Monoamine transporters (MATs) carry out neurotransmitter reuptake from the synaptic cleft, a key step in neurotransmission, which is targeted in the treatment of neurological disorders. Cholesterol (CHOL), a major component of the synaptic plasma membrane, has been shown to exhibit a modulatory effect on MATs. Recent crystal structures of the dopamine transporter (DAT) revealed the presence of two conserved CHOL-like molecules, suggesting a functional protein-CHOL direct interaction. Here, we present extensive atomistic molecular dynamics (MD) simulations of DAT in an outward-facing conformation. In the absence of bound CHOL, DAT undergoes structural changes reflecting early events of dopamine transport: transition to an inward-facing conformation. In contrast, in the presence of bound CHOL, these conformational changes are inhibited, seemingly by an immobilization of the intracellular interface of transmembrane helix 1a and 5 by CHOL. We also provide evidence, from coarse grain MD simulations that the CHOL sites observed in the DAT crystal structures are preserved in all human monoamine transporters (dopamine, serotonin and norepinephrine), suggesting that our findings might extend to the entire family.

The human monoamine transporters (MATs) facilitate the reuptake of the neurotransmitters serotonin, dopamine, and norepinephrine from the synaptic cleft. Imbalance in monoaminergic neurotransmission is linked to various diseases including major depression, attention deficit hyperactivity disorder, schizophrenia, and Parkinson’s disease. Inhibition of the MATs is thus an important strategy for treatment of such diseases. The MATs are sodium-coupled transport proteins belonging to the neurotransmitter/Na+ symporter (NSS) family, and the publication of the first high-resolution structure of a NSS family member, the bacterial leucine transporter LeuT, in 2005, proved to be a major stepping stone for understanding this family of transporters. Structural data allows for the use of computational methods to study the MATs, which in turn has led to a number of important discoveries. The process of substrate translocation across the membrane is an intrinsically dynamic process. Molecular dynamics simulations, which can provide atomistic details of molecular motion on ns to ms timescales, are therefore well-suited for studying transport processes. In this review, we outline how molecular dynamics simulations have provided insight into the large scale motions associated with transport of the neurotransmitters, as well as the presence of external and internal gates, the coupling between ion and substrate transport, and differences in the conformational changes induced by substrates and inhibitors.

Selective inhibitors of the human serotonin transporter (hSERT) have been first-line treatment against depression for several decades. Recently, vortioxetine was approved as a new therapeutic option for the treatment of depression. Vortioxetine represents a new class of antidepressant drugs with a multimodal pharmacological profile that in addition to potent inhibition of hSERT include agonistic or antagonistic effects at different serotonin receptors. We used a combination of computational, chemical, and biological methods to decipher the molecular basis for high affinity binding of vortioxetine in hSERT. X-ray crystal structures of the bacterial amino acid transporter LeuT and the Drosophila melanogaster dopamine transporter were used to build homology models of hSERT. Comparative modeling and ligand docking suggest that vortioxetine can adopt several distinct binding modes within the central binding site of hSERT. To distinguish between the identified binding modes, we determined the effect of 57 functional hSERT point mutants on vortioxetine potency and characterized seven structurally related analogs of vortioxetine in a subset of the point mutants. This allowed us to determine the orientation of vortioxetine within the central binding site and showed that only one of the proposed binding modes is functionally relevant. The findings provide important new insight about the molecular basis for high affinity recognition of vortioxetine in hSERT, which is essential for future structure-based drug discovery of novel multimodal drugs with fine-tuned selectivity across different transporter and receptor proteins in the human brain.

5-Hydroxytryptamine 3 (5-HT 3) receptors are ligand-gated ion channels that mediate neurotransmission by serotonin in the central nervous system. Pharmacological inhibition of 5-HT 3 receptor activity has therapeutic potential in several psychiatric diseases, including depression and anxiety. The recently approved multimodal antidepressant vortioxetine has potent inhibitory activity at 5-HT 3 receptors. Vortioxetine has an inhibitory mechanism that differs from classic 5-HT 3 receptor competitive antagonists despite being believed to bind in the same binding site. Specifically, vortioxetine shows partial agonist activity followed by persistent and insurmountable inhibition. We have investigated the binding mode of vortioxetine at the human 5-HT 3A receptor through computational and in vitro experiments to provide insight into the molecular mechanisms behind the unique pharmacological profile of the drug. We find that vortioxetine binds in a manner different from currently known 5-HT 3A orthosteric ligands. Specifically, while the binding pattern of vortioxetine mimics some aspects of both the setron class of competitive antagonists and 5-hydroxytryptamine (5-HT) with regards to interactions with residues of the aromatic box motif in the orthosteric binding site, vortioxetine also forms interactions with residues not previously described to be important for the binding of either setrons or 5-HT such as Val202 on Loop F. Our results expand the framework for understanding how orthosteric ligands drive 5-HT 3 receptor function, which is of importance for the potential future development of novel classes of 5-HT 3 receptor antagonists.

The human serotonin transporter (hSERT) terminates serotonergic signaling through reuptake of neurotransmitter into presynaptic neurons and is a target for many antidepressant drugs. We describe here the development of a photoswitchable hSERT inhibitor, termed azo-escitalopram, that can be reversibly switched between trans and cis configurations using light of different wavelengths. The dark-adapted trans isomer was found to be significantly less active than the cis isomer, formed upon irradiation.