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Monoamine transporters (MATs) regulate neurotransmission via the reuptake of dopamine, serotonin and norepinephrine from extra-neuronal regions and thus maintain neurotransmitter homeostasis. As targets of a wide range of compounds, including antidepressants, substances of abuse and drugs for neuropsychiatric and neurodegenerative disorders, their mechanism of action and their modulation by small molecules have long been of broad interest. Recent advances in the structural characterization of dopamine and serotonin transporters have opened the way for structure-based modeling and simulations, which, together with experimental data, now provide mechanistic understanding of their transport function and interactions. Here we review recent progress in the elucidation of the structural dynamics of MATs and their conformational landscape and transitions, as well as allosteric regulation mechanisms.

The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants ( S )-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of ( S )-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design. X-ray crystal structures of the human serotonin transporter (SERT) bound to the antidepressants ( S )-citalopram or paroxetine show that the antidepressants lock the protein in an outward-open conformation, and directly block serotonin from entering its binding site; the structures define the mechanism of antidepressant action in SERT and pave the way for future drug design. Antidepressant structure/activity relationships Serotonin modulates the activity of the central nervous system, as well as many other processes throughout the body. These authors have solved X-ray structures of the human serotonin transporter (SERT) in complex with the selective serotonin reuptake inhibitors (SSRIs) ( S )-citalopram and paroxetine — two of the most widely prescribed antidepressants. The resulting structures reveal that the antidepressants lock the protein in an outward-open conformation, and directly block the entry of serotonin into its binding site. A previously unknown allosteric site is seen in the extracellular vestibule; binding of ligands to this site prevents dissociation from the central site, establishing a mechanism of antidepressant action in SERT and pointing the way for future drug design.

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.

We studied seven genes that reflect events relevant to antidepressant action at four sequential levels: (1) entry into the brain, (2) binding to monoaminergic transporters, and (3) distal effects at the transcription level, resulting in (4) changes in neurotrophin and neuropeptide receptors. Those genes are ATP-binding cassette subfamily B member 1 ( ABCB1 ), the noradrenaline, dopamine, and serotonin transporters ( SLC6A2, SLC6A3 and SLC6A4 ), cyclic AMP-responsive element binding protein 1 ( CREB1 ), corticotropin-releasing hormone receptor 1 ( CRHR1 ) and neurotrophic tyrosine kinase type 2 receptor ( NTRK2 ). Sequence variability for those genes was obtained in exonic and flanking regions. A total of 56 280 000 bp across were sequenced in 536 unrelated Mexican Americans from Los Angeles (264 controls and 272 major depressive disorder (MDD)). We detected in those individuals 419 single nucleotide polymorphisms (SNPs); the nucleotide diversity was 0.00054±0.0001. Of those, a total of 204 novel SNPs were identified, corresponding to 49% of all previously reported SNPs in those genes: 72 were in untranslated regions, 19 were in coding sequences of which 7 were non-synonymous, 86 were intronic and 27 were in upstream/downstream regions. Several SNPs or haplotypes in ABCB1, SLC6A2, SLC6A3, SLC6A4, CREB1 and NTRK2 were associated with MDD, and in ABCB1, SLC6A2 and NTRK2 with antidepressant response. After controlling for age, gender and baseline 21-item Hamilton Depression Rating Scale (HAM-D21) score, as well as correcting for multiple testing, the relative reduction of HAM-D21 score remained significantly associated with two NTRK2 -coding SNPs (rs2289657 and rs56142442) and the haplotype CAG at rs2289658 (splice site), rs2289657 and rs2289656. Further studies in larger independent samples will be needed to confirm these associations. Our data indicate that extensive assessment of sequence variability may contribute to increase understanding of disease susceptibility and drug response. Moreover, these results highlight the importance of direct re-sequencing of key candidate genes in ethnic minority groups in order to discover novel genetic variants that cannot be simply inferred from existing databases.

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons 1 – 3 . Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors—small molecules that competitively block substrate binding and thereby prolong neurotransmitter action 2 , 4 . The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines 2 , 3 , and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder 2 , 5 . Studies of bacterial NSS homologues—including LeuT—have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane 1 , 6 – 12 . However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties 13 , 14 . Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors 15 , 16 . Here we report cryo-electron microscopy structures of SERT–ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition. Cryo-electron microscopy reveals three conformations of the serotonin transporter in complex with ibogaine, detailing the structural rearrangements that occur between the different stages of its transport cycle.

Sperm–oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm–oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm–oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm–oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm–oocyte fusion.

In many animals, females respond to mating with changes in physiology and behavior that are triggered by molecules transferred by males during mating. In Drosophila melanogaster, proteins in the seminal fluid are responsible for important female postmating responses, including temporal changes in egg production, elevated feeding rates and activity levels, reduced sexual receptivity, and activation of the immune system. It is unclear to what extent these changes are mutually beneficial to females and males or instead represent male manipulation. Here we use an experimental evolution approach in which females are randomly paired with a single male each generation, eliminating any opportunity for competition for mates or mate choice and thereby aligning the evolutionary interests of the sexes. After >150 generations of evolution, males from monogamous populations elicited a weaker postmating stimulation of egg production and activity than males from control populations that evolved with a polygamous mating system. Males from monogamous populations did not differ from males from polygamous populations in their ability to induce refractoriness to remating in females, but they were inferior to polygamous males in sperm competition. Mating-responsive genes in both the female abdomen and head showed a dampened response to mating with males from monogamous populations. Males from monogamous populations also exhibited lower expression of genes encoding seminal fluid proteins, which mediate the female response to mating. Together, these results demonstrate that the female postmating response, and the male molecules involved in eliciting this response, are shaped by ongoing sexual conflict.

BackgroundThe retinoic acid (RA) pathway plays a crucial role in both eye morphogenesis and the visual cycle. Individuals with monoallelic and biallelic pathogenic variants in retinol-binding protein 4 (RBP4), encoding a serum retinol-specific transporter, display variable ocular phenotypes. Although few families have been reported worldwide, recessive inherited variants appear to be associated with retinal degeneration, while individuals with dominantly inherited variants manifest ocular development anomalies, mainly microphthalmia, anophthalmia and coloboma (MAC).MethodsWe report here seven new families (13 patients) with isolated and syndromic MAC harbouring heterozygous RBP4 variants, of whom we performed biochemical analyses.ResultsFor the first time, malformations that overlap the clinical spectrum of vitamin A deficiency are reported, providing a link with other RA disorders. Our data support two distinct phenotypes, depending on the nature and mode of inheritance of the variants: dominantly inherited, almost exclusively missense, associated with ocular malformations, in contrast to recessive, mainly truncating, associated with retinal degeneration. Moreover, we also confirm the skewed inheritance and impact of maternal RBP4 genotypes on phenotypical expression in dominant forms, suggesting that maternal RBP4 genetic status and content of diet during pregnancy may modify MAC occurrence and severity. Furthermore, we demonstrate that retinol-binding protein blood dosage in patients could provide a biological signature crucial for classifying RBP4 variants. Finally, we propose a novel hypothesis to explain the mechanisms underlying the observed genotype–phenotype correlations in RBP4 mutational spectrum.ConclusionDominant missense variants in RBP4 are associated with MAC of incomplete penetrance with maternal inheritance through a likely dominant-negative mechanism.

The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement 1 . Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, the human dopamine transporter (hDAT) and mechanisms by which it is inhibited by small molecules and Zn 2+ are without a high-resolution structural context. Here we determine the structure of hDAT in a tripartite complex with the competitive inhibitor and cocaine analogue, (–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane 2 (β-CFT), the non-competitive inhibitor MRS7292 3 and Zn 2 + (ref. 4 ). We show how β-CFT occupies the central site, approximately halfway across the membrane, stabilizing the transporter in an outward-open conformation. MRS7292 binds to a structurally uncharacterized allosteric site, adjacent to the extracellular vestibule, sequestered underneath the extracellular loop 4 (EL4) and adjacent to transmembrane helix 1b (TM1b), acting as a wedge, precluding movement of TM1b and closure of the extracellular gate. A Zn 2+ ion further stabilizes the outward-facing conformation by coupling EL4 to EL2, TM7 and TM8, thus providing specific insights into how Zn 2+ restrains the movement of EL4 relative to EL2 and inhibits transport activity. Cryo-electron microscopy structure of the human dopamine transporter in complex with multiple inhibitors illuminates mechanisms of allosteric inhibition.

The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology 1 . Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder 2 . Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders. Cryo-electron microscopy structures of the noradrenaline transporter in the apo state, bound to noradrenaline and bound to various antidepressants shed light on the substrate transport, molecular recognition and dimeric architecture of this protein.