The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter release

A longstanding hypothesis is that ion channels are present in the membranes of synaptic vesicles and might affect neurotransmitter release. Here we demonstrate that TRPM7, a member of the transient receptor potential (TRP) ion channel family, resides in the membrane of synaptic vesicles of sympathetic neurons, forms molecular complexes with the synaptic vesicle proteins synapsin I and synaptotagmin I, and directly interacts with synaptic vesicular snapin. In sympathetic neurons, changes in TRPM7 levels and channel activity alter acetylcholine release, as measured by EPSP amplitudes and decay times in postsynaptic neurons. TRPM7 affects EPSP quantal size, an intrinsic property of synaptic vesicle release. Targeted peptide interference of TRPM7's interaction with snapin affects the amplitudes and kinetics of postsynaptic EPSPs. Thus, vesicular TRPM7 channel activity is critical to neurotransmitter release in sympathetic neurons.     

Synaptotagmin-7 controls the size of the reserve and resting pools of synaptic vesicles in hippocampal neurons

Continuous neurotransmitter release is subjected to synaptic vesicle availability, which in turn depends on vesicle recycling and the traffic of vesicles between pools. We studied the role of Synaptotagmin-7 (Syt-7) in synaptic vesicle accessibility for release in hippocampal neurons in culture. Synaptic boutons from Syt-7 knockout (KO) mice displayed normal basal secretion with no alteration in the RRP size or the probability of release. However, stronger stimuli revealed an increase in the size of the reserve and resting vesicle pools in Syt-7 KO boutons compared with WT. These data suggest that Syt-7 plays a significant role in the vesicle pool homeostasis and, consequently, in the availability of vesicles for synaptic transmission during strong stimulation, probably, by facilitating advancing synaptic vesicles to the readily releasable pool.     

Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.     

v-SNARE composition distinguishes synaptic vesicle pools

Synaptic vesicles belong to two distinct pools, a recycling pool responsible for the evoked release of neurotransmitter and a resting pool unresponsive to stimulation. The uniform appearance of synaptic vesicles has suggested that differences in location or cytoskeletal association account for these differences in function. We now find that the v-SNARE tetanus toxin-insensitive vesicle-associated membrane protein (VAMP7) differs from other synaptic vesicle proteins in its distribution to the two pools, providing evidence that they differ in molecular composition. We also find that both resting and recycling pools undergo spontaneous release, and when activated by deletion of the longin domain, VAMP7 influences the properties of release. Further, the endocytosis that follows evoked and spontaneous release differs in mechanism, and specific sequences confer targeting to the different vesicle pools. The results suggest that different endocytic mechanisms generate synaptic vesicles with different proteins that can endow the vesicles with distinct properties.     

Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles.

A highly purified preparation of synaptic vesicles was prepared to study the relationship between calcium-dependent neurotransmitter release and protein phosphorylation. Calcium ions simultaneously produced significant increases in both the endogenous release of norepinephrine from the synaptic vesicles and the endogenous incorporation of [³²p] phosphate into specific synaptic vesicle proteins. The results are compatible with the hypothesis that the action of calcium on the phosphorylation of specific synaptic vesicle proteins is the molecular mechanism mediating some of the effects of calcium on neurotransmitter release and synaptic vesicle function.     

Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.     

The coupling between synaptic vesicles and Ca2+ channels determines fast neurotransmitter release

In order to release neurotransmitter synchronously in response to a presynaptic action potential, synaptic vesicles must be both release competent and located close to presynaptic Ca2+ channels. It has not been shown, however, which of the two is the more decisive factor. We tested this issue at the calyx of Held synapse by combining Ca2+ uncaging and electrophysiological measurements of postsynaptic responses. After depletion of the synaptic vesicles that are responsible for synchronous release during action potentials, uniform elevation of intracellular Ca2+ by Ca2+ uncaging could still elicit rapid release. The Ca2+ sensitivity of remaining vesicles was reduced no more than 2-fold, which is insufficient to explain the slow-down of the kinetics of release (10-fold) observed during a depolarizing pulse. We conclude that recruitment of synaptic vesicles to sites where Ca2+ channels cluster, rather than fusion competence, is a limiting step for rapid neurotransmitter release in response to presynaptic action potentials.     

Neurotransmitter release: the dark side of the vacuolar-H+ATPase

Vacuolar-H⁺ATPase (V-ATPase) is a complex enzyme with numerous subunits organized in two domains. The membrane domain V0 contains a proteolipid hexameric ring that translocates protons when ATP is hydrolysed by the catalytic cytoplasmic sector (V1). In nerve terminals, V-ATPase generates an electrochemical proton gradient that is acid and positive inside synaptic vesicles. It is used by specific neurotransmitter-proton antiporters to accumulate neurotransmitters inside their storage organelles. During synaptic activity, neurotransmitters are released from synaptic vesicles docked at specialized portions of the presynaptic plasma membrane, the active zones. A fusion pore opens that allows the neurotransmitter to be released from the synaptic vesicle lumen into the synaptic cleft. We briefly review experimental data suggesting that the membrane domain of V-ATPase could be such a fusion pore.We also discuss the functional implications for quantal neurotransmitter release of the sequential use of the same V-ATPase membrane domain in two different events, neurotransmitter accumulation in synaptic vesicles first, and then release from these organelles during synaptic activity.     

The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.     

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.     

Ultrastructural organization of presynaptic terminals

In response to calcium influx, synaptic vesicles fuse very rapidly with the plasma membrane to release their neurotransmitter content. An important mechanism for sustained release includes the formation of new vesicles by local endocytosis. How synaptic vesicles are trafficked from the sites of endocytosis to the sites of release and how they are maintained at the release sites remain poorly understood. Recent studies using fast freezing immobilization and electron tomography have led to insights on the ultrastructural organization of presynaptic boutons and how these structural elements may maintain synaptic vesicles and organize their exocytosis at particular areas of the plasma membrane.     

Protein-protein interactions and protein modules in the control of neurotransmitter release

Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release.     

Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release

During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, we monitored an endosomal compartment in Drosophila neuromuscular synapses. We showed that in conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and only recovers from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. We demonstrate that the small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance.     

Ca2+/calmodulin-mediated neurotransmitter release and neurobehavioural deficits following lead exposure

The present study was designed to investigate the effect of in vitro and in vivo lead exposure on calmodulin-mediated neurotransmitter release from synaptic vesicles with a view to explain the mechanism involved in its behavioural effects. It was observed that lead stimulated calmodulin, in terms of its ability to activate cAMP phosphodiesterase, following in vitro and in vivo exposure. Lead was also seen to enhance calmodulin-mediated synaptic vesicle protein phosphorylation. The increase in lead-induced synaptic vesicle protein phosphorylation was accompanied by enhanced release of acetylcholine from synaptic vesicles following in vitro lead exposure by a calmodulin-dependent mechanism. The ability of Ca(2+)/calmodulin to evoke acetylcholine release was reduced in the synaptic vesicles isolated from lead-exposed animals. Concomitantly, the levels of acetylcholine were found to decrease by 37.8% in the lead-treated animals as compared to the controls. The neurochemical alterations following lead exposure were accompanied by neurobehavioural deficits in terms of impaired motor and cognitive functions. The results from the present study clearly suggest that lead exerts its neurotoxic effects by interfering with Ca(2+)/calmodulin-mediated neurotransmitter release that is eventually responsible for behavioural impairment.     

Vesicular and Plasma Membrane Transporters for Neurotransmitters

The regulated exocytosis that mediates chemical signaling at synapses requires mechanisms to coordinate the immediate response to stimulation with the recycling needed to sustain release. Two general classes of transporter contribute to release, one located on synaptic vesicles that loads them with transmitter, and a second at the plasma membrane that both terminates signaling and serves to recycle transmitter for subsequent rounds of release. Originally identified as the target of psychoactive drugs, these transport systems have important roles in transmitter release, but we are only beginning to understand their contribution to synaptic transmission, plasticity, behavior, and disease. Recent work has started to provide a structural basis for their activity, to characterize their trafficking and potential for regulation. The results indicate that far from the passive target of psychoactive drugs, neurotransmitter transporters undergo regulation that contributes to synaptic plasticity.The speed and potency of synaptic transmission depend on the immediate availability of synaptic vesicles filled with high concentrations of neurotransmitter. In this article, we focus on the mechanisms responsible for packaging transmitter into synaptic vesicles and for reuptake from the extracellular space that both terminates synaptic transmission and recycles transmitter for future rounds of release. Collectively, we refer to this entire process as the neurotransmitter cycle.The recycling of neurotransmitter illustrates a general, conceptual problem for the mechanism of vesicular release. At the plasma membrane, more active reuptake should help to replenish the pool of releasable transmitter, but may also reduce the extent and duration of signaling to the postsynaptic cell. Conversely, loss of reuptake increases the activation of receptors but results in the depletion of stores (Jones et al. 1998). At the vesicle, steeper concentration gradients release more transmitter per vesicle but reduce the cytosolic transmitter available for refilling, whereas more shallow gradients facilitate refilling but reduce the transmitter available for release. The way in which the nerve terminal balances these competing factors thus has profound consequences for synaptic transmission.     

Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.     

A minimal domain responsible for Munc13 activity

Munc13 proteins are essential in neurotransmitter release, controlling the priming of synaptic vesicles to a release-ready state. The sequences responsible for this priming activity are unknown. Here we identify a large alpha-helical domain of mammalian Munc13-1 that is autonomously folded and is sufficient to rescue the total arrest in neurotransmitter release observed in hippocampal neurons lacking Munc13s.