Structural basis of promiscuous substrate transport by Organic Cation Transporter 1.

Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.

The twisting elevator mechanism of glutamate transporters reveals the structural basis for the dual transport-channel functions.

Glutamate transporters facilitate the removal of this excitatory neurotransmitter from the synapse. Increasing evidence indicates that this process is linked to intrinsic chloride channel activity that is thermodynamically uncoupled from substrate transport. A recent cryo-EM structure of GltPh - an archaeal homolog of the glutamate transporters - in an open channel state has shed light on the structural basis for channel opening formed at the interface of two domains within the transporter which is gated by two clusters of hydrophobic residues. These transporters cycle through several conformational states during the transport process, including the chloride conducting state, which appears to be stabilised by protein-membrane interactions and membrane deformation. Several point mutations that perturb the chloride conductance can have detrimental effects and are linked to the pathogenesis of the neurological disorder, episodic ataxia type 6.

Characterizing unexpected interactions of a glutamine transporter inhibitor with members of the SLC1A transporter family.

The solute carrier 1A family comprises a group of membrane proteins that act as dual-function amino acid transporters and chloride (Cl-) channels and includes the alanine serine cysteine transporters (ASCTs) as well as the excitatory amino acid transporters. ASCT2 is regarded as a promising target for cancer therapy, as it can transport glutamine and other neutral amino acids into cells and is upregulated in a range of solid tumors. The compound L-γ-glutamyl-p-nitroanilide (GPNA) is widely used in studies probing the role of ASCT2 in cancer biology; however, the mechanism by which GPNA inhibits ASCT2 is not entirely clear. Here, we used electrophysiology and radiolabelled flux assays to demonstrate that GPNA activates the Cl- conductance of ASCT2 to the same extent as a transported substrate, whilst not undergoing the full transport cycle. This is a previously unreported phenomenon for inhibitors of the solute carrier 1A family but corroborates a body of literature suggesting that the structural requirements for transport are distinct from those for Cl- channel formation. We also show that in addition to its currently known targets, GPNA inhibits several of the excitatory amino acid transporters. Together, these findings raise questions about the true mechanisms of its anticancer effects.

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Cl- channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified what we believe is a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Acetyl-CoA-Mediated Post-Biosynthetic Modification of Desferrioxamine B Generates N- and N-O-Acetylated Isomers Controlled by a pH Switch.

Biosynthesis of the hydroxamic acid siderophore desferrioxamine D1 (DFOD1, 6), which is the N-acetylated analogue of desferrioxamine B (DFOB, 5), has been delineated. Enzyme-independent Ac-CoA-mediated N-acetylation of 5 produced 6, in addition to three constitutional isomers containing an N-O-acetyl group installed at either one of the three hydroxamic acid groups of 5. The formation of N-Ac-DFOB (DFOD1, 6) and the composite of N-O-acetylated isomers N-O-Ac-DFOB[001] (6a), N-O-Ac-DFOB[010] (6b), and N-O-Ac-DFOB[100] (6c) (defined as the N-O-Ac motif positioned within the terminal amine, internal, or N-acetylated region of 5, respectively), was pH-dependent, with 6a-6c dominant at pH < 8.5 and 6 dominant at pH > 8.5. The trend in the pH dependence was consistent with the pKa values of the NH3+ (pKa ∼ 10) and N-OH (pKa ∼ 8.5-9) groups in 5. The N- and N-O-acetyl motifs can be conceived as a post-biosynthetic modification (PBM) of a nonproteinaceous secondary metabolite, akin to a post-translational modification (PTM) of a protein. The pH-labile N-O-acetyl group could act as a reversible switch to modulate the properties and functions of secondary metabolites, including hydroxamic acid siderophores. An alternative (most likely minor) biosynthetic pathway for 6 showed that the nonribosomal peptide synthetase-independent siderophore synthetase DesD was competent in condensing N'-acetyl-N-succinyl-N-hydroxy-1,5-diaminopentane (N'-Ac-SHDP, 7) with the dimeric hydroxamic acid precursor (AHDP-SHDP, 4) native to 5 biosynthesis to generate 6. The strategy of diversifying protein structure and function using PTMs could be paralleled in secondary metabolites with the use of PBMs.

Regulation of Glutamate, GABA and Dopamine Transporter Uptake, Surface Mobility and Expression.

Neurotransmitter transporters limit spillover between synapses and maintain the extracellular neurotransmitter concentration at low yet physiologically meaningful levels. They also exert a key role in providing precursors for neurotransmitter biosynthesis. In many cases, neurons and astrocytes contain a large intracellular pool of transporters that can be redistributed and stabilized in the plasma membrane following activation of different signaling pathways. This means that the uptake capacity of the brain neuropil for different neurotransmitters can be dynamically regulated over the course of minutes, as an indirect consequence of changes in neuronal activity, blood flow, cell-to-cell interactions, etc. Here we discuss recent advances in the mechanisms that control the cell membrane trafficking and biophysical properties of transporters for the excitatory, inhibitory and modulatory neurotransmitters glutamate, GABA, and dopamine.

Glutamate transporters have a chloride channel with two hydrophobic gates.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity1. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2-5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6-8. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family.

Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics.

The treatment of chronic pain is poorly managed by current analgesics, and there is a need for new classes of drugs. We recently developed a series of bioactive lipids that inhibit the human glycine transporter GlyT2 (SLC6A5) and provide analgesia in animal models of pain. Here, we have used functional analysis of mutant transporters combined with molecular dynamics simulations of lipid-transporter interactions to understand how these bioactive lipids interact with GlyT2. This study identifies a novel extracellular allosteric modulator site formed by a crevice between transmembrane domains 5, 7, and 8, and extracellular loop 4 of GlyT2. Knowledge of this site could be exploited further in the development of drugs to treat pain, and to identify other allosteric modulators of the SLC6 family of transporters.

Development of an N-Acyl Amino Acid That Selectively Inhibits the Glycine Transporter 2 To Produce Analgesia in a Rat Model of Chronic Pain.

Inhibitors that target the glycine transporter 2, GlyT2, show promise as analgesics, but may be limited by their toxicity through complete or irreversible binding. Acyl-glycine inhibitors, however, are selective for GlyT2 and have been shown to provide analgesia in animal models of pain with minimal side effects, but are comparatively weak GlyT2 inhibitors. Here, we modify the simple acyl-glycine by synthesizing lipid analogues with a range of amino acid head groups in both l- and d-configurations, to produce nanomolar affinity, selective GlyT2 inhibitors. The potent inhibitor oleoyl-d-lysine (33) is also resistant to degradation in both human and rat plasma and liver microsomes, and is rapidly absorbed following an intraperitoneal injection to rats and readily crosses the blood-brain barrier. We demonstrate that 33 provides greater analgesia at lower doses, and does not possess the severe side effects of the very slowly reversible GlyT2 inhibitor, ORG25543 (2).

Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A.

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.

Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5.

Cancer cells undergo a shift in metabolism where they become reliant on nutrients such as the amino-acid glutamine. Glutamine enters the cell via the alanine/serine/cysteine transporter 2 (ASCT2) that is upregulated in several cancers to maintain an increased supply of this nutrient and are therefore an attractive target in cancer therapeutic development. ASCT2 belongs to the glutamate transporter (SLC1A) family but is the only transporter in this family able to transport glutamine. The structural basis for glutamine selectivity of ASCT2 is unknown. Here, we identify two amino-acid residues in the substrate-binding site that are responsible for conferring glutamine selectivity. We introduce corresponding mutations into a prokaryotic homologue of ASCT2 and solve four crystal structures, which reveal the structural basis for neutral amino acid and inhibitor binding in this family. This structural model of ASCT2 may provide a basis for future development of selective ASCT2 inhibitors to treat glutamine-dependent cancers.

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuTAa, and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuTAa, contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC50 of 9 µM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 µM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

Structural Optimization and Pharmacological Evaluation of Inhibitors Targeting Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases (DYRK) and CDC-like kinases (CLK) in Glioblastoma.

The DYRK family contains kinases that are up-regulated in malignancy and control several cancer hallmarks. To assess the anticancer potential of inhibitors targeting DYRK kinases, we developed a series of novel DYRK inhibitors based on the 7-azaindole scaffold. All compounds were tested for their ability to inhibit DYRK1A, DYRK1B, DYRK2, and the structurally related CLK1. The library was screened for anticancer efficacy in established and stem cell-like glioblastoma cell lines. The most potent inhibitors (IC50 ≤ 50 nM) significantly decreased viability, clonogenic survival, migration, and invasion of glioblastoma cells. Target engagement was confirmed with genetic knockdown and the cellular thermal shift assay. We demonstrate that DYRK1A's thermal stability in cells is increased upon compound treatment, confirming binding in cells. In summary, we present synthesis, structure-activity relationship, and efficacy in glioblastoma-relevant models for a library of novel 7-azaindoles.

The amino acid transporter, SLC1A3, is plasma membrane-localised in adipocytes and its activity is insensitive to insulin.

The hormone insulin coordinates the catabolism of nutrients by protein phosphorylation. Phosphoproteomic analysis identified insulin-responsive phosphorylation of the Glu/Asp transporter SLC1A3/EAAT1 in adipocytes. The role of SLC1A3 in adipocytes is not well-understood. We show that SLC1A3 is localised to the plasma membrane and the major regulator of acidic amino acid uptake in adipocytes. However, its localisation and activity were unaffected by insulin or mutation of the insulin-regulated phosphosite. The latter was also observed using a heterologous expression system in Xenopus laevis oocytes. Thus, SLC1A3 maintains a constant import of acidic amino acids independently of nutritional status in adipocytes.

Characterization of the Inward- and Outward-Facing Substrate Binding Sites of the Prokaryotic Aspartate Transporter, GltPh.

Crystal structures of the prokaryotic aspartate transporter, GltPh, have provided important insights into the mechanism of amino acid transport by GltPh and related eukaryotic members of the glutamate transporter family (SLC1A family). Identification of inhibitors of GltPh can provide valuable tools for understanding the molecular basis for substrate and inhibitor specificity and selectivity of SLC1A members, but at present, few inhibitors of GltPh have been identified. We have screened a collection of commercially available aspartate analogues and identified new transportable and nontransportable GltPh inhibitors. We have explored the inhibition profile of GltPh by utilizing a thiol modification assay that isolates sided populations of the transporters reconstituted in liposomes to determine if any aspartate analogues display a preference for either the inwardly or outwardly directed binding sites. Here, we have characterized several new inhibitors of GltPh and identified three ß-carbon-substituted molecules that display a strong preference for the outwardly directed binding site of GltPh.

Correction: Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

[This corrects the article DOI: 10.1371/journal.pone.0157583.].

Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.

Tuning the ion selectivity of glutamate transporter-associated uncoupled conductances.

The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). In addition to their primary role in clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl(-) conductance. This conductance is activated by the binding of substrate and Na(+), but the direction of Cl(-) flux is independent of the rate or direction of substrate transport; thus, the two processes are thermodynamically uncoupled. A recent molecular dynamics study of the archaeal EAAT homologue GltPh (an aspartate transporter from Pyrococcus horikoshii) identified an aqueous pore at the interface of the transport and trimerization domains, through which anions could permeate, and it was suggested that an arginine residue at the most restricted part of this pathway might play a role in determining anion selectivity. In this study, we mutate this arginine to a histidine in the human glutamate transporter EAAT1 and investigate the role of the protonation state of this residue on anion selectivity and transporter function. Our results demonstrate that a positive charge at this position is crucial for determining anion versus cation selectivity of the uncoupled conductance of EAAT1. In addition, because the nature of this residue influences the turnover rate of EAAT1, we reveal an intrinsic link between the elevator movement of the transport domain and the Cl(-) channel.