Monoamine transporters are transmembrane proteins located in plasma membranes of monoaminergic neurons, including the dopamine transporter (DAT), serotonin transporter (SERT, also expressed in platelets), and norepinephrine transporter (NET). These proteins use ion (Na+, Cl-) gradients as energy sources to move monoamines into or out of neurons. The major function of these transporters is to terminate monoamine transmission by inward transport of substrates away from the synaptic cleft. In the membrane of intracellular synaptic vesicles is the vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2), which use a proton gradient as the energy source to sequester cytosolic monoamines into the vesicles and then release the monoamines into synaptic cleft by exocytosis.
Vesicular monoamine transporter (VMAT) proteins are proton-driven, ATPase-dependent transporters with 12 transmembrane domains. There are two VMAT isoforms: VMAT1 is found in the adrenal medulla, in the membranes of chromaffin granules (vesicles), where it sequesters epinephrine and norepinephrine for release by the adrenal gland. In this context, these monoamines act as hormones, affecting organs such as the heart, vasculature, and respiratory system. VMAT2, in contrast, is primarily found integrated into the membranes of pre-synaptic vesicles of monoaminergic (serotonergic and dopaminergic) neurons of the central nervous system (CNS), where it transports monoamines from the cytoplasm into synaptic vesicles. In this context, these monoamines act as neurotransmitters, relaying signals between neurons in the brain.
VMAT Transport Substrates
Transport by VMAT has been demonstrated for a surprisingly wide range of substances. These include serotonin, dopamine, norepinephrine, epinephrine, L-DOPA, histamine, tyramine, octopamine, and others. The discovery of several permanently charged substrates for VMAT, including meta-iodobenzylguanidine, N-methyl-4-phenylpyridinium (MPP+), and tetraphenylphosphonium have led to acceptance of the view that the charged form of amine substrate is the form which is transported, despite previous studies interpreted to suggest otherwise. The generally accepted stoichiometry of transport is two lumenal protons exchanged for one positively-charged amine. Transport of each of these substrates has not been demonstrated for both VMAT isoforms or by VMAT from all available species. For example, octopamine transport has only been reported to date for C. elegans VMAT. Perhaps the best example of VMAT isoform substrate specificity is the transport of histamine. VMAT1 and VMAT2 differ only slightly in their ability to transport serotonin, dopamine, and norepinephrine, however only VMAT2 binds and transports histamine. Furthermore, while the inhibitor reserpine competes with serotonin, dopamine, and norepinephrine for VMAT binding, histamine and reserpine do not compete for a binding site. Therefore, it seems quite possible that there are at least two binding sites and perhaps distinct translocation pathways for different VMAT substrates. A similar isoform binding specificity exists for the inhibitor tetrabenazine, and it has been hypothesized that high-affinity histamine and tetrabenazine binding may be structurally related properties.
Reserpine is a high-affinity inhibitor of both VMAT1 and VMAT2. It is a large molecule with an end-to-end span (in an extended conformation) approximately the length of an entire TM segment. Reserpine binds with two different affinities to VMAT. Very high affinity binding (Kd = 30 pM for bovine chromaffin granule membranes) occurs only in the presence of, and is accelerated by, an electrochemical proton gradient. Binding in the absence of this gradient is still high affinity, with a Kd in the 20-200 nM range. Reserpine has a serotonin-like substructure pharmacophore, and competes with serotonin, norepinephrine, and dopamine for VMAT binding. These monoamines inhibit serotonin with KiS in the same range as their KmS for transport, and reserpine competitively inhibits substrate transport with a subnanomolar Ki corresponding to the higher of its two binding affinities. Notably, reserpine inhibits histamine transport much less effectively than serotonin transport, with a 200-fold higher IC50, for histamine transport inhibition. Conversely, histamine does not inhibit reserpine binding even at concentrations far above its Km. This suggests that histamine does not bind to the same site as reserpine, nor is it bound or transported in the same fashion as the other endogenous monoamine neurotransmitters. Reserpic acid, a presumably membrane impermeant derivative of reserpine, inhibits norepinephrine uptake into chromaffin vesicles when applied from the outside of the vesicle, but not from the inside. This suggests that the reserpic acid binding site is on the cystoplasmic rather than the lumenal side of the transporter. However, reserpic acid only has a Ki for inhibition of norepinephrine transport of 10 μm, orders of magnitude less than reserpine, suggesting that transport inhibition may occur by a mechanism different from that of reserpine.
Bender, C. N. (2011). In Vitro Expression and Characterization of Polymorphic Variants of Mouse Vesicular Monoamine Transporter-2 (VMAT2) (Doctoral dissertation, University of the Sciences in Philadelphia).
Thiriot, D. S. (2002). Engineering the human vesicle monoamine transporter to study structure, function, and ligand binding sites.