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Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter.

ABSTRACT: The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied by cholesterol and functionally relevant. In the present study, we focus on the conserved cholesterol site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5, and TM7. By molecular dynamics simulations, we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments, we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation, and, vice versa, mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity, and turnover rates. Taken together, we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium, which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.

SUBMITTER: Laursen L

PROVIDER: S-EPMC5846164 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter.

Laursen Louise L Severinsen Kasper K Kristensen Kristina Birch KB Periole Xavier X Overby Malene M Müller Heidi Kaastrup HK Schiøtt Birgit B Sinning Steffen S

The Journal of biological chemistry 20180119 10

The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied by cholesterol and functionally relevant. In the present st ...[more]

PMID: 29352106

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Project description:The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons1-3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action2,4. 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 amphetamines2,3, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder2,5. 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 membrane1,6-12. 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 properties13,14. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors15,16. 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.

Project description:BackgroundPreviously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes.MethodsTo explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity.ResultsOne consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior.ConclusionsOur findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans.

Dataset Information

Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter.

Publications

Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter.

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