The vesicular monoamine transporter (VMAT) is a transport protein integrated into the membranes of synaptic vesicles of presynaptic neurons. It transports monoamine neurotransmitters – such as dopamine, serotonin, norepinephrine, epinephrine, and histamine – into the vesicles, which release the neurotransmitters into synapses, as chemical messages to postsynaptic neurons. VMATs utilize a proton gradient generated by V-ATPases in vesicle membranes to power monoamine import.
Pharmaceutical drugs that target VMATs have possible applications for many conditions, leading to a plethora of biological research, including hypertension, drug addiction, psychiatric disorders, Parkinson's disease, and other neurological disorders. Many drugs that target VMATs act as inhibitors and alter the kinetics of the protein. Much research regarding the effects of altered VMATs on biological systems is still ongoing. Monoamines Monoamines transported by VMATs are mainly noradrenaline, adrenaline, dopamine, serotonin, histamine, and trace amines. Exogenous substrates include guanethidine and MPP+.
Discovery VMAT research began in 1958 when Nils-Åke Hillarp discovered secretory vesicles. In the 1970s, scientists like Arvid Carlsson recognized the need to understand how transport systems and ion gradients work in different organisms in order to explore new treatment options such as reserpine (RES). Researchers discovered inhibitors that blocked the uptake of neurotransmitters into vesicles, suggesting the existence of VMATs. A decade later, molecular genetic tools have improved methods for protein identification. Scientists have used these tools to analyze DNA and amino acid sequences, and discovered that transporters in bacteria and humans were very similar, which emphasized the importance and universality of transporters. The transporters were first structurally identified by cloning VMATs in rats. The VMAT were first isolated and purified in bovine chromaffin granules, in both its native and denatured forms.
Location There are two types of VMATs expressed in humans: VMAT1 and VMAT2. VMAT1 is expressed mainly in large dense-core vesicles (LDCVs) of the peripheral nervous system. VMAT1 may be found in neuroendocrine cells, particularly chromaffin and enterochromaffin granules, which are primarily found in the medulla of the adrenal glands.
VMAT2 favors expression in a variety of monoaminergic cells of the central nervous system, such as the brain, sympathetic nervous system, mast cells, It is prevalent in β-cells, expressed in blood platelets, and co-expressed in chromaffin cells. Expression of the two transporters in internal organs seems to differ between species: only VMAT1 is expressed in rat adrenal medulla cells, whereas VMAT2 is the major transporter in bovine adrenal medulla cells.
Structure and function
VMAT1 and VMAT2 are acidic glycoproteins with a molecular weight of approximately 70 kDa. Both isoforms are transmembrane proteins with 12 transmembrane domains (TMDs).
VMATs function by loading monoamines—dopamine, serotonin, histamine, norepinephrine, and epinephrine—into transport vesicles.Purves, Dale, et al. Neuroscience. Sinauer Associates. 087893646 VMATs use the same transport mechanism for all types of monoamines, and transport them from the cytosol into high-concentration storage vesicles. Transport vesicles are released into the space between neurons, called the synaptic cleft, where they convey a chemical message to the next neuron. VMATs also function in sorting, storing, and releasing neurotransmitters, and are believed to participate in protecting these neurotransmitters from autoxidation. The transporters are also known to continue biochemical modification after loading certain neurotransmitters.
Vesicle packing requires a large energy source to store large quantities of neurotransmitters into a small vesicular space at high concentrations. VMAT transport relies on the pH and electrochemical gradient generated by a vesicular H+-ATPase. The current model of VMAT function proposes that the efflux of two protons (H+) against the H+ gradient is coupled with influx of one monoamine. The first H+ efflux generates a transporter conformation associated with a high-affinity amine-binding site in the cytosolic phase, and the second H+ efflux is coupled with a second large conformational change that leads to amine transport from the cytosolic side into the vesicle, reducing amine-binding affinity.
Studies indicate that the amino acid residue His419, located on the domain between TMDs X and XI of rat VMAT1, plays a role in energy coupling to the amine transport by assisting the first proton-dependent conformational change.Shirvan A, Laskar O, Steiner-Mordoch S, Schuldiner S. (1994). "Histidine-419 plays a role in energy coupling in the vesicular monoamine transporter from rat." FEBS Lett, 356:145–150. It has been proposed that RES inhibits VMAT by interacting with this conformation.
VMAT gene sequence analysis demonstrates that four aspartic acid residues in the middle region of TMDs I, VI, X, and XI and one lysine residue in TMD II have highly conserved gene sequences, suggesting these residues play a critical role in transporter structure and function. Specifically, the residues Lys139 and Asp427 are thought to compose an ion pair that promotes high-affinity interaction with VMAT substrates and inhibitors. The Asp431 residue located in TMD XI is believed to be critical for amine transport, but does not interact with RES binding; it is thought to complete the substrate transport cycle.
Kinetics VMATs have a relatively low Vmax, with an estimated rate of 5–20/sec depending on the substrate. Vesicle filling may limit monoamine release from neurons with high rates of firing.
Specific amine-binding affinity varies by VMAT isoform; studies indicate that catecholamines dopamine, norepinephrine, and epinephrine have a threefold higher affinity for VMAT2 than VMAT1 binding and uptake. The imidazoleamine histamine has a thirtyfold higher affinity for VMAT2 compared to VMAT1, and is thought to bind to a different site than other monoamines. Unlike catecholamines and histamine, the indoleamine serotonin binds to VMAT1 and VMAT2 with a similar affinity for both transporter isoforms.
VMAT1 has a lower turnover number and a lower affinity for most monoamine substrates than VMAT2, which may be because of VMAT2's location in the central nervous system, which demands fast recovery from neurotransmitter release in order to prepare for subsequent releases. The uptake efficiencies of each VMAT substrate can be ranked in order of efficiency as: serotonin, dopamine, epinephrine, and norepinephrine.
Methamphetamines decrease Vmax, while cocaine increases Vmax reversibly in rat brain.
Inhibition The effects of VMAT inhibition have been studied in-depth in animal models. Mutant homozygous VMAT(-/-) mice move little, feed poorly, and die within a few days of birth.
More specifically, inhibition of VMAT2 may cause an increase in cytosolic catecholamine levels, which can result in an increase in efflux of catecholamines through the cell membrane, depleting catecholamine concentrations and causing increased oxidative stress and oxidative damage to the neuron.
Heterozygous VMAT mutants display hypersensitivity to amphetamine, cocaine, and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the latter being a substance causally linked to Parkinson's disease (PD) in rodents. This suggests a protective role of VMATs against oxidative stress through removal of such substances from the cytosol.
VMAT inhibitors include: Reserpine (RES), bietaserpine, and ketanserin (KET) (potent inhibitors of VMAT2 mediated serotonin transport) Tetrabenazine (TBZ) (specific to VMAT2) Phenylethylamine Amphetamine MDMA N-Methyl-4-phenylpyridinium (MPP+) (very potent inhibitors of VMAT2 mediated serotonin transport) Fenfluramine (specific to VMAT1 ) Non-hydrolysable GTP-analogue guanylyllimidodiphosphate GMP-P(NH)P (VMAT2 only)
Binding site structures Ligand-binding affinities and structures Two known binding sites for VMAT inhibitors include the RES binding site and the TBZ binding site. Some evidence suggests these two sites may overlap or exist as two separate conformations of the same binding site. VMAT inhibitors tend to fall into two classes: those that interact with the RES binding site and those that interact with the TBZ binding site.
RES, methoxytetrabenazine (MTBZ), and the drug amiodarone bind to the RES binding site. TBZ (also called Nitoman and Xenazine), dihydrotetrabenazine (DTBZOH), ketanserin (KET), and the drug lobeline bind to the TBZ binding site. Amphetamine, methamphetamine and GZ-7931 are also known to interact with VMAT2.Vesicular monoamine transporter 2#Binding sites and ligands
Inhibitor affinity varies among VMAT isoforms. RES and KET have higher inhibitory affinity for VMAT2–mediated 5HT transport than for that of VMAT1; TBZ seems to inhibit VMAT2 exclusively.
The residues asp33 and ser180, 181, and 182 are believed to be involved in substrate recognition, and interact with the protonated amino group and hydroxyl group on the catechol or indole rings.
Cocaine and methylphenidate (MPD, also known as Ritalin and Concerta) are believed to interact with VMAT2 to cause a shift in VMAT2 "from a plasmalemmal membrane-associated fraction to a vesicle-enriched, nonmembrane-associated fraction."
RES binding site Consistent with catecholamine-binding affinity, RES has a threefold higher affinity for VMAT2 than for VMAT1. The RES binding site is known to be hydrophobic, which is thought to contribute to ligand binding affinity. Methamphetamine binds to the RES site on VMATs.
The current working model proposes that RES and the substrate bind to a single site in a pH-gradient modulated conformational structure of the transporter. The conformation occurs after the transport of one H+ across the membrane and into the vesicle; proton transport drives the substrate recognition site from the lumen to the cytoplasmic surface of the vesicle for RES and substrate binding. Methoxytetrabenazine (MTBZ) may bind to the RES binding site, based on studies indicating that RES significantly inhibited MTBZ-binding. Amiodarone is also believed to inhibit monoamine vesicular uptake by binding to the RES binding site.
TBZ binding site TBZ and dihydrotetrabenazine (DTBZOH) are believed to bind to a different binding site from the RES/substrate binding site, or to a different conformation of the RES/substrate binding site. This site is believed to be located at the N-terminus, based on studies done in bovine VMAT2. Tyr434 and asp461 are identified as being responsible for the high-affinity interaction of TBZ, serotonin, and histamine in VMAT2. Unlike methamphetamine, amphetamine binds to the TBZ site on hVMAT2.
Unlike RES inhibition, TBZ inhibition is only affected by very high concentrations of monoamines; however, single injections of RES can inhibit TBZ binding. ketanserin (KET) and lobeline also bind to the TBZ binding site conformation.
Glycosylation sites: N- and C-linked terminals Three to four glycosylation sites exist in the vesicular matrix on a loop between TMD I and TMD II. In biology, the vesicle matrix refers to the material or tissue between cells in which more specialized structures are embedded. Two of the glycosylation sites, the N-linked glycosylation terminal and C-linked terminal, are located in the cytosolic portion of the vesicle.Erickson, Eiden, & Hoffman, 1992
The highest amount of genetic variance between VMAT1 and VMAT2 exists near the N- and C- terminals in the cytosolic phase, and in the glycosylated loop between TMDs I and II.
C-terminus and VMAT trafficking cycle