or fusion (or both) of secretory vesicles with the acceptor compartment during protein secretion.     

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites

Vesicular glutamate transporters (VGLUTs) 1 and 2 show a mutually exclusive distribution in the adult brain that suggests specialization for synapses with different properties of release. Consistent with this distribution, inactivation of the VGLUT1 gene silenced a subset of excitatory neurons in the adult. However, the same cell populations exhibited VGLUT1-independent transmission early in life. Developing hippocampal neurons transiently coexpressed VGLUT2 and VGLUT1 at distinct synaptic sites with different short-term plasticity. The loss of VGLUT1 also reduced the reserve pool of synaptic vesicles. Thus, VGLUT1 plays an unanticipated role in membrane trafficking at the nerve terminal.