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LeuT, a bacterial homologue of eukaryotic biogenic amine transporters (BATs), is engineered to harbour human BAT-like pharmacology by the mutation of key residues around the primary binding pocket; this mutant is able to bind several classes of antidepressant drug with high affinity, helping to define their common mechanisms of action. Structural approach to antidepressant activity Neurotransmitter sodium symporters (NSSs) 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). An X-ray crystal structure of a eukaryotic NSS is not available, hindering our understanding of the mechanism of action of these antidepressants. In this manuscript, the authors used a bacterial homologue of NSSs as a scaffold to generate a hybrid protein with a pharmacological profile very similar to that of biogenic amine transporters. They solved X-ray crystal structures of these 'LeuBAT' variants in the presence of four SSRIs, two SNRIs, a TCA and the stimulant mazindol. Even though these compounds have very different chemical structures, they all bind at the same site of LeuBAT, thereby enabling the authors to better understand how SSRIs, SNRIs and TCAs bind to their eukaryotic NSS targets. 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) 1 , 2 . 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 3 . 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 4 , it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT 5 , 6 , 7 and act as non-competitive inhibitors of transport 5 . By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 . 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.

Tricyclic antidepressants exert their pharmacological effect--inhibiting the reuptake of serotonin, norepinephrine, and dopamine--by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 angstroms of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters.

Creatine is a critical metabolite used to buffer cellular energy demands in highly energetic tissues such as the brain and muscle. Genetic defects in endogenous creatine synthesis or transport across cellular membranes lead to a common set of phenotypes referred to as Cerebral Creatine Deficiency Syndrome (CCDS). The most common form of CCDS is Creatine Transporter 1 (CT1) Deficiency (CTD). It accounts for ~ 70% of cases and results from loss-of-function mutations in the X-linked gene SLC6A8 . Affected individuals suffer from intellectual disability, autistic-like behaviors, and epilepsy. There are currently no effective therapies for this disorder, but gene therapy has emerged as a potential approach. The two enzymes which comprise the endogenous creatine synthetic pathway (AGAT and GAMT) are selectively expressed by specific cell types throughout the body. However, after synthesized, creatine uptake relies on the protein product of SLC6A8 , CT1, to transport creatine into target cell types. We hypothesized that gene delivery of GATM (encoding AGAT) and GAMT into end-user cell types would bypass the need for CT1, allowing for intracellular synthesis of creatine. We tested this strategy in two human cell types: HEK293T cells and primary fibroblasts. Co-delivery of GATM and GAMT increased internal creatine concentrations by 7.6-fold in HEK293T cells and 12.3-fold in healthy control fibroblasts. We then employed this approach to primary fibroblasts from patients with CTD. This resulted in an up to 11.6-fold increase in intracellular creatine concentrations, far exceeding the intracellular concentration of creatine in healthy control fibroblasts. Importantly, overexpression of AGAT and GAMT resulted in proper targeting of these enzymes to their natural cellular compartment and did not impair the growth of patient fibroblasts. These findings establish gene therapy with GATM and GAMT as a potential strategy for patients with CTD.

Creatine transporter deficiency (CTD) caused by mutations in SLC6A8 encoding the creatine transporter (CRT), leads to cerebral creatine deficiency syndromes; however, the cellular impact of CRT loss remains unclear. In this study, we investigated the consequences of the G561R mutation by examining fibroblasts using proteomics and functional assays. We observed severe intracellular creatine deficiency (> 90% reduction), leading to impaired energy metabolism (low ATP and high ADP/ATP). Proteomic analysis revealed significant alterations in the mitochondrial and extracellular vesicle pathways. Our investigation revealed impaired mitochondrial oxidative phosphorylation, reduced spare respiratory capacity, elevated oxidative stress, and significant alterations in amino acid transporter activity. Protein misfolding associated with G561R exacerbated these deficits compared to the deletion model. These findings elucidate the key pathological mechanisms induced by the CRT-G561R mutation—including energy metabolic reprogramming, mitochondrial dysfunction, and cellular stress—which significantly contribute to our understanding of the pathogenesis of creatine transporter deficiency and suggest potential therapeutic targets.

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L -Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na + binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins. Neurotransmitter:sodium symporters (NSS) serve as targets for drugs including antidepressants and psychostimulants. Here authors report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation which is a key intermediate in the LeuT transport cycle.

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. Whereas the non-homologous glutamate transporters mediate chloride conductance, in the eukaryotic NSS chloride is transported together with the neurotransmitter. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions, and is highly relevant for the neurotransmitter transporters. However, the precise role of chloride in neurotransmitter transport and the location of its binding site remain elusive. Here we show that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (gamma-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux, but not of exchange, was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) also result in chloride-independent transport, whereas the reciprocal mutations in LeuT and Tyt1 render substrate binding and/or uptake by these bacterial NSS chloride dependent. Our data indicate that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.

One in five couples who wish to conceive is infertile, and half of these couples have male infertility. However, the causes of male infertility are still largely unknown. Creatine is stored in the body as an energy buffer, and the testes are its second-largest reservoir after muscles. Further, even though intratesticular creatine levels have long been known to decrease in male patients with infertility, its role in the testis is unknown. We investigated the intratesticular role of creatine, specifically in the context of the creatine synthesizing enzyme Gamt , and the creatine transporter Slc6a8 . The Slc6a8 knockout mice showed no abnormalities in spermatogenesis. While Gamt knockout mice formed spermatozoa, they demonstrated reduced sperm count and decreased sperm motility and fertilization rate. Additionally, intratesticular creatine in Gamt knockout mice was significantly decreased, resulting in the disruption of tight junctions, which could be rectified by creatine supplementation, as was evidenced by the improved sperm count and fertilization rate in these mice. In conclusion, we identified creatine as being required for the maintenance of the tight junction in the testis.

The norepinephrine transporter (NET) transports norepinephrine from the synapse into presynaptic neurons, where norepinephrine regulates signaling pathways associated with cardiovascular effects and behavioral traits via binding to various receptors (e.g., β2-adrenergic receptor). NET is a known target for a variety of prescription drugs, including antidepressants and psychostimulants, and may mediate off-target effects of other prescription drugs. Here, we identify prescription drugs that bind NET, using virtual ligand screening followed by experimental validation of predicted ligands. We began by constructing a comparative structural model of NET based on its alignment to the atomic structure of a prokaryotic NET homolog, the leucine transporter LeuT. The modeled binding site was validated by confirming that known NET ligands can be docked favorably compared to nonbinding molecules. We then computationally screened 6,436 drugs from the Kyoto Encyclopedia of Genes and Genomes (KEGG DRUG) against the NET model. Ten of the 18 high-scoring drugs tested experimentally were found to be NET inhibitors; five of these were chemically novel ligands of NET. These results may rationalize the efficacy of several sympathetic (tuaminoheptane) and antidepressant (tranylcypromine) drugs, as well as side effects of diabetes (phenformin) and Alzheimer’s (talsaclidine) drugs. The observations highlight the utility of virtual screening against a comparative model, even when the target shares less than 30% sequence identity with its template structure and no known ligands in the primary binding site.

Neurotransmitter:Na + symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na + gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport. Conformational states of a membrane transporter The neurotransmitter:Na + symporter (NSS) family stops cellular signalling by recapturing released neurotransmitters from the synapse in a reuptake process driven by the Na + gradient. Drugs that interfere with this mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. The conformational changes associated with substrate binding have been studied in the prokaryotic NSS homologue LeuT using single-molecule fluorescence imaging experiments and molecular dynamics simulations. The observed single-molecule transitions reflect the structural dynamics of the intracellular region of the transporter, and can be interpreted in the context of an allosteric mechanism coupling ion and substrate binding to transport. Neurotransmitter:Na + symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na + gradient. Here, single-molecule fluorescence imaging assays have been combined with molecular dynamics simulations to probe the conformational changes that are associated with substrate binding and transport by a prokaryotic NSS homologue, LeuT. The findings are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na⁺ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na⁺ transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na⁺, allowing intracellular substrate and Na⁺ release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na⁺ on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT in Escherichia coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na⁺, these results identify mechanistic components underlying ion–substrate coupling in NSS transporters.