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The serotonin transporter (SERT)2 is an integral membrane protein that facilitates transport of serotonin (5-hydroxytryptamine, 5HT) across cellular membranes (1). In addition to peripheral endocrine functions, 5HT is a neurotransmitter in the brain; it is involved in control of several important physiological functions such as mood, appetite, and sexual behavior. Expressed mainly in the membrane of serotonergic neurons, SERT utilizes energetically favorable cotransport of Na ϩ to remove released 5HT from the extracellular space. Human SERT (hSERT) belongs to the solute carrier 6 (SLC6) transporter family along with highly homologous transporters for the neurotransmitters ␥-aminobutyric acid, glycine, dopamine, and norepinephrine (2-4). These transporters are important drug targets for treatment of a wide range of neurological diseases. In particular, hSERT is the molecular target for widely used drugs for treatment of depression and anxiety. Also, psychostimulants such as amphetamine and 3,4-methylenedioxy-N-methylamphetamine ("ecstasy") have hSERT as the molecular target (5-7). The selective serotonin re-uptake inhibitors (SSRIs) are a class of antidepressant and anti-anxiety drugs that function as highly selective competitive inhibitors of hSERT (8). Although SSRIs are highly important for treatment of affective disorders (6), the molecular basis for their function, including location and structure of drug binding pockets, is largely unknown and a matter of debate (9, 10). Such information is important for understanding essential aspects of drug action, ranging from selectivity profile to therapeutic efficacy. Moreover, such information is indispensable for the development of new and improved drugs targeting hSERT. The primary impediment for elucidation of the structural mechanisms of hSERT inhibition is the lack of a three-dimensional structure of the protein. Still, several residues in SERT have been identified mainly by mutagenesis studies that modulate antidepressants potency (11)(12)(13)(14)(15)(16)(17). The use of comparative molecular modeling to create structural models of ligand-hSERT interactions has previously been hampered by the low phylogenetic and functional similarity between hSERT and available template proteins (18 -21). However, high resolution crystal structures of a bacterial homolog to mammalian SLC6 transporters, LeuT (22,23), have proven excellent templates for constructing experimentally validated models of substrate and drug binding pockets in human SLC6 transporters, including the human transporters for dopamine and ␥-aminobutyric acid (24 -32).In this study, we provide an experimentally validated threedimensional model of the binding site in hSERT for the SSRI (S)-citalopram (Lexapro) using mutational analysis of hSERT paired with structure-activity data for (S)-citalopram analogs. LeuT structures are used to create homology models of hSERT, followed by docking of (S)-citalopram. Validation of binding models was performed based on the mutational dataset from 64 hSERT point mutants ...

Background: SERT and NET are important targets for antidepressants. Results: Antidepressants are differentially affected by mutations within the central S1 pocket of SERT and NET. Conclusion: Our data indicate that many antidepressants bind within the S1 pocket, and inhibitor selectivity is determined by residues within this site. Significance: This study provides a framework for modeling of drug binding, which may be used in future structure-based drug design.

Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated. A systematic structureactivity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R-and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R-and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity.monoamine | neurotransmitter | SLC6 transporter I mbalances in neurotransmission involving the monoamines serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) are implicated in depression and anxiety disorders (1). In the brain, specific monoamine transporters, the 5-HT transporter (SERT) and the NE transporter (NET), curtail the lifetime of extracellular monoamines by performing active uptake (or reuptake) from the extracellular space into neurons. Medications for the treatment of depression and anxiety disorders act by increasing the extracellular concentration of 5-HT and/or NE by inhibiting SERT and/or NET mediated transmitter reuptake (2). SERT and NET belong to the solute carrier 6 (SLC6) transporter family, and they are integral membrane proteins that use cotransport of sodium as an energy source to convey neurotransmitters from the extracellular space to the cytoplasm (3). The first generation of drugs targeting SERT and NET were the tricyclic antidepressants (TCAs), but their activity across a variety of other neurotransmitter receptor systems (4) associate their use with severe side effects. Development of newer generations of monoamine transporter drugs have focused on compounds with an improved selectivity toward SERT and/or NET, exemplified by the selective 5-HT reuptake inh...

The serotonin transporter (SERT) regulates extracellular levels of serotonin (5-hydroxytryptamine, 5HT) in the brain by transporting 5HT into neurons and glial cells. The human SERT (hSERT) is the primary target for drugs used in the treatment of emotional disorders, including depression. hSERT belongs to the solute carrier 6 family that includes a bacterial leucine transporter (LeuT), for which a high resolution crystal structure has become available. LeuT has proved to be an excellent model for human transporters and has advanced the understanding of solute carrier 6 transporter structure-function relationships. However, the precise structural mechanism by which antidepressants inhibit hSERT and the location of their binding pockets are still elusive. We have identified a residue (Ser-438) located within the 5HT-binding pocket in hSERT to be a critical determinant for the potency of several antidepressants, including the selective serotonin reuptake inhibitor citalopram and the tricyclic antidepressants imipramine, clomipramine, and amitriptyline. A conservative mutation of Ser-438 to threonine (S438T) selectively increased the K i values for these antidepressants up to 175-fold. The effects of introducing a protein methyl group into the 5HT-binding pocket by S438T were absent or reduced for analogs of these antidepressants lacking a single methyl group. This suggests that these antidepressants interact directly with Ser-438 during binding to hSERT, implying an overlapping localization of substrateand inhibitor-binding sites in hSERT suggesting that antidepressants function by a mechanism that involves direct occlusion of the 5HT-binding site.

The biogenic monoamine transporters are integral membrane proteins that perform active transport of extracellular dopamine, serotonin and norepinephrine into cells. These transporters are targets for therapeutic agents such as antidepressants, as well as addictive substances such as cocaine and amphetamine. Seminal advances in the understanding of the structure and function of this transporter family have recently been accomplished by structural studies of a bacterial transporter, as well as medicinal chemistry and pharmacological studies of mammalian transporters. This feature article focuses on antidepressant drugs that act on the serotonin and/or the norepinephrine transporters. Specifically, we focus on structure-activity relationships of these drugs with emphasis on relationships between their molecular properties and the current knowledge of transporter structure.

Inhibitors of the serotonin transporter (SERT) are widely used antidepressant agents, but the structural mechanism for inhibitory activity and selectivity over the closely related norepinephrine transporter (NET) is not well understood. Here we use a combination of chemical, biological, and computational methods to decipher the molecular basis for high-affinity recognition in SERT and selectivity over NET for the prototypical antidepressant drug fluoxetine (Prozac; Eli Lilly, Indianapolis, IN). We show that fluoxetine binds within the central substrate site of human SERT, in agreement with recent X-ray crystal structures of LeuBAT, an engineered monoamine-like version of the bacterial amino acid transporter LeuT. However, the binding orientation of fluoxetine is reversed in our experimentally supported model compared with the LeuBAT structures, emphasizing the need for careful experimental verification when extrapolating findings from crystal structures of bacterial transporters to human relatives. We find that the selectivity of fluoxetine and nisoxetine, a NET selective structural congener of fluoxetine, is controlled by residues in different regions of the transporters, indicating a complex mechanism for selective recognition of structurally similar compounds in SERT and NET. Our findings add important new information on the molecular basis for SERT/NET selectivity of antidepressants, and provide the first assessment of the potential of LeuBAT as a model system for antidepressant binding in human transporters, which is essential for future structure-based drug development of antidepressant drugs with fine-tuned transporter selectivity.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is up-regulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs, and noncanonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is primarily mediated through electrostatic interactions of basic amino acids in the BigDyn N terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the noncanonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn, and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.