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Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsy, and neurodegenerative diseases, among others. EAAT2 is the main subtype responsible for glutamate clearance in the brain, having a key role in regulating transmission and preventing excitotoxicity. Therefore, compounds that increase the expression or activity of EAAT2 have therapeutic potential for neuroprotection. Previous studies identified molecular determinants for EAAT2 transport stimulation in a structural domain that lies at the interface of the rigid trimerization domain and the central substrate binding transport domain. In this work, a hybrid structure based approach was applied, based on this molecular domain, to create a high-resolution pharmacophore. Subsequently, virtual screening of a library of small molecules was performed, identifying 10 hit molecules that interact at the proposed domain. Among these, three compounds were determined to be activators, four were inhibitors, and three had no effect on EAAT2-mediated transport in vitro. Further characterization of the two best ranking EAAT2 activators for efficacy, potency, and selectivity for glutamate over monoamine transporters subtypes and NMDA receptors and for efficacy in cultured astrocytes is demonstrated. Mutagenesis studies suggest that the EAAT2 activators interact with residues forming the interface between the trimerization and transport domains. These compounds enhance the glutamate translocation rate, with no effect on substrate interaction, suggesting an allosteric mechanism. The identification of these novel positive allosteric modulators of EAAT2 offers an innovative approach for the development of therapies based on glutamate transport enhancement.

The serotonin transporter (SERT) is the principal mechanism for terminating serotonin (5HT) signals in the nervous system and is a site of action for a variety of psychoactive drugs including antidepressants, amphetamines, and cocaine. Here we show that human SERTs (hSERTs) and rat SERTs are capable of robust dopamine (DA) uptake through a process that differs mechanistically from 5HT transport in several unanticipated ways. DA transport by hSERT has a higher maximum velocity than 5HT transport, requires significantly higher Na+ and Cl− concentrations to sustain transport, is inhibited non-competitively by 5HT and is more sensitive to SERT inhibitors, including selective serotonin reuptake inhibitors (SSRIs). We use a thiol reactive methane thiosulfonate (MTS) reagent to modify a conformationally-sensitive cysteine residue to demonstrate that hSERT spends more time in an outward facing conformation when transporting DA than when transporting 5HT. Co-transfection of an inactive or an MTS-sensitive SERT with wild type SERT subunits reveals an absence of cooperative interactions between subunits during DA, but not 5HT transport. To establish the physiological relevance of this mechanism for DA clearance, we show using in vivo high-speed chronoamperometry that SERT has the capacity to clear extracellularly applied DA in the hippocampal CA3 region of anesthetized rats. Together, these observations suggest the possibility that SERT serves as a DA transporter in vivo and highlight the idea that there can be distinct modes of transport of alternative physiological substrates by SERT.

The dopamine (DAT), serotonin (SERT), and norepinephrine (NET) transporters, which are collectively referred to as monoamine transporters (MATs), play significant roles in regulating the neuronal response to these neurotransmitters. MATs terminate the action of these neurotransmitters by translocating them from the synaptic space into the presynaptic neurons. These three transmitters are responsible for controlling a number of physiological, emotional, and behavioral functions, with their transporters being the site of action of drugs employed for the treatment of a variety of conditions, including depression, anxiety, ADHD, schizophrenia, and psychostimulant abuse. Provided in this unit is information on the localization and regulation of MATs and the structural components of these proteins most responsible for the translocation process. Also included is a brief description of the evolution of ligands that interact with these transporters, as well as current theories concerning the pharmacological effects of substances that interact with these sites, including the molecular mechanisms of action of uptake inhibitors and allosteric modulators. Data relating to the presence, structure, and functions of allosteric modulators are included as well. The aim of this review is to provide background information on MATs to those who are new to this field, with a focus on the therapeutic potential of compounds that interact with these substrate transport sites. © 2017 by John Wiley & Sons, Inc.

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The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/ DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several wellstudied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.

The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.