Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.     

A Tyrosine-based Motif Localizes a Drosophila Vesicular Transporter to Synaptic Vesicles in Vivo

Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.     

The loading of neurotransmitters into synaptic vesicles

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.     

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters

Studies of synapsin-deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild-type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double-labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.     

Molecular analysis of vesicular amine transporter function and targeting to secretory organelles.

Vesicular transporters are responsible for the loading of neurotransmitters into specialized secretory organelles in neurons and neuroendocrine cells to make them available for regulated neurosecretion. The exocytotic release of neurotransmitter therefore depends on the functional activity of the vesicular transporters and their efficient sorting to these secretory organelles. Molecular analysis of vesicular transport proteins has revealed important information regarding structural domains responsible for their functional properties, including substrate specificity and trafficking to various classes of secretory vesicles. These studies have established the existence of an important functional relationship between transporter activity and presynaptic quantal neurosecretion.     

The vesicular monoamine transporter 2 contains trafficking signals in both its N-glycosylation and C-terminal domains

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family that packages acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or SVs, respectively. These transporters share similarities in sequence and structure with their N- and C-terminal domains located in the cytoplasm. When expressed in PC12 cells, VMAT2 localizes to LDCV, whereas VAChT is found mainly on synaptic-like microvesicles. Previous studies have shown that the cytoplasmic C-terminal domain of VAChT contains signals targeting this transporter to SVs. However, the targeting signals for VMAT have not been completely elucidated. To identify signals targeting VMAT2 to LDCV, the subcellular localization of VMAT2-VAChT chimeras was analyzed in PC12 cells. Chimeras having either the N-terminal region through transmembrane domain 2 of VMAT2 or the C-terminal domain of VMAT2 do not traffic to LDCV efficiently. In contrast, chimeras having both of these regions, or the luminal glycosylated loop in conjunction with transmembrane domains 1 and 2 and the C-terminal domain of VMAT2, traffic to LDCV. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, causes VMAT2 to localize to synaptic-like microvesicles. The results indicate that both mature N-linked glycosylation and the C-terminus are important for proper trafficking of VMAT2 and that the locations of trafficking signals in VMAT2 and VAChT are surprisingly different.     

A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.     

Differential sorting of the vesicular glutamate transporter 1 into a defined vesicular pool is regulated by light signaling involving the clock gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.     

Diversity of function and mechanism in a family of organic anion transporters

Originally identified as transporters for inorganic phosphate, solute carrier 17 (SLC17) family proteins subserve diverse physiological roles. The vesicular glutamate transporters (VGLUTs) package the principal excitatory neurotransmitter glutamate into synaptic vesicles (SVs). In contrast, the closely related sialic acid transporter sialin mediates the flux of sialic acid in the opposite direction, from lysosomes to the cytoplasm. The two proteins couple in different ways to the H⁺ electrochemical gradient driving force, and high-resolution structures of the Escherichia coli homolog d-galactonate transporter (DgoT) and more recently rat VGLUT2 now begin to suggest the mechanisms involved as well as the basis for substrate specificity.     

Dileucine motif is sufficient for internalization and synaptic vesicle targeting of vesicular acetylcholine transporter

Efficient cholinergic transmission requires accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs). However, the signals that regulate this vesicular targeting are not well characterized. Although previous studies suggest that the C-terminus of the transporter is required for its SV targeting, it is not clear whether this region is sufficient for this process. Furthermore, a synaptic vesicle-targeting motif (SVTM) within this sequence remains to be identified. Here we use a chimeric protein, TacA, between an unrelated plasma membrane protein, Tac, and the C-terminus of VAChT to demonstrate the sufficiency of the C-terminus for targeting to synaptic vesicle-like vesicles (SVLVs) in PC12 cells. TacA shows colocalization and cosedimentation with the SV marker synaptophysin. Deletion mutation analysis of TacA demonstrates that a short, dileucine motif-containing sequence is required and sufficient to direct this targeting. Dialanine mutation analysis within this sequence suggests indistinguishable signals for both internalization and SV sorting. Using additional chimeras as controls, we confirm the specificity of this region for SVLVs targeting. Therefore, we suggest that the dileucine-containing motif is sufficient as a dual signal for both internalization and SV targeting during VAChT trafficking.     

The vesicular monoamine content regulates VMAT2 activity through Galphaq in mouse platelets. Evidence for autoregulation of vesicular transmitter uptake

Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go(2), which down-regulates the activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5'-(beta(i)gamma-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1-/-) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1-/- mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1-/- platelets with serotonin or noradrenaline. Galpha(q) mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Galpha(q) deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A(2) receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Galpha(q).     

The components required for amino acid neurotransmitter signaling are present in adipose tissues

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABA(A)Ralpha2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues.     

Regulation of vesicular neurotransmitter transporters

Neurotransmitters are key molecules of neurotransmission. They are concentrated first in the cytosol and then in small synaptic vesicles of presynaptic terminals by the activity of specific neurotransmitter transporters of the plasma and the vesicular membrane, respectively. It has been shown that postsynaptic responses to single neurotransmitter packets vary over a wide range, which may be due to a regulation of vesicular neurotransmitter filling. Vesicular filling depends on the availability of transmitter molecules in the cytoplasm and the active transport into secretory vesicles relying on a proton gradient. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Go2 and Gq, which regulate VMAT activities in brain and platelets, respectively, and may also be involved in the regulation of VGLUTs. It appears that the vesicular content activates the G protein, suggesting a signal transduction form the luminal site which might be mediated by a vesicular G-protein coupled receptor or, as an alternative, possibly by the transporter itself. These novel functions of G proteins in the control of transmitter storage may link regulation of the vesicular content to intracellular signal cascades.     

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.     

Vesicular neurotransmitter transporters

Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

A splice variant of the Drosophila vesicular monoamine transporter contains a conserved trafficking domain and functions in the storage of dopamine, serotonin, and octopamine

Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.     

Transport mechanisms in acetylcholine and monoamine storage.

Sequence-related vesicular acetylcholine transporter (VAChT) and vesicular monoamine transporter (VMAT) transport neurotransmitter substrates into secretory vesicles. This review seeks to identify shared and differentiated aspects of the transport mechanisms. VAChT and VMAT exchange two protons per substrate molecule with very similar initial velocity kinetics and pH dependencies. However, vesicular gradients of ACh in vivo are much smaller than the driving force for uptake and vesicular gradients of monoamines, suggesting the existence of a regulatory mechanism in ACh storage not found in monoamine storage. The importance of microscopic rather than macroscopic kinetics in structure-function analysis is described. Transporter regions affecting binding or translocation of substrates, inhibitors, and protons have been found with photoaffinity labeling, chimeras, and single-site mutations. VAChT and VMAT exhibit partial structural and mechanistic homology with lactose permease, which belongs to the same sequence-defined superfamily, despite opposite directions of substrate transport. The vesicular transporters translocate the first proton using homologous aspartates in putative transmembrane domain X (ten), but they translocate the second proton using unknown residues that might not be conserved between them. Comparative analysis of the VAChT and VMAT transport mechanisms will aid understanding of regulation in neurotransmitter storage.     

Vesicular Monogamy?

Vesicular neurotransmitter transporters package transmitter into the lumen of synaptic vesicles for quantal release. However, the number of transporters that localize to each vesicle is not known. In this issue of Neuron, a study by Daniels et al. using the Drosophila neuromuscular junction and mutations of the vesicular glutamate transporter suggests that one transporter may suffice to fill each vesicle.     

A putative vesicular transporter expressed in Drosophila mushroom bodies that mediates sexual behavior may define a neurotransmitter system

Vesicular transporters are required for the storage of?all classical and amino acid neurotransmitters in synaptic vesicles. Some neurons lack known vesicular transporters, suggesting additional neurotransmitter systems remain unidentified. Insect mushroom bodies (MBs) are critical for several behaviors, including learning, but the neurotransmitters released by the intrinsic Kenyon cells (KCs) remain unknown. Likewise, KCs do not express a known vesicular transporter. We report the identification of a novel Drosophila gene portabella (prt) that is structurally similar to known vesicular transporters. Both larval and adult brains express PRT in the KCs of the MBs. Additional PRT cells project to the central complex and optic ganglia. prt mutation causes an olfactory learning deficit and an unusual defect in the male's position during copulation that is rescued by expression in KCs. Because prt is expressed in neurons that lack other known vesicular transporters or neurotransmitters, it may define a previously unknown neurotransmitter system responsible for sexual behavior and a component of olfactory learning.