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.     

Ultrastructural localization of vesicle-associated membrane protein(s) to specialized membrane structures in human pericytes, vascular smooth muscle cells, endothelial cells, neutrophils, and eosinophils.

Vesicle-associated membrane proteins (VAMPs) are important to the trafficking of vesicles between membrane-bound intracytoplasmic organelles, in the facilitation of neurosecretion, and in constitutive and regulated secretion in non-neuronal cells. We used a pre-embedding ultrastructural immunonanogold method to localize VAMPs to subcellular sites in human cells of five lineages known to have cytoplasmic vesicles that may function in vesicular transport. We found VAMPs localized to caveolae in pericytes, vascular smooth muscle cells, and endothelial cells of venules, to the vesiculo-vacuolar organelle, recently defined in venular endothelial cells, to the vesicle-rich intergranular cytoplasm and secretory granule membranes of neutrophils, and to perigranular cytoplasmic secretory vesicles and secretory granule membranes in eosinophils. These specific localizations in five human vascular and granulocyte lineages support the notion that VAMPs have vesicle-associated functions in these cells.     

Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters

Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.     

Vesicular Ca(2+) mediates granule motion and exocytosis

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis.     

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.     

Chemical neuroanatomy of the vesicular amine transporters.

Acetylcholine, catecholamines, serotonin, and histamine are classical neurotransmitters. These small molecules also play important roles in the endocrine and immune/inflammatory systems. Serotonin secreted from enterochromaffin cells of the gut epithelium regulates gut motility; histamine secreted from basophils and mast cells is a major regulator of vascular permeability and skin inflammatory responses; epinephrine is a classical hormone released from the adrenal medulla. Each of these molecules is released from neural, endocrine, or immune/inflammatory cells only in response to specific physiological stimuli. Regulated secretion is possible because amines are stored in secretory vesicles and released via a stimulus-dependent exocytotic event. Amine storage-at concentrations orders of magnitude higher than in the cytoplasm-is accomplished in turn by specific secretory vesicle transporters that recognize the amines and move them from the cytosol into the vesicle. Immunohistochemical visualization of specific vesicular amine transporters (VATs) in neuronal, endocrine, and inflammatory cells provides important new information about how amine-handling cell phenotypes arise during development and how vesicular transport is regulated during homeostatic response events. Comparison of the chemical neuroanatomy of VATs and amine biosynthetic enzymes has also revealed cell groups that express vesicular transporters but not enzymes for monoamine synthesis, and vice versa: their function and regulation is a new topic of investigation in mammalian neurobiology. The chemical neuroanatomy of the vesicular amine transporters is reviewed here. These and similar data emerging from the study of the localization of the recently characterized vesicular inhibitory and excitatory amino acid transporters will contribute to understanding chemically coded synaptic circuitry in the brain, and amine-handling neuroendocrine and immune/inflammatory cell regulation.     

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein

Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase-activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.     

Vesicular and non-vesicular lipid export from the ER to the secretory pathway

The endoplasmic reticulum is the site of synthesis of most glycerophospholipids, neutral lipids and the initial steps of sphingolipid biosynthesis of the secretory pathway. After synthesis, these lipids are distributed within the cells to create and maintain the specific compositions of the other secretory organelles. This represents a formidable challenge, particularly while there is a simultaneous and quantitatively important flux of membrane components stemming from the vesicular traffic of proteins through the pathway, which can also vary depending on the cell type and status. To meet this challenge cells have developed an intricate system of interorganellar contacts and lipid transport proteins, functioning in non-vesicular lipid transport, which are able to ensure membrane lipid homeostasis even in the absence of membrane trafficking. Nevertheless, under normal conditions, lipids are transported in cells by both vesicular and non-vesicular mechanisms. In this review we will discuss the mechanism and roles of vesicular and non-vesicular transport of lipids from the ER to other organelles of the secretory pathway.     

Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

Trafficking of vesicular neurotransmitter transporters

Vesicular neurotransmitter transporters are required for the storage of all classical and amino acid neurotransmitters in secretory vesicles. Transporter expression can influence neurotransmitter storage and release, and trafficking targets the transporters to different types of secretory vesicles. Vesicular transporters traffic to synaptic vesicles (SVs) as well as large dense core vesicles and are recycled to SVs at the nerve terminal. Some of the intrinsic signals for these trafficking events have been defined and include a dileucine motif present in multiple transporter subtypes, an acidic cluster in the neural isoform of the vesicular monoamine transporter (VMAT) 2 and a polyproline motif in the vesicular glutamate transporter (VGLUT) 1. The sorting of VMAT2 and the vesicular acetylcholine transporter to secretory vesicles is regulated by phosphorylation. In addition, VGLUT1 uses alternative endocytic pathways for recycling back to SVs following exocytosis. Regulation of these sorting events has the potential to influence synaptic transmission and behavior.     

Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles

Much current work on the mechanism of neurosecretion has focused on proteins specific to neural secretory vesicles (synaptic vesicles). We report a calcium-stimulated lipid kinase that co-purifies with rat brain synaptic vesicles. This enzyme activity is found only in membrane fractions that contain synaptic vesicle markers. Based on identification of the lipid product as ceramide 1-phosphate and on the finding that ceramide kinase activity co-purifies with synaptic vesicles, the enzyme is proposed to be a ceramide kinase. Kinase activity is stimulated by micromolar concentrations of calcium. Calcium increases the apparent Vmax of the reaction with little effect on the Km for ceramide. The vesicular localization of this enzyme, the requirement for ATP, and the stimulation of enzyme activity by micromolar calcium suggest that ceramide phosphorylation may be associated with neurotransmitter release.     

Differential Localization of Vesicular Acetylcholine and Monoamine Transporters in PC12 Cells but Not CHO Cells

Previous studies have indicated that neuro-endocrine cells store monoamines and acetylcholine (ACh) in different secretory vesicles, suggesting that the transport proteins responsible for packaging these neurotransmitters sort to distinct vesicular compartments. Molecular cloning has recently demonstrated that the vesicular transporters for monoamines and ACh show strong sequence similarity, and studies of the vesicular monoamine transporters (VMATs) indicate preferential localization to large dense core vesicles (LDCVs) rather than synaptic-like microvesicles (SLMVs) in rat pheochromocytoma PC12 cells. We now report the localization of the closely related vesicular ACh transporter (VAChT). In PC12 cells, VAChT differs from the VMATs by immunofluorescence and fractionates almost exclusively to SLMVs and endosomes by equilibrium sedimentation. Immunoisolation further demonstrates colocalization with synaptophysin on SLMVs as well as other compartments. However, small amounts of VAChT also occur on LDCVs. Thus, VAChT differs in localization from the VMATs, which sort predominantly to LDCVs. In addition, we demonstrate ACh transport activity in stable PC12 transformants overexpressing VAChT. Since previous work has suggested that VAChT expression confers little if any transport activity in non-neural cells, we also determined its localization in transfected CHO fibroblasts. In CHO cells, VAChT localizes to the same endosomal compartment as the VMATs by immunofluorescence, density gradient fractionation, and immunoisolation with an antibody to the transferrin receptor. We have also detected ACh transport activity in the transfected CHO cells, indicating that localization to SLMVs is not required for function. In summary, VAChT differs in localization from the VMATs in PC12 cells but not CHO cells.     

The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na⁺-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15 mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H⁺-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.     

Book reviews

Eukaryotic cells contain hundreds of different lipid species that are not uniformly distributed among their membranes. For example, sphingolipids and sterols form gradients along the secretory pathway with the highest levels in the plasma membrane and the lowest in the endoplasmic reticulum. Moreover, lipids in late secretory organelles display asymmetric transbilayer arrangements with the aminophospholipids concentrated in the cytoplasmic leaflet. This lipid heterogeneity can be viewed as a manifestation of the fact that cells exploit the structural diversity of lipids in organizing intracellular membrane transport. Lipid immiscibility and the generation of phase-separated lipid domains provide a molecular basis for sorting membrane proteins into specific vesicular pathways. At the same time, energy-driven aminophospholipid transporters participate in membrane deformation during vesicle biogenesis. This review will focus on how selective membrane transport relies on a dynamic interplay between membrane lipids and proteins.     

Intravesicular calcium release mediates the motion and exocytosis of secretory organelles: a study with adrenal chromaffin cells

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.     

Lipid microdomains,lipid translocation and the organization of intracellular membrane transport (Review)

Eukaryotic cells contain hundreds of different lipid species that are not uniformly distributed among their membranes. For example, sphingolipids and sterols form gradients along the secretory pathway with the highest levels in the plasma membrane and the lowest in the endoplasmic reticulum. Moreover, lipids in late secretory organelles display asymmetric transbilayer arrangements with the aminophospholipids concentrated in the cytoplasmic leaflet. This lipid heterogeneity can be viewed as a manifestation of the fact that cells exploit the structural diversity of lipids in organizing intracellular membrane transport. Lipid immiscibility and the generation of phase-separated lipid domains provide a molecular basis for sorting membrane proteins into specific vesicular pathways. At the same time, energy-driven aminophospholipid transporters participate in membrane deformation during vesicle biogenesis. This review will focus on how selective membrane transport relies on a dynamic interplay between membrane lipids and proteins.     

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.     

The Role of Clathrin in Post-Golgi Trafficking in Toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle.     

Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae

Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form. Here, we show that Rho1p in secretory vesicles cannot activate 1,3-beta-glucan synthase, a cell wall synthesizing enzyme, during vesicular transport to the plasma membrane. Analyses with an antibody preferentially reacting with the GTP-bound form of Rho1p revealed that Rho1p remains in the inactive form in secretory vesicles. Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked. Overexpression of Rom2p results in delocalization of Rom2p and accumulation of 1,3-beta-glucan in secretory vesicles. Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.     

Walking to work: roles for class V myosins as cargo transporters

Cells use molecular motors, such as myosins, to move, position and segregate their organelles. Class V myosins possess biochemical and structural properties that should make them ideal actin-based cargo transporters. Indeed, studies show that class V myosins function as cargo transporters in yeast, moving a range of organelles, such as the vacuole, peroxisomes and secretory vesicles. There is also increasing evidence in vertebrate cells that class V myosins not only tether organelles to actin but also can serve as short-range, point-to-point organelle transporters, usually following long-range, microtubule-dependent organelle transport.