Reserpine And Tetrabenazine

[Varinia Swicegood]

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Tetrabenazine (TBZ) is used in the treatment of hyperkinetic movement disorders. Its effect is thought to be mediated by depletion of dopamine (DA) stores. We studied other possible mechanisms of action of this drug. TBZ decreased DA concentration in rat striatum and nucleus accumbens in a dose-dependent manner with an IC50 of approximately 1.2 mg.kg-1. Maximal depletion was obtained within 30 min with only partial recovery at 8 hr. TBZ induced (at 40 mg . kg-1) 5- to 8-fold increases in 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations in both brain regions. Unlike reserpine, TBZ completely abolished the apomorphine-induced inhibition of DA synthesis under conditions in which this effect is mediated by presynaptic DA receptors. Both TBZ (5 mg . kg-1) and reserpine (5 mg . kg-1) depleted, at 1 hr, striatal DA content by approximately 90%. However, TBZ, but not reserpine, significantly stimulated in vivo tyrosine hydroxylase activity. TBZ also inhibited [3H]spiperone binding in the striatum with Ki = 2.1 X 10(-6) M. In rats, with unilateral destruction of the nigrostriatal pathway with 6-hydroxydopamine, pretreatment with TBZ significantly reduced the number of rotations induced by apomorphine. Finally, in rats treated with either TBZ (5 mg . kg-1) or reserpine (5 mg . kg-1), prolactin levels significantly increased as compared to control values. TBZ, but not reserpine, blocked apomorphine inhibition of prolactin secretion. We conclude that, in addition to depleting monoamines, TBZ also blocks both presynaptic and postsynaptic DA receptors in rat brain.

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VMAT2 and its close paralog VMAT1 (also known as SLC18A2 and SLC18A1, respectively) belong to the major facilitator superfamily (MFS).21 Despite a shared sequence identity of over 60%, the two VMATs differ significantly in cellular distribution, substrate recognition and pharmacological profiles.22 VMAT1 is primarily found in neuroendocrine cells in the sympathetic and peripheral nervous system, whereas VMAT2 is more broadly distributed in both central and peripheral nervous systems. Specifically, central, peripheral and enteric neurons only express VMAT2. Of note, VMAT2 co-exists with VMAT1 in the adrenal glands, and its expression is induced by stress, while VMAT1 level remains constant.23 Both VMAT2 and VMAT1 have a similar affinity for serotonin, but VMAT2 exhibits a preference for catecholamines (e.g., DA, NE, epinephrine) with a 3-fold higher affinity, and even more so for histamine with a 30-fold higher affinity. While the competitive inhibitors reserpine (RES) and ketanserin only show a slight preference for VMAT2 over VMAT1, the non-competitive inhibitors tetrabenazine (TBZ) and its derivatives selectively target VMAT2.22

Although VMAT2 holds significant physiological and pharmaceutical importance, the precise molecular mechanisms governing its recognition and transport of monoamines remain elusive. Furthermore, the distinct pharmacological effects exerted by the competitive inhibitor RES and the non-competitive inhibitor TBZ warrant in-depth investigation. Here we set to address these questions by performing single-particle cryo-electron microscopy (cryo-EM) analysis on human VMAT2 complexed with substrate serotonin, inhibitors RES and TBZ.

TBZOH and TBZ differ only in the position of carbonyl oxygen, with TBZOH acquiring a hydroxyl group as a result of TBZ metabolism. In the TBZ-bound VMAT2 structure, the carbonyl oxygen is oriented towards Asn34 on TM1. The N34A substitution resulted in a 7-fold reduction in TBZ binding affinity (Supplementary information, Fig. S8a), suggesting a similar and important interplay between Asn34 residue and both TBZ and its metabolite TBZOH. An additional polar interaction is noted between Arg189 and the two methoxyl groups on TBZ, which may further contribute to the stable coordination network.

The intensified polar and non-polar interaction between TBZ and VMAT2 likely triggers the aforementioned conformational shift. Specifically, Trp318 inserts its indole group deeply towards the TBZ location from the luminal side as a result of the helix-to-loop transformation. This insertion is buttressed by several hydrophobic/non-polar residues such as Leu44, Pro45, Val131, Pro313, Leu315, Leu317 and Leu330 (Fig. 3e). While not in direct contact with TBZ in the occluded state, W318A mutant showed a substantial reduction in TBZ binding (Supplementary information, Fig. S9a). Similarly, mutagenetic perturbations on the TM2 luminal apical (Leu124 and Leu125), which are distant from the translocation funnel and TBZ binding pocket, largely diminished TBZ association (Supplementary information, Fig. S9a). Consistently, both the W318A and L124R/L125R mutations nearly abolished the FFN uptake activity (Fig. 2e). These observations indicate that the drastic conformational change, particularly in TM2 and TM7, is critical for TBZ-induced closure of the VMAT2 luminal exit.

RES, a natural indole alkaloid, has been a first-line therapy in treating hypertension since 1955 but is currently considered a second-line treatment due to its potential depression side effects. RES binds with high affinity to both VMAT2 and VMAT1, at the substrate-binding site on the cytoplasmic side.37 As mentioned earlier, we were unable to obtain the RES-bound complex with WT VMAT2 protein (Supplementary information, Fig. S2).

a The cytosolic gate. Gate residues from TMs 4, 5, 10 and 11 from the VMAT2A structure (semi-transparent gray cartoon) are shown in sticks. Left, expanded view from membrane plane; right, expanded view from cytosol. The hydrophobic Met-layer and hydrophilic Arg-layer are highlighted by dashed rhomboids. b The luminal gate. Gate residues from TMs 1, 2, 7 and 8 from the cytosol-facing RES-bound VMAT2YCR structure are shown. Left, expanded view from membrane plane; right, expanded view from lumen. The Phe-layer and Pro-layer are highlighted.

Notably, when the VMAT2YC protein was incubated with the substrate 5-HT, the resulting reconstructed VMAT2YCS map adopted the same lumen-facing conformation as VMAT2S (Supplementary information, Fig. S2). These findings strongly suggest that 5-HT binding in the background of this weakened cytosolic gate is not sufficient to transform the presumed resting lumen-facing state for substrate reception in current experimental conditions. It would possibly reflect the critical role of the cytosol-directed proton flux in driving the conformational switch of VMAT2.35

To gain more insights into the transportation cycle of VMAT2-mediated monoamine loading, we performed in silico molecular docking and MD simulation. In the cytosol-facing VMAT2 structure, 5-HT predominately adopts a single conformation, with its primary amine facing Asp399 near the cytosol side (Supplementary information, Fig. S6d). Interestingly, in the lumen-facing state, 5-HT appears in two different locations, with its primary amine pointing to either Asp399, or Glu312 near the luminal exit (Supplementary information, Fig. S6e). Experimenting with the protonation state of Asp399 and Glu312 revealed a sequential binding and release of 5-HT in the translocation funnel (Supplementary information, Fig. S6f, g). Incorporating the general alternating access mechanism,38,40 we propose the working model for VMAT2 as follows (Fig. 6).

Schematic representation of the alternating access transport cycle. The 7 states are derived from direct experimental structures (States 4 and 6), docking poses (States 1, 2 and 5), and AlphaFold2 prediction (States 3 and 7). For clarity, only TMs 1, 2, 7 and 8 are shown as empty tubes in color. States that are not experimentally determined are shown in faint shades. Two negative residues D399 and E312 along the translocation pathway (empty circles, non-protonated; solid circles filled with green, protonated) facilitate substrate movement by alternating protonation states. Significant conformational shifts, particularly in TM2 and TM7, triggered by TBZ (green) entrance at the luminal side induce a dead-end occluded state.

VMAT2 is responsible for packaging bioactive monoamine substances including serotonin and dopamine into presynaptic vesicles in neurons. This process primes the neurotransmitters for subsequent synaptic quantal release upon stimulation and also serves as a protective mechanism for neurons against toxic substances. Our results have uncovered the structural basis for substrate recognition, and mechanism of competitive and non-competitive inhibition by clinical drugs including RES and TBZ. These inhibitors exploit a forgiving central binding site to achieve their potency.

In conclusion, our VMAT2 structures, in association with substrate serotonin and inhibitors of diverse chemical structures, along with biochemical evidence and MD simulation analyses, provide new insights into the mechanism underlying the vesicular packaging of monoamine neurotransmitters, offering a platform for the development of improved pharmaceutical strategies in the future.

Initial VMAT2 model was retrieved from AphaFold30 database which is predicted as occluded conformation (ID: AF-Q05940). The predicted model was rigid-body docked into VMAT2A cryo-EM density map in ChimeraX (v.1.6),45 followed by iterative manual adjustment in COOT (v.0.9.8)46 and real-space refinement in Phenix (v.1.19).47,48 Models and geometry restraints for 5-HT, TBZ, and RES were generated by the eLBOW tool from Phenix. The model statistics were validated by Molprobity. Side-chains that do not have well-defined density were trimmed for deposition. The final refinement statistics are provided in Supplementary information, Table S1. Structural figures were prepared in ChimeraX or PyMOL (PyMOL Molecular Graphics SYtem, v.2.3.4, Schrdinger) ( ).

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