Human Vesicular Monoamine Transporter Research Articles

Neurotransmitter recognition by human vesicular monoamine transporter 2

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design.

Humanized substitutions of Vmat1 in mice alter amygdala-dependent behaviors associated with the evolution of anxiety

SummaryThe human vesicular monoamine transporter 1 (VMAT1) harbors unique substitutions (Asn136Thr/Ile) that affect monoamine uptake into synaptic vesicles. These substitutions are absent in all known mammals, suggesting their contributions to distinct aspects of human behavior modulated by monoaminergic transmissions, such as emotion and cognition. To directly test the impact of these human-specific mutations, we introduced the humanized residues into mouse Vmat1 via CRISPR/Cas9-mediated genome editing and examined changes at the behavioral, neurophysiological, and molecular levels. Behavioral tests revealed reduced anxiety-related traits of Vmat1Ile mice, consistent with human studies, and electrophysiological recordings showed altered oscillatory activity in the amygdala under anxiogenic conditions. Transcriptome analyses further identified changes in gene expressions in the amygdala involved in neurodevelopment and emotional regulation, which may corroborate the observed phenotypes. This knock-in mouse model hence provides compelling evidence that the mutations affecting monoaminergic signaling and amygdala circuits have contributed to the evolution of human socio-emotional behaviors.

Synthesis and Discovery of Arylpiperidinylquinazolines: New Inhibitors of the Vesicular Monoamine Transporter.

Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate, releases dopamine, serotonin, and norepinephrine from vesicles into the cytosol of presynaptic neurons and induces reverse transport by the monoamine transporters to increase extracellular neurotransmitters. Currently available radioligands for VMAT2 have considerable liabilities: The binding of [3H]dihydrotetrabenazine ([3H]DHTB) to a site on VMAT2 is not dependent on ATP, and [3H]reserpine binds almost irreversibly to VMAT2. Herein we demonstrate that several arylpiperidinylquinazolines (APQs) are potent inhibitors of [3H]reserpine binding at recombinant human VMAT2 expressed in HEK-293 cells. These compounds are biodiastereoselective and bioenantioselective. The lead radiolabeled APQ is unique because it binds reversibly to VMAT2 but does not bind the [3H]DHTB binding site. Furthermore, experimentation shows that several novel APQ ligands have high potency for inhibition of uptake by both HEK-VMAT2 cells and mouse striatal vesicles and may be useful tools for characterizing drug-induced effects on human VMAT2 expression and function.

The profile of mephedrone on human monoamine transporters differs from 3,4-methylenedioxymethamphetamine primarily by lower potency at the vesicular monoamine transporter

Mephedrone (4-methylmethcathinone, MMC) and 3,4-methylenedioxymethamphetamine (MDMA) are constituents of popular party drugs with psychoactive effects. Structurally they are amphetamine-like substances with monoamine neurotransmitter enhancing actions. We therefore compared their effects on the human monoamine transporters using human cell lines stably expressing the human noradrenaline, dopamine and serotonin transporter (NET, DAT and SERT); preparations of synaptic vesicles from human striatum in uptake experiments; and a superfusion system where releasing effects can be reliably measured. MMC and MDMA were equally potent in inhibiting noradrenaline uptake at NET, with IC50 values of 1.9 and 2.1µM, respectively. Compared to their NET inhibition potency, both drugs were weaker uptake inhibitors at DAT and SERT, with MMC being more potent than MDMA at DAT (IC50: 5.9 vs 12.6µM) and less potent than MDMA at SERT (IC50: 19.3 vs 7.6µM). MMC and MDMA both induced concentration-dependently [3H]1-methyl-4-phenylpyridinium-release from NET-, DAT or SERT-expressing cells which was clearly transporter-mediated release as demonstrated by the selective inhibitory effects of nmolar to low µmolar concentrations of desipramine, GBR 12909 and fluoxetine, respectively. MMC and MDMA differed most in their inhibition of [3H]dopamine uptake by synaptic vesicles from human striatum with MDMA being 10-fold more potent than MMC (IC50: 20 vs 223µM) and their ability to release [3H]dopamine from human vesicular monoamine transporter expressing SH-SY5Y neuroblastoma cells in which MDMA seems to have a stronger effect. Our findings give a molecular explanation to the lower long-term neurotoxicity of MMC compared to MDMA.

Tetrabenazine

Oral tetrabenazine is currently the only drug approved by the US FDA for the treatment of chorea associated with Huntington's disease (HD). Although the precise antichorea mechanism of action is unknown, it most likely involves reversible depletion of monoamines, particularly dopamine, from presynaptic terminals via inhibition of human vesicular monoamine transporter type 2. In a 12-week, double-blind, placebo-controlled trial conducted in the US in patients with HD, oral tetrabenazine (≤100 mg/day; n = 54) was significantly (p = 0.0001) more efficacious than placebo (n = 30) at improving adjusted mean Unified HD Rating Scale (UHDRS) total maximum chorea scores (reduced from baseline by 5 vs 1.5) [primary endpoint]. After 12 weeks, improvements in UHDRS total maximum chorea scores of >3 were achieved by significantly (p < 0.0001) more patients in the tetrabenazine group than in the placebo group. The antichorea efficacy of tetrabenazine was maintained in an 80-week extension study (n = 75), with the adjusted mean UHDRS total maximum chorea score significantly (p < 0.001) reduced from baseline (score of 14.9) by 4.6 points (primary outcome). In the 12-week trial and 80-week extension study, treatment-emergent adverse events in the tetrabenazine group mainly occurred during the dosage-titration phase, a period during which the dosage was individually optimized. Most of these events were mild to moderate and were manageable with dosage adjustments or discontinuation of study drug.

Effects of methamphetamine and lobeline on vesicular monoamine and dopamine transporter-mediated dopamine release in a cotransfected model system.

Dopamine (DA) retention and drug-induced release kinetics were characterized in human embryonic kidney (HEK)-293 cells stably coexpressing the human DA transporter (hDAT) and human vesicular monoamine transporter (hVMAT2). Cofunction of hDAT and hVMAT2 caused greater retention of [3H]DA at 20 min (37 degrees C), or 45 min (22 degrees C) compared with cells that were treated with dihydrotetrabenazene (DHTB) to block the hVMAT2. In hDAT- and hVMAT2-coexpressing cells treated with DHTB during [3H]DA loading, methamphetamine (METH)-induced efflux was only 20% of preloaded [3H]DA, compared with 50 to 60% efflux in the absence of DHTB. Interestingly, the presence of DHTB (during release only) increased the potency and efficacy of METH at inducing [3H]DA release (without DHTB: EC50=33.8 microM, maximal release 51%; release with DHTB: EC50=3.2 microM, maximal release 61%), suggesting that the effects of METH and DHTB on vesicular storage are additive. High concentrations of lobeline induced a statistically significant release of [3H]DA from HEK-hDAT-hVMAT2 cells, but only in the absence of DHTB, suggesting an hVMAT2-mediated effect. Likewise, lobeline did not induce a significant release of [3H]DA from HEK-hDAT cells. The substrates DA and p-tyramine induced robust release of preloaded [3H]DA from cotransfected cells. Cocaine was somewhat effective at blocking substrate-induced [3H]DA efflux. These results suggest that coexpression of the hDAT and hVMAT2 can be used as a model system to distinguish functional pools of DA and to quantify differences in drug effects on DA disposition. In addition, cotransfected cells can be used to determine mechanisms of simultaneous drug interactions at multiple sites.

Dissociation of the vesicular acetylcholine transporter domains important for high-affinity transport recognition, binding of vesamicol and targeting to synaptic vesicles

Chimeras between the human vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the human vesicular monoamine transporter (hVMAT2) have been constructed and stably expressed in a rat pheochromocytoma cell line (PC12) in an effort to identify cholinergic-specific domains of VAChT. Examination of the transport properties of a chimera in which the N-terminal portion (up to putative transmembrane domain II and including the lumenal glycosylated loop) of hVAChT was replaced with hVMAT2 sequences (2/V@ NheI) revealed that its apparent affinity for acetylcholine (ACh) was reduced approximately seven-fold compared to wild-type. However, the affinity of this chimera for vesamicol did not significantly differ from hVAChT. Similarly, the 2/V@ NheI chimera retained its preferential targeting to the small synaptic-like vesicles found in PC12 cells in agreement with our recently reported observations that the synaptic vesicle targeting domain resides in the cytoplasmic tail of VAChT.

Structural organization of the human vesicular monoamine transporter type-2 gene and promoter analysis using the jelly fish green fluorescent protein as a reporter.

The genomic structure of a human vesicle monoamine transporter, type-2 (hVMAT2) was determined from two overlapping cosmids, phVMAT2-cos1 and phVMT2-cos2, spanning more than 35 kb. The hVMAT2 open reading frame is encoded by 16 exons, with translation initiation and termination in exon 2 and exon 16, respectively. Several potential binding sites for transcriptional regulatory factors, including a cAMP response element (CRE) were identified in the 5'-upstream region of the gene. A promoter construct using the jellyfish green fluorescent protein (GFP) as reporter has been made and transfected into the human neuroblastoma cell line, SHSY-5Y. The cellular expression of the GFP was readily detected by fluorescence microscopy and cells expressing GFP could be sorted using a fluorescence-activated cell sorter (FACS), allowing the level of GFP expression in transfected SHSY-5Y cells to be quickly and reliably determined.

A Chimeric Vesicular Monoamine Transporter Dissociates Sensitivity to Tetrabenazine and Unsubstituted Aromatic Amines

Publisher Summary Two isoforms of the human vesicular monoamine transporter (hVMAT1 and hVMAT2) have recently been cloned and shown to differ with respect to their tissue distribution, substrate affinity, and sensitivity toward various inhibitors, including neurotoxic and psychoactive compounds. Functional chimeras have been constructed between hVMATl and hVMAT2 to identify domains responsible for the observed pharmacologic differences between these proteins. Tetrabenazine (TBZ) and two unsubstituted aromatic amines—amphetamine and histamine—have been evaluated because of the marked differences in their interaction with hVMAT1 and hVMAT2. The ability of these compounds to inhibit or compete with [ 3 H]serotonin (SHT) for uptake by hVMAT1 or hVMAT2 was measured after expression in digitonin-permeabilized fibroblastic (CV-1) cells. The affinity of hVMAT2 and hVMAT1 for [ 3 H]SHT was similiar. The specific interactions of substrates, inhibitors, neurotoxins, and psychoactive substances with VMAT1 and VMAT2 may facilitate a basic understanding of the molecular mechanisms of vesicular H + /monoamine antiport. Conserved Asp residues are predicted to lie within putative transmembrane domain (TMD) I, VI, X, and XI. It is likely that these putative TMDs, and perhaps others, participate in the formation of a gated pore through which protonated substrates and H + ions are exchanged.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Synaptic vesicular monoamine transporter expression: distribution and pharmacologic profile

The human vesicular monoamine transporter (hSVMT) cDNA predicts a protein of 515 amino acids that shares 92% amino acid identity with the rat cDNA. Northern analyses reveal expression of 4.3 kb SVMT mRNAs in rat hypothalamus, midbrain and brainstem, a 3 kb hSVMT mRNA in human brainstem and a 4.8 kb hSVMT mRNA in human hypothalamus. In situ hybridization documents significant SVMT expression in human nigra compacta neurons and in rat hypothalamic neurons whose distribution patterns are identical to those previously reported to display histaminergic markers. COS cell hSVMT expression yielded nanomolar affinities for tetrabenazine and reserpine, micromolar affinities for haloperidol, GBR12909, serotonin, mazindol, nomifensin and d-amphetamine, while dopamine, epinephrine, norepinephrine and histamine each displayed millimolar affinities. These observations extend the pharmacological characterization of hSVMT and studies of its distribution, and indicate likely physiological roles for SVMT in packaging monoamine transmitters including histamine.

A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs

A human vesicular monoamine transporter cDNA has been identified by screening a human brainstem library using sequences from the rat brain synaptic vesicle monoamine transporter (SVMT) [(1992) Cell 70, 539-551; (1992) Proc. Natl. Acad. Sci. USA 89, 10993-10997]. The hSVMT shares 92% amino acid identity with the rat sequence, but displays one less consensus site for asparagine N-linked glycosylation and one more consensus site for phosphorylation by protein kinase C. The human SVMT gene maps to chromosome 10q25 using Southern blotting analysis of human/rodent hybrid cell lines and fluorescent in situ hybridization approaches. The cDNA, and a subclone, recognize TaqI polymorphisms that may prove useful to assess this gene's involvement in neuropsychiatric disorders involving monoaminergic brain systems.

Multiple system degeneration with glutamate dehydrogenase deficiency: pathology and biochemistry.

The neuropathological findings in a patient with antemortem diagnosis of olivopontocerebellar atrophy (OPCA) and reduced leucocytic glutamate dehydrogenase (GDH) activity included cerebellar cortical degeneration, most marked in the superior vermis, mild atrophy of the pons and the inferior olivary nucleus, marked reduction of anterior horn cells at all levels and gliosis in both lateral columns. GDH activities and their thermolability in "soluble" and "particulate" fractions in the cerebral cortex, cerebellar hemisphere and vermis were not significantly different from the values in two control brains. GDH mRNA in the patient's brain was not altered in size or amount.