Insights into the mechanism and pharmacology of neurotransmitter sodium symporters.

Neurotransmitter sodium symporters (NSS) belong to the SLC6 family of solute carriers and play an essential role in neurotransmitter homeostasis throughout the body. In the past decade, structural studies employing bacterial orthologs of NSSs have provided insight into the mechanism of neurotransmitter transport. While the overall architecture of SLC6 transporters is conserved among species, in comparison to the bacterial homologs, the eukaryotic SLC6 family members harbor differences in amino acid sequence and molecular structure, which underpins their functional and pharmacological diversity, as well as their ligand specificity. Here, we review the structures and mechanisms of eukaryotic NSSs, focusing on the molecular basis for ligand recognition and on transport mechanism.

Toxicokinetics of the Synthetic Cathinone α-Pyrrolidinohexanophenone.

Synthetic cathinones inhibit monoamine transporters, such as serotonin, norepinephrine, and dopamine transporters, and act on the central nervous system via increasing synaptic concentrations of monoamines. These compounds, which are highly addictive and potentially poisonous, are new psychoactive substances. In this study, we investigated the toxicokinetics of the synthetic cathinone, α-pyrrolidinohexanophenone (α-PHP), and assessed the relationship between the toxicokinetics and the long-term clinical symptoms induced by α-PHP in a male patient. The patient (39 years old) suddenly started uttering inarticulate words and demonstrating incomprehensible behavior in his house, and was brought to the emergency department of Iwate Medical University hospital. He presented with psychotic symptoms, such as hallucinations and delusion; however, his vital signs were normal. The hallucinations and delusion improved by the third day of hospitalization. Toxicological analysis was performed using liquid chromatography-tandem mass spectrometry with QuEChERS extraction. α-PHP was detected in his serum at a concentration of 175 ng/mL on his arrival at the hospital. His serum concentrations of α-PHP were serially determined and their natural logarithms were plotted against time after arrival. Although serum concentrations at early time points were lacking, the obtained curve was consistent with a two-compartment model and indicated a serum elimination half-life of 37 h. The long-lasting psychotic symptoms induced by synthetic cathinones appear to be correlated with their toxicokinetic characteristics, such as their long half-lives. Finally, interpreting the toxicokinetics of synthetic cathinones may provide useful information for the toxicological assessment of new psychoactive substances for forensic and clinical purposes.

Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

BACKGROUND: The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. METHODS: CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. RESULTS: Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 µg/kg, 80 µg/kg, or 100 µg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. CONCLUSION: We report for the first time that CHT1 may be involved in pain modulation in CP, as it plays an important role in pain inhibition. Increased CHT1 activity or the up-regulation of its expression may be used to treat pain in patients with CP.

Synthesis and biological evaluation of 3-phenethylazetidine derivatives as triple reuptake inhibitors.

We report the synthesis of 3-phenethylazetidine derivatives 2 and their biological activities against 5-HT, NE and DA transporters as well as microsomal stability, CYP inhibition, and hERG inhibition profiles. Compound 2at showed most potent triple reuptake inhibitor with good selectivity as a candidate for depression.

The transport mechanism of the human sodium/myo-inositol transporter 2 (SMIT2/SGLT6), a member of the LeuT structural family.

The sodium/myo-inositol transporter 2 (SMIT2) is a member of the SLC5A gene family, which is believed to share the five-transmembrane segment inverted repeat of the LeuT structural family. The two-electrode voltage-clamp (TEVC) technique was used to measure the steady-state and the pre-steady-state currents mediated by human SMIT2 after expression in Xenopus laevis oocytes. Phlorizin is first shown to be a poor inhibitor of pre-steady-state currents for depolarizing voltage pulse. From an up to threefold difference between the apparent ON and OFF transferred charges during a voltage pulse, we also show that a fraction of the transient current recorded for very negative potentials is not a true pre-steady-state current coming from the cotransporter conformational changes. We suggest that this transient current comes from a time-dependent leak current that can reach large amplitudes when external Na(+) concentration is reduced. A kinetic model was generated through a simulated annealing algorithm. This algorithm was used to identify the optimal connectivity among 19 different kinetic models and obtain the numerical values of the associated parameters. The proposed 5-state model includes cooperative binding of Na(+) ions, strong apparent asymmetry of the energy barriers, a rate-limiting step that is likely associated with the translocation of the empty transporter, and a turnover rate of 21 s(-1). The proposed model is a proof of concept for a novel approach to kinetic modeling of electrogenic transporters and allows insight into the transport mechanism of members of the LeuT structural family at the millisecond timescale.

5-hydroxytryptamine-mediated neurotransmission modulates spontaneous and vagal-evoked glutamate release in the nucleus of the solitary tract effect of uptake blockade.

The effect of blockade of either 5-hydroxytryptamine (5-HT)/serotonin transporter (SERT) with citalopram or the organic cation transporter 3 (OCT3)/plasma membrane monoamine transporter (PMAT) with decynium-22 (D-22) on spontaneous and evoked release of 5-HT in the nucleus tractus solitarius (NTS) was investigated in rat brainstem slices treated with gabazine. 5-HT release was measured indirectly by changes in the frequency and amplitude of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) [in the presence of tetrodotoxin (TTX)] and evoked EPSCs. Blockade of 5-HT3 receptors with granisetron reduced, whereas the 5-HT3 agonist phenylbiguanide increased, the frequency of mEPSCs. 5-HT decreased mEPSC frequency at low concentrations and increased frequency at high concentrations. This inhibition was blocked by the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635), which was ineffective on its own, whereas the excitation was reversed by granisetron. The addition of citalopram or D-22 caused inhibition, which was prevented by 5-HT1A blockade. Thus, in the NTS, the spontaneous release of 5-HT is able to activate 5-HT3 receptors, but not 5-HT1A receptors, as the release in their vicinity is removed by uptake. The ineffectiveness of corticosterone suggests that the low-affinity, high-capacity transporter is PMAT, not OCT3. For evoked 5-HT release, only D-22 caused an increase in the amplitude of EPSCs, with a decrease in the paired pulse ratio, and increased the number of spontaneous EPSCs after 20-Hz stimulation. Thus, for the evoked release of 5-HT, the low-affinity, high-capacity transporter PMAT, but not 5-HT transporter (5-HTT)/SERT, is important in the regulation of changes in 5-HT extracellular concentration.

Lipopolysaccharide increases degradation of central monoamines: an in vivo microdialysis study in the nucleus accumbens and medial prefrontal cortex of mice.

Peripheral administration of lipopolysaccharide (LPS) in rodents induces anhedonia, i.e. the inability to experience pleasure. Recently, we reported that serotonin transporter (SERT) function is required for LPS-induced anhedonia. Less is known about the effect of LPS on the biological activity of dopamine transporters (DAT) and norepinephrine transporters (NET). Therefore, in vivo microdialysis was performed in the nucleus accumbens and medial prefrontal cortex of C57BL6/J mice exposed to saline or LPS (133 µg/kg i.p.). To investigate the possible involvement of different monoamine transporters, the triple reuptake inhibitor DOV 216,303 or saline was i.p. injected 30 min before the saline/LPS injection. The dose of LPS, shown to decrease responding for brain stimulation reward in mice, significantly increased extracellular levels of monoamine metabolites (5-HIAA, DOPAC and HVA) in the nucleus accumbens and medial prefrontal cortex. Remarkably, DOV 216,303 abolished LPS-induced DOPAC and HVA formation in the nucleus accumbens, suggesting that LPS increases DAT activity in this brain area. DOV 216,303 also inhibited LPS-induced DOPAC and HVA formation in the medial prefrontal cortex. Since DAT density is very low in this brain structure, reuptake of DA predominantly takes place via NET, suggesting that LPS increases DAT and NET activity in the medial prefrontal cortex. Furthermore, DOV 216,303 pretreatment prevented LPS-induced 5-HIAA formation only in the medial prefrontal cortex, indicating that LPS increases prefrontal SERT activity. In conclusion, the present findings suggest that peripheral LPS increases DAT activity in the nucleus accumbens and increases NET and SERT activity in the medial prefrontal cortex of mice.

Monoamine transporter and receptor interaction profiles of a new series of designer cathinones.

Psychoactive ß-keto amphetamines (cathinones) are sold as "bath salts" or "legal highs" and recreationally abused. We characterized the pharmacology of a new series of cathinones, including methedrone, 4-methylethcathinone (4-MEC), 3-fluoromethcathinone (3-FMC), pentylone, ethcathinone, buphedrone, pentedrone, and N,N-dimethylcathinone. We investigated norepinephrine (NE), dopamine (DA), and serotonin (5-HT) uptake inhibition using human embryonic kidney 293 (HEK 293) cells that express the respective human monoamine transporter, the drug-induced efflux of NE, DA, and 5-HT from monoamine-preloaded cells, and binding affinity to monoamine transporters and receptors. All of the cathinones were potent NE uptake inhibitors but differed in their DA vs. 5-HT transporter inhibition profiles and monoamine release effects. Methedrone was a more potent 5-HT than DA transporter inhibitor and released NE and 5-HT similar to para-methoxymethamphetamine (PMMA), para-methoxyamphetamine (PMA), 4-methylthioamphetamine (4-MTA), and 3,4-methylenedioxymethamphetamine (MDMA). 4-MEC and pentylone equipotently inhibited all of the monoamine transporters and released 5-HT. Ethcathinone and 3-FMC inhibited NE and DA uptake and released NE, and 3-FMC also released DA similar to N-ethylamphetamine and methamphetamine. Pentedrone and N,N-dimethylcathinone were non-releasing NE and DA uptake inhibitors as previously shown for pyrovalerone cathinones. Buphedrone preferentially inhibited NE and DA uptake and also released NE. None of the cathinones bound to rodent trace amine-associated receptor 1, in contrast to the non-ß-keto-amphetamines. None of the cathinones exhibited relevant binding to other monoamine receptors. In summary, we found considerable differences in the monoamine transporter interaction profiles among different cathinones and compared with related amphetamines.

Structural basis for action by diverse antidepressants on biogenic amine transporters.

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

[Simultaneous determination of guanidinoacetate, creatine and creatinine by liquid chromatography-tandem mass spectrometry: a diagnostic tool for creatine deficiency syndromes in body fluids and a perspective use on cultured fibroblasts]. / Détermination simultanée du guanidinoacétate, de la créatine et de la créatinine par chromatographie liquide couplée à la spectrométrie de masse en tandem : outil de diagnostic biochimique pour les syndromes de déficit en créatine et perspectives d'utilisation sur les fibroblastes.

Guanidinoacetate (GAA) and creatine (Cr) are creatine deficiency syndromes (CDS) biochemical markers. We describe a liquid chromatography - tandem mass spectrometry method (LC/MSMS) performing simultaneous analysis of GAA, Cr and creatinine (Crn). Study of Cr uptake by fibroblasts for Cr transporter defect diagnosis is also assessed. The three butylated compounds were separated by liquid chromatography and MSMS quantification was achieved by isotopic dilution with electrospray positive ion mode. Linearity was demonstrated from 0 to 600, 675 and 4500 µmol/L and limit of quantification was 0.1, 0.04 and 0.9 µmol/L for GAA, Cr, and Crn respectively. Intra- and inter-assay precision for each analyte was better than 11%, and standard recoveries ranged from 83 to 109%. Reference values in cerebrospinal fluid samples for subjects ≥14 years were also established for GAA and Cr. Five fibroblast cell lines were used for Cr uptake study. Cr uptake by fibroblasts increased with the Cr media concentrations and was significantly inhibited by 3-guanidinopropionate (500 µmol/L), a Cr transporter inhibitor (96h incubation, [Cr media] = 25 µmol/L, p<0.05). A reliable LC/MSMS method for the diagnosis of CDS was developed in different biological fluids. Finally, results of the Cr uptake study reinforce the interest of this technique to diagnose Cr transporter deficiencies.

Endogenous ACh tonically stimulates ANP secretion in rat atria.

Exogenous acetylcholine (ACh) is known to stimulate atrial natriuretic peptide (ANP) secretion concomitantly with a decrease in atrial pulse pressure. However, the role of intrinsic ACh in the regulation of ANP secretion remains unknown. Recently, it was shown that nonneuronal and neuronal ACh is present in the cardiac atria. From this finding we hypothesize that endogenously released ACh is involved in the regulation of ANP secretion in an autocrine or paracrine manner in the atria. Experiments were performed in isolated beating rat atria. ANP was measured using radioimmunoassay. To increase the availability of the ACh in the extracellular space of the atrium, its degradation was inhibited with an inhibitor of acetylcholinesterase. Acetylcholinesterase inhibition with physostigmine increased ANP secretion concomitantly with a decrease in atrial dynamics in a concentration-dependent manner. Inhibitors of M2 muscarinic ACh receptor (mAChR), methoctramine, and ACh-activated K(+) (KACh(+)) channels, tertiapin-Q, abolished the physostigmine-induced changes. The effects were not observed in the atria from rats treated with pertussis toxin. Furthermore, the physostigmine-induced effects were attenuated by an inhibitor of high-affinity choline transporter, hemicholinium-3, which is a rate-limiting step of ACh synthesis. Inhibitors of the mAChR signaling pathway and ACh synthesis also attenuated the basal levels of ANP secretion and accentuated atrial dynamics. These findings suggest that endogenously released ACh tonically stimulates ANP secretion from atrial cardiomyocytes via activation of M2 mAChR-Gi/o-KACh(+) channel signaling. It is also suggested that the ACh-ANP signaling is implicated in cardiac physiology and pathophysiology.

Differential regulation of MeCP2 phosphorylation in the CNS by dopamine and serotonin.

Systemic administration of amphetamine (AMPH) induces phosphorylation of MeCP2 at Ser421 (pMeCP2) in select populations of neurons in the mesolimbocortical brain regions. Because AMPH simultaneously activates multiple monoamine neurotransmitter systems, here we examined the ability of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) to induce pMeCP2. Selective blockade of the DA transporter (DAT) or the 5-HT transporter (SERT), but not the NE transporter (NET), was sufficient to induce pMeCP2 in the CNS. DAT blockade induced pMeCP2 in the prelimbic cortex (PLC) and nucleus accumbens (NAc), whereas SERT blockade induced pMeCP2 only in the NAc. Administration of selective DA and 5-HT receptor agonists was also sufficient to induce pMeCP2; however, the specific combination of DA and 5-HT receptors activated determined the regional- and cell-type specificity of pMeCP2 induction. The D(1)-class DA receptor agonist SKF81297 induced pMeCP2 widely; however, coadministration of the D(2)-class agonist quinpirole restricted the induction of pMeCP2 to GABAergic interneurons of the NAc. Intra-striatal injection of the adenylate cyclase activator forskolin was sufficient to induce pMeCP2 in medium-spiny neurons, suggesting that the combinatorial regulation of cAMP by different classes of DA and 5-HT receptors may contribute to the cell-type specificity of pMeCP2 induction. Consistent with the regulation of pMeCP2 by multiple monoamine neurotransmitters, genetic disruption of any single monoamine transporter in DAT-, SERT-, and NET-knockout mice failed to eliminate AMPH-induced pMeCP2 in the NAc. Together, these studies indicate that combinatorial signaling through DA and 5-HT receptors can regulate the brain region- and cell-type specific pMeCP2 in the CNS.

SLC6 neurotransmitter transporters: structure, function, and regulation.

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

Is slow-onset long-acting monoamine transport blockade to cocaine as methadone is to heroin? Implication for anti-addiction medications.

The success of methadone in treating opiate addiction has suggested that long-acting agonist therapies may be similarly useful for treating cocaine addiction. Here, we examined this hypothesis, using the slow-onset long-acting monoamine reuptake inhibitor 31,345, a trans-aminotetralin analog, in a variety of addiction-related animal models, and compared it with methadone's effects on heroin's actions in the same animal models. Systemic administration of 31,345 produced long-lasting enhancement of electrical brain-stimulation reward (BSR) and extracellular nucleus accumbens (NAc) dopamine (DA). Pretreatment with 31,345 augmented cocaine-enhanced BSR, prolonged cocaine-enhanced NAc DA, and produced a long-term (24-48 h) reduction in cocaine self-administration rate without obvious extinction pattern, suggesting an additive effect of 31,345 with cocaine. In contrast, methadone pretreatment not only dose-dependently inhibited heroin self-administration with an extinction pattern but also dose-dependently inhibited heroin-enhanced BSR and NAc DA, suggesting functional antagonism by methadone of heroin's actions. In addition, 31,345 appears to possess significant abuse liability, as it produces dose-dependent enhancement of BSR and NAc DA, maintains a low rate of self-administration behavior, and dose-dependently reinstates drug-seeking behavior. In contrast, methadone only partially maintains self-administration with an extinction pattern, and fails to induce reinstatement of drug-seeking behavior. These findings suggest that 31,345 is a cocaine-like slow-onset long-acting monoamine transporter inhibitor that may act as an agonist therapy for cocaine addiction. However, its pattern of action appears to be significantly different from that of methadone. Ideal agonist substitutes for cocaine should fully emulate methadone's actions, that is, functionally antagonizing cocaine's action while blocking monoamine transporters to augment synaptic DA.

Synthesis of 1,1-[1-naphthyloxy-2-thiophenyl]-2-methylaminomethylcyclopropanes and their evaluation as inhibitors of serotonin, norepinephrine, and dopamine transporters.

Stereodefined trisubstituted cyclopropanes bearing naphthyloxy, thiophenyl, and (N-methylamino)methyl groups were synthesized in enantiopure form employing asymmetric cyclopropanation of (E)- and (Z)-allylic alcohols as the key step. In vitro assays of the synthesized cyclopropanes revealed that the K(i) of one of the enantiomers as a dual inhibitor of serotonin and norepinephrine transporters is in the low nanomolar range and is comparable to that of duloxetine.

Effects of the endocannabinoid transport inhibitors AM404 and UCM707 on diabetic neuropathy in rats.

1. Diabetic rats display increased pain responses following injection of formalin into the paw, suggesting the presence of hyperalgesia. In the present study, we investigated the efficacy of the systemic administration of the endocannabinoid transport inhibitors UCM707 and AM404 (1, 10 and 50 mg/kg, i.p.) on hyperalgesia during the formalin test in streptozocin (STZ)-induced diabetic rats. 2. Nociceptive testing was performed in male adult Wistar rats 4 weeks after the onset of hyperglycaemia. At the end of the experiment, all rats were weighed and then underwent plasma glucose measurements. 3. Diabetes caused significant hyperalgesia during both phases of the formalin test. At 10 and 50 mg/kg, both UCM707 and AM404 reversed chemical hyperalgesia in diabetic rats. UCM707 (10 and 50 mg/kg) caused less intensive nociceptive behaviour during both phases of the test, whereas AM404 (10 and 50 mg/kg) only affected pain scores during Phase 1 of the formalin test. At 1 mg, neither drug had any effect on pain behaviour in control and diabetic groups compared with their respective controls. Neither UCM707 nor AM404 had any effect on bodyweight or plasma glucose levels of treated compared with non-treated rats at any of the doses tested. 4. The results of the present study indicate that systemic administration of UCM707 and AM404 is effective in ameliorating chemical hyperalgesia in STZ-diabetic rats. Thus, endocannabinoid transport inhibitors may have potential in the treatment of painful diabetic neuropathy.

Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.

Overexpression of wild-type creatine transporter (SLC6A8) restores creatine uptake in primary SLC6A8-deficient fibroblasts.

In the study reported, we prove that mutations in the SLC6A8 gene are responsible for SLC6A8 deficiency, a cerebral creatine deficiency syndrome (CCDS), since overexpression of the wild-type SLC6A8 open reading frame (ORF) restores the creatine uptake profile in SLC6A8-deficient fibroblasts.