René Weissensteiner

The N Terminus of Monoamine Transporters Is a Lever Required for the Action of Amphetamines

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin Transporter

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants.

Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

Probing the Selectivity of Monoamine Transporter Substrates by Means of Molecular Modeling.

The structurally similar serotonin and dopamine transporter (resp. SERT and DAT) play an important role in neuronal transmission. Although the concept of their function, i.e. the re-uptake of neurotransmitters from the synaptic cleft, has been extensively studied,1–4 the exact mechanism for their substrate selectivity is still unknown. Phenylethylamines (PEAs) are ligands of SERT and DAT and many induce reverse transport (efflux) of the protein’s natural substrate (the neurotransmitters 5-hydroxytryptamine and dopamine) in varying degrees and with different kinetics.2,5–7 Thus, studying the interplay of bioactivity values and certain structural features of selected PEAs can lead to new insights about monoamine transporter selectivity. The broadest SAR data currently available for PEAs and their interaction with SERT and DAT has been measured in rat synaptosomes by Baumann and colleagues.8,9 Thus, we used this data set to figure out important features which contribute towards selectivity and to guide the selection of a probe compound for subsequent structure-based studies. Consequently, pEC50 values of 28 compounds for SERT and DAT (Table ​(Table1)1) were plotted against each other, providing a clear picture of the PEA′s selectivity profile (Figure ​(Figure1).1). Out of this, a couple of detailed SARs can be drawn: Table 1 Monoamine transporter substrate structure-activity relationships Figure 1 Selectivity plot with numbers corresponding to Table 1. Compounds with similar SERT/DAT affinity are located around the middle diagonal line, while compounds in the upper left corner and lower right corner are DAT and SERT-selective, respectively. Chirality of the α-methylene atom of amphetamines does not influence SERT/DAT selectivity. The (S)-enantiomer is the most active in both transporters. DAT selective substrates seem smaller in size and therefore, their conformational flexibility in the binding pocket is expected to be relatively high and interactions with the target less defined. N-Methyl substitution slightly increases activity in SERT (compare compounds 4, 8, 20 and 21), and is somewhat unchanged in DAT (compare compounds 16, 17, 18 and 19). The only exception is for the naphtylisopropylamine (NIPA, 23) which is not selective for both transporters and shows a slight decrease in SERT activity (24). N-Ethyl substitution is generally more favorable in SERT as compared to methyl substitution or no substitution, while it decreases activity in DAT (see compounds 19, 22 and 25). para-Chlorine, meta-CF3 or meta-methyl substitution dramatically increases SERT affinity (compare 9, 11, 12, 4, 17). β-Hydroxyl substitution (R4, Table ​Table1)1) decreases affinity in both SERT and DAT (compare 1, 3, 5, 7). para-Methyl substitution increases SERT affinity and slightly decreases DAT affinity (compare 4, 10, 26, 27). The highest SERT/DAT selectivity is shown by (S)-fenfluramine (SFF) and because of its relatively large size, docking studies with this ligand are expected to result in a more restricted amount of poses as compared to the smaller analogs. Subsequently, we used SFF as a probe compound in order to study the molecular basis of the high affinity and selectivity of this compound towards SERT by means of a structure-based approach. Conveniently, sequence identity between the human and rat transporters is very high (92 % with SERT; 93 % with DAT), and local alignment of the primary substrate binding site (S14) shows even 100 % sequence identity between both species.10 Thus, in order to build upon our already established protein homology models for human SERT11, we switched to human proteins for subsequent studies. To show that data derived from rat transporters indeed can be transferred to the human transporters, we confirmed the high selectivity of SFF for SERT employing an uptake inhibition assay on HEK cells expressing human SERT and DAT (IC50=5.89 µM in SERT and 118 µM in DAT, see Figure ​Figure22). Figure 2 Uptake inhibition by (S)-fenfluramine in HEK293 cells stably expressing YFP-tagged DAT and SERT. Uptake was inhibited by increasing concentrations of fenfluramine as indicated. The concentration of tritiated substrates was 0.15 µM in the case ... Docking of a set of diverse high-affinity SERT substrates (see Methods) into a homology model of hSERT followed by common scaffold clustering revealed a binding mode for SFF which is in accordance to previously published studies.12,13 In addition, SFF was docked into an analogously constructed homology model of hDAT. Results showed that this ligand fits nicely into the S1 site, meaning that steric hindrance caused by the trifluoromethyl or N-ethyl group could not serve as an explanation for its low DAT affinity (see Figure ​Figure3).3). In addition, scoring functions could not show a preference of SFF for SERT or DAT (see Table ​Table2)2) and hence are not able to capture the activity determining factors. Since SFF′s trifluoromethyl moiety seems to be driving the selectivity, we further analysed the pocket between the TM3 and TM8 helical domains where this moiety is located: local alignment of SERT and DAT showed that five of the seven residues within this pocket are different. In general, the SERT pocket has more lipophilic side chains in its binding site, except for Thr439 in SERT which is more hydrophilic than the corresponding Ala423 in DAT (see Table ​Table33). Figure 3 Overlay of the selected fenfluramine (SFF) poses in the substrate binding site of hSERT and hDAT with a T439(O)-F(SFF) distance of 3.5 A. Table 2 Average scoring values after docking and evaluation of (S)-fenfluramine in the substrate binding site of homology models of SERT and DAT Table 3 Local alignment of the helical domains TM3 and TM8 of hSERT and hDAT showing more lipophilic side chains in SERT, except that for Thr439 This indicates a potential role of the CF3 group and Thr439 for SERT selectivity. Furthermore, as shown in Table ​Table1,1, (S)-amphetamine and (S)-norfenfluramine only have a trifluoromethyl moiety dissimilar, and their Ki values for rSERT are 3830 nM and 214 nM, respectively.5,8 Since the ratio of these values should be similar to the KD ratio (and since Ki is comparable to KD14), the binding free energy formula can be applied: with T=298.15 K5 and Hence, from a ligand-based point of view, a more favorable binding energy of about 1.72 kcal/mol is calculated for (S)-norfenfluramine. Considering the inhibitory values of (S)-fenfluramine from our human DAT and SERT uptake inhibition assay, we obtain a binding free energy difference of about 1.75 kcal/mol:15 with T=293.15 K Both calculated energy values are close to each other, strengthening the evidence that the trifluoromethyl group is responsible for SERT/DAT selectivity and high SERT affinity. Moreover, these values are relatively close to the ΔG value of a sp3-fluorine hydrogen bond (−2.38 kcal/mol).16 It is thus tempting to speculate that an interaction of Thr439 with the CF3 group triggers both affinity and selectivity of SFF in SERT. In addition, lipophilic dispersion forces with the SERT specific side chains (Ala169, Ile172, Ala173) that surround the trifluoromethyl moiety might contribute. Further evidence for the potential role of the lipophilicity of this pocket can be deduced from the increase in activity of the more lipophilic meta-methyl-substituted compound 9 and a decrease in activity of the hydrophilic meta-hydroxy-substituted dopamine (1) and norepinephrine (3) in this protein. Finally, when comparing phentermine and chlorphentermine, the halide increases the SERT affinity 13 900/338=41 times,5 which corresponds to more favorable energy of about 2.21 kcal/mol. Whether this can be ascribed to an interaction between the chlorine and Thr439, or simply to lipophilic contributions, is a point of discussion. With this study we have shown that combining ligand- and structure based studies are a powerful tool to probe substrate selectivity of monoamine transporters leading to preliminary evidence for the potential role of halogen atoms and Thr439 in SERT. Synthesis of additional PEAs combined with biochemical studies in both wild type and T439A mutants are obvious further steps towards this direction.

Development of Refined Homology Models: Adding the Missing Information to the Medically Relevant Neurotransmitter Transporters

Neurotransmitter:sodium symporters are located on presynaptic neurons and terminate neurotransmission by removing the monoamine substrates from the synaptic cleft. Until very recently, only several conformational snapshots/structures of a bacterial homolog of neurotransmitter:sodium symporters, namely, the leucine/alanine transporter LeuT from Aquifex aeolicus, were available. However, this transporter shares only 21b % overall sequence identity with its human homologs. In this chapter, we describe how a model can be developed from a template with such low identity. The effort of model building will strongly depend on the purpose. We discuss this process and focus on the important steps that allowed us to obtain a model which can be used for molecular dynamics simulations. Furthermore, we also highlight the inherent limitations of the proposed approaches. Prediction of ligand binding brings in additional complexity. Therefore, experimental scrutiny of the resulting models is a key component to successful validation. We describe two specific examples: model building of the dopamine transporter and ligand docking to the serotonin transporter. We evaluate our modeling approach by direct comparison of our models to the recently published first eukaryotic neurotransmitter:sodium symporter, the drosophila melanogaster DAT transporter.

Towards an understanding of the psychostimulant action of amphetamine and cocaine

Cocaine and amphetamine are psychostimulant drugs that are illicitly used; they affect sensory perception by targeting the neurotransmitter: sodium symporters (NSS) at the synapses between neurons. They both increase the concentration of the neurotransmitter in the synaptic cleft but by different means.

Amphetamine actions rely on the availability of phosphatidylinositol-4,5-bisphosphate

Background Neuronal functions, such as excitability or endoand exocytosis, require phosphatidylinositol-4,5-bisphosphate (PIP2) since ion channels and other proteins involved in these processes are regulated by PIP2. Monoamine transporters control neurotransmission by removing monoamines from the extracellular space. They also display channel properties, but their regulation by PIP2 has not been reported. The psychostimulant amphetamine acts on monoamine transporters to stimulate transportermediated currents and efflux and thereby increases the levels of extracellular monoamines.