The timing of synaptic vesicle endocytosis.

Alternative models to describe the endocytosis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of 20 s (24 degrees C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2+ during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.     

Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons.

Synaptophysin is an abundant synaptic vesicle protein without a definite synaptic function. Here, we examined a role for synaptophysin in synapse formation in mixed genotype micro-island cultures of wild-type and synaptophysin-mutant hippocampal neurons. We show that synaptophysin-mutant synapses are poor donors of presynaptic terminals in the presence of competing wild-type inputs. In homogenotypic cultures, however, mutant neurons display no apparent deficits in synapse formation compared with wild-type neurons. The reduced extent of synaptophysin-mutant synapse formation relative to wild-type synapses in mixed genotype cultures is attenuated by blockers of synaptic transmission. Our findings indicate that synaptophysin plays a previously unsuspected role in regulating activity-dependent synapse formation.     

Role of epsin 1 in synaptic vesicle endocytosis

Epsin has been suggested to act as an alternate adaptor in several endocytic pathways. Its role in synaptic vesicle recycling remains, however, unclear. Here, we examined the role of epsin in this process by using the lamprey reticulospinal synapse as a model system. We characterized a lamprey ortholog of epsin 1 and showed that it is accumulated at release sites at rest and also at clathrin-coated pits in the periactive zone during synaptic activity. Disruption of epsin interactions, by presynaptic microinjection of antibodies to either the epsin-N-terminal homology domain (ENTH) or the clathrin/AP2 binding region (CLAP), caused profound loss of vesicles in stimulated synapses. CLAP antibody-injected synapses displayed a massive accumulation of distorted coated structures, including coated vacuoles, whereas in synapses perturbed with ENTH antibodies, very few coated structures were found. In both cases coated pits on the plasma membrane showed a shift to early intermediates (shallow coated pits) and an increase in size. Moreover, in CLAP antibody-injected synapses flat clathrin-coated patches occurred on the plasma membrane. We conclude that epsin is involved in clathrin-mediated synaptic vesicle endocytosis. Our results support a model, based on in vitro studies, suggesting that epsin coordinates curvature generation with coat assembly and further indicating that epsin limits clathrin coat assembly to the size of newly formed vesicles. We propose that these functions of epsin 1 provide an additional mechanism for generation of uniformly sized synaptic vesicles.     

Readily releasable pool of synaptic vesicles measured at single synaptic contacts

To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.     

Endosomal sorting of readily releasable synaptic vesicles

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.     

Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis

The ability of synapses to sustain signal propagation relies on rapid recycling of transmitter-containing presynaptic vesicles. Clathrin- and dynamin-mediated retrieval of vesicular membrane has an undisputed role in synaptic vesicle recycling. There is also evidence for other modes of vesicle retrieval, including bulk retrieval and the so-called kiss-and-run recycling. Whether dynamin in required for these other modes of synaptic vesicle endocytosis remains unclear. Here, we have tested the role of dynamin in synaptic vesicle endocytosis by using a small molecule called dynasore, which rapidly inhibits the GTPase activity of dynamin with high specificity. Endocytosis after sustained or brief stimuli was completely and reversibly blocked by dynasore in cultured hippocampal neurons expressing the fluorescent tracer synaptopHluorin. By contrast, dynasore had no effect on exocytosis. In the presence of dynasore, low-frequency stimulation led to sustained accumulation of synaptopHluorin and other vesicular proteins on the surface membrane at a rate predicted from net exocytosis. These vesicular components remained on surface membranes even after the stimulus was terminated, suggesting that all endocytic events rely on dynamin during low-frequency activity as well as in the period after it. Ultrastructural analysis revealed a reduction in the density of synaptic vesicles and the presence of endocytic structures only at synapses that were stimulated in the presence of dynasore. In sum, our data indicate that dynamin is essential for all forms of compensatory synaptic vesicle endocytosis including any kiss-and-run events.     

Nitric oxide regulates endocytosis by S-nitrosylation of dynamin

The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the beta(2) adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both beta(2) adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both beta(2) adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.     

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.     

Protein tyrosine phosphorylation in synaptic vesicles.

Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2+ and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosphotyrosine-containing proteins distributed in the same manner as synaptophysin in all subcellular fractions. Like synaptophysin, the two high molecular weight phosphotyrosine proteins (105 and 94 kDa) were found to be glycoproteins by lectin chromatography. Tyrosine phosphorylation of synaptophysin was an intravesicular reaction and reached 50% of maximal level within 3 min. Triton X-100, a nonionic detergent, inhibited tyrosine phosphorylation of endogenous protein substrates but not the phosphorylation of an exogenous substrate, poly(Glu80,-Tyr20). Tyrosine phosphorylation of synaptophysin was also demonstrated in synaptosomes, indicating that tyrosine phosphorylation of synaptic vesicle proteins occurs in intact nerve terminals.     

Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release.

Synaptophysin (syp I) is a synaptic vesicle membrane protein that constitutes approximately 7% of the total vesicle protein. Multiple lines of evidence implicate syp I in a number of nerve terminal functions. To test these, we have disrupted the murine Syp I gene. Mutant mice lacking syp I were viable and fertile. No changes in the structure and protein composition of the mutant brains were observed except for a decrease in synaptobrevin/VAMP II. Synaptic transmission was normal with no detectable changes in synaptic plasticity or the probability of release. Our data demonstrate that one of the major synaptic vesicle membrane proteins is not essential for synaptic transmission, suggesting that its function is either redundant or that it has a more subtle function not apparent in the assays used.     

Hidden proteome of synaptic vesicles in the mammalian brain

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

The functions of eukaryotic cells, in all their complexity, depend upon highly specific compartmentalization into subcellular domains, including organelles. These compartments represent functional units characterized by specific supramolecular protein complexes. A major goal of modern biology is to establish an exhaustive, quantitative inventory of the protein components of each intracellular compartment. Such inventories are points of departure, not only for functional understanding and reconstruction of biological systems, but also for a multitude of investigations, such as evolutionary diversification and derivation of general principles of biological regulation and homeostasis.Essential to communication within the nervous system, chemical synapses constitute highly specific compartments that are connected by axons to frequently distant neuronal cell bodies. Common to all chemical synapses are protein machineries that orchestrate exocytosis of synaptic vesicles (SVs) filled with neurotransmitters in response to presynaptic action potentials (APs), resulting in activation of postsynaptic receptors. Moreover, synapses are composed of structurally and functionally distinct subcompartments, such as free and docked SVs, endosomes, active zones (AZs) at the presynaptic side, and receptor-containing membranes with associated scaffold proteins on the postsynaptic side. Thus, it is not surprising that mass spectrometry (MS)-based proteomics, combined with subcellular fractionation, yields protein inventories of high complexity. For instance, >2,000 protein species were identified in synaptosomes (1), ∼400 in the SV fraction (2), ∼1,500 in postsynaptic densities (3), and ∼100 in an AZ-enriched preparation (4).While these studies provide insights into the protein composition of synaptic structures, they are still inherently limited for two reasons. First, synapses are functionally diverse with respect to the chemical nature of their neurotransmitters, as well as their synaptic strength, kinetics, and plasticity properties (5). Therefore, analyzed subcellular fractions represent “averages” of a great diversity of synapses (6) or SVs (2). The second limitation is that proteins known to be present in specific subsets were not found in these studies, despite the unprecedented sensitivity of modern mass spectrometers. In fact, many functionally critical synaptic proteins have remained undetected. For example, the synaptotagmin (Syt) family, major Ca²⁺ sensors of SV exocytosis, comprises >15 members, of which only 5 had been identified in previous SV proteomics (2, 4, 7). Missing isoforms included Syt7, involved in asynchronous transmitter release (8), synaptic plasticity (9), and SV recycling (10). Likewise, the vesicular transporters for monoamines (VMATs) and acetylcholine (VAChT) neurotransmitters were missing in these studies. Clearly, known components of the diversified synaptic proteome have been missing, and it is not possible to predict how many more such proteins remain hidden.What are the reasons for the continuing incompleteness of the synaptic protein inventory? Proteome identification and quantification rely heavily on MS detectability of peptides generated by digestion of extracted proteins with sequence-specific enzymes, such as trypsin. However, in MS analysis of complex biological samples, peptide signals from a few abundant proteins often mask those that are less abundant. Additionally, the probability of obtaining peptides with similar masses, but different amino acid sequences, increases with increasing sample complexity (11, 12). To overcome these limitations, we have elaborated a workflow with dual-enzymatic protein digestion in sequence combined with an extensive peptide separation prior to MS analysis. As proof of concept, we have utilized purified SV fractions from rat whole brain, which serve as a benchmark for quantitative organellar proteomics (2). As a result, we detected ∼1,500 proteins in the SV fraction, three times more than reported previously. This proteome not only covers all known canonical SV proteins but also contains proteins previously overlooked, such as the low-abundance Syts and SV transporters. Moreover, peptide quantification allowed for differentiating “SV-resident” from “SV-visitor” proteins. In fact, most “SV-resident” proteins revealed in our SV proteomics are of low abundance, with an average copy number of less than 1 per SV, suggesting a larger molecular and functional diversity of SVs than previously thought. Remarkably, more than 200 proteins detected in the SV fraction are genetically associated with brain disorders, 76% of which were previously hidden.     

CD24 is expressed by myofiber synaptic nuclei and regulates synaptic transmission

The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further. We show that CD24, which encodes a small, variably and highly glycosylated, glycosylphosphatidylinositol (GPI)-linked protein, is expressed preferentially by myofiber synaptic nuclei in embryonic and adult muscle, and that CD24 expression is restricted to the central region of muscle independent of innervation. Moreover, we show that CD24 has a role in presynaptic differentiation, because synaptic transmission is depressed and fails entirely, in a cyclical manner, after repetitive stimulation of motor axons in CD24 mutant mice. These deficits in synaptic transmission, which are accompanied by aberrant stimulus-dependent uptake of AM1-43 from axons, indicate that CD24 is required for normal presynaptic maturation and function. Because CD24 is also expressed in some neurons, additional experiments will be required to determine whether pre- or postsynaptic CD24 mediates these effects on presynaptic development and function.     

Saturable acetylcholine transport into purified cholinergic synaptic vesicles.

The uptake of [3H[acetylcholine ([3H]AcCho) into cholinergic synaptic vesicle ghosts purified from Torpedo electric organ was studied at concentration of [3H]AcCho ranging from 0.1 to 10 mM. The accumulated [3H]AcCho can be released either by hypoosmotic buffer or by low levels of the detergent Triton X-100. Kinetic analysis of the initial rate of [3H]AcCho uptake reveals temperature-dependent saturation kinetics which are best fitted by high-affinity (KTh approximately 0.3 mM) and low-affinity (KT) approximately 10 mM) vesicular [3H]AcCho transport systems. Several lines of evidence suggest that [3H]AcCho transport is mediated by vesicle-associated transport systems and not by a contaminant of other subcellular moieties such as the plasma membrane choline transport system. (i) The specific activity of the [3H]AcCho transport systems is higher in the purest vesicular fraction than in the less-pure fractions. (ii) Ghosts prepared from isolated synaptosomes manifest only low levels of low-affinity [3H]AcCho transport and no high-affinity [3H]AcCho transport. (iii) The vesicular AcCho transport systems lack some of the typical characteristics of synaptosomal choline transport, such as Na+ activation. (iv) The ratio of uptakes of [3H]AcCho and [3H]choline (10 microM) is about 5-fold higher in the pure vesicles than in isolated synaptosomal membranes. Addition of Mg2+-ATP decreases the rate of vesicular [3H]AcCho uptake by about 50%. The simultaneous addition of NaHCO3 and Mg2+-ATP results in activation of [3H]AcCho uptake to about 125% (relative to control), which is a 2.5-fold enhancement relative to the rate observed with Mg2+-ATP. The present findings demonstrate the presence of novel vesicle-associated AcCho transport systems. Their physiological role in the life cycle of the cholinergic synaptic vesicle and nerve terminal are discussed.     

A small pool of vesicles maintains synaptic activity in vivo

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1-5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ≈ 5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper.     

Tissue factor-mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor

Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue factor (TF) was investigated in baby hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell surface expression of TF on BHK cells was required for VIIa internalization and degradation. Approximately 50% of cell surface-bound VIIa was internalized in one hour, and a majority of the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino acid residue 245 (C245S). Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibit the internalization via clathrin-coated pits, did not affect the internalization of VIIa. Furthermore, receptor-associated protein, known to block binding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Addition of tissue factor pathway inhibitor/factor Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approximately 10% of available cell surface TF/VIIa sites, it accounts for 65% of the maximal activation of factor X by the cell surface TF/VIIa. In summary, the present data provide evidence that TF-dependent internalization of VIIa in kidney cells occurs through a clathrin-independent mechanism and does not require the cytoplasmic domain of TF.     

Ion channels in synaptic vesicles from Torpedo electric organ.

A simple method has been developed for fusing synaptic vesicles into spherical structures 20-50 micron in diameter. The method has been applied to purified cholinergic synaptic vesicles from Torpedo electric organ, and the membrane properties of these fused structures have been studied by the "cell"-attached version of the patch clamp technique. A large conductance potassium-preferring channel, termed the P channel, was consistently observed in preparations of fused synaptic vesicles. The selectivity of the channel for potassium over sodium was approximately equal to 2.8-fold. Two major conductance levels were observed during P-channel activity, and their relative proportion was dependent on the voltage applied to the membrane through the patch pipette. P channels were not seen in fused preparations of purified Torpedo lipids, nor was the frequency of their occurrence increased in preparations enriched with plasma membrane or nonvesicular membranes. We suggest, therefore, that the P channels are components of the synaptic vesicle membrane. Their function in synaptic transmission physiology is still unknown.     

Differential requirements for clathrin in receptor-mediated endocytosis and maintenance of synaptic vesicle pools

Clathrin is a coat protein involved in vesicle budding from several membrane-bound compartments within the cell. Here we present an analysis of a temperature-sensitive (ts) mutant of clathrin heavy chain (CHC) in a multicellular animal. As expected Caenorhabditis elegans chc-1(b1025ts) mutant animals are defective in receptor-mediated endocytosis and arrest development soon after being shifted to the restrictive temperature. Steady-state clathrin levels in these mutants are reduced by more than 95% at all temperatures. Hub interactions and membrane associations are lost at the restrictive temperature. chc-1(b1025ts) animals become paralyzed within minutes of exposure to the restrictive temperature because of a defect in the nervous system. Surprisingly synaptic vesicle number is not reduced in chc-1(b1025ts) animals. Consistent with the normal number of vesicles, postsynaptic miniature currents occur at normal frequencies. Taken together, these results indicate that a high level of CHC activity is required for receptor-mediated endocytosis in nonneuronal cells but is largely dispensable for maintenance of synaptic vesicle pools.     

Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis

The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.     

Active transport of gamma-aminobutyric acid and glycine into synaptic vesicles.

Although gamma-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study we have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. We present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity (Km in the millimolar range) similar to vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.     

The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

Presynaptic nerve terminals contain between several hundred vesicles (for example in small CNS synapses) and several tens of thousands (as in neuromuscular junctions). Although it has long been assumed that such high numbers of vesicles are required to sustain neurotransmission during conditions of high demand, we found that activity in vivo requires the recycling of only a few percent of the vesicles. However, the maintenance of large amounts of reserve vesicles in many evolutionarily distinct species suggests that they are relevant for synaptic function. We suggest here that these vesicles constitute buffers for soluble accessory proteins involved in vesicle recycling, preventing their loss into the axon. Supporting this hypothesis, we found that vesicle clusters contain a large variety of proteins needed for vesicle recycling, but without an obvious function within the clusters. Disrupting the clusters by application of black widow spider venom resulted in the diffusion of numerous soluble proteins into the axons. Prolonged stimulation and ionomycin application had a similar effect, suggesting that calcium influx causes the unbinding of soluble proteins from vesicles. Confirming this hypothesis, we found that isolated synaptic vesicles in vitro sequestered soluble proteins from the cytosol in a process that was inhibited by calcium addition. We conclude that the reserve vesicles support neurotransmission indirectly, ensuring that soluble recycling proteins are delivered upon demand during synaptic activity.