The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux

Sealover, Natalie R.; Felts, Bruce; Kuntz, Charles P.; Jarrard, Rachel E.; Hockerman, Gregory H.; Barker, Eric L.; Henry, L. Keith

doi:10.1016/j.bcp.2016.09.006

Cited by 5 publications

(1 citation statement)

References 52 publications

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“…Futher validation of the strength of this approach is found in agreement between identified mutants and previous focused studies characterizing the same residues. For instance, our previous study investigating the molecular determinants necessary for transport of 3,4 methylendioxymethamphetamine (MDMA) and 5-HT by SERT ( 69 ) show that outer gate residue Arg104 is intolerant of substitutions (as also observed in our mutational scan for APP+ import, in which only substitution R104K had a log 2 enrichment value > −1) and that only acidic residues at Glu493 are permissive for MDMA transport but other substitutions of Glu493 are tolerated for 5-HT ( 69 ) and APP + transport based on our findings here. Furthermore, residues Ile179 and Asp400 are highly sensitive to substitution in regards to APP + transport, V489 can only tolerate substitution by hydrophobics Leu, Met, Ile, and Phe, and K490 can accept multiple substitutions, which is consistent with the findings of Anderson et al ( 60 ).…”

Section: Resultsmentioning

confidence: 99%

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

Young

Chan

Selvam

et al. 2021

Preprint

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The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate analogue APP+, identifying many mutations that enhance APP+ import. Comprehensive simulations of the entire ion-coupled import process reveal that while binding of the native substrate, serotonin, reduces free energy barriers between conformational states to promote SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na+ bound-SERT, with a higher free energy barrier for transitioning to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ finds mutations that promote the necessary conformational changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor opening of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which the neurotransmitter and a symported sodium share a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface trafficking, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.

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Section: Resultsmentioning

confidence: 99%

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

Young

Chan

Selvam

et al. 2021

Preprint

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Post-translational mechanisms in psychostimulant-induced neurotransmitter efflux

Vaughan,

Henry,

Foster

et al. 2024

Pharmacological Advances in Central Nervous System Stimulants

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Induced fit, ensemble binding space docking and Monte Carlo simulations of MDMA ‘ecstasy’ and 3D pharmacophore design of MDMA derivatives on the human serotonin transporter (hSERT)

Islas

Moreno

Scior

2021

Heliyon

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The popular recreational drug MDMA (3,4-methylenedioxy-methamphetamine) has a documented potential as a psychopharmacological clinical and research tool. This is due to its unique ability to promote reprocessing of traumatic memories, empathetic and pro-social states. Although it is established that MDMA exerts its behavioural effects via the serotonin transporter (SERT), the ligand-protein molecular interplay remains elusive. In order to shed light on the binding of MDMA and its primary congeneric entactogens (MDA, MBDB and MDAI), we first combined induced fit with Monte Carlo simulations. The computed interaction energies of the models correlated well with experimental activities ( adj R 2 ¼ 0.78). Then we carried out 'ensemble binding space docking' on trajectories generated by interpolation of experimentally derived structures of the hSERT from the outward-open, and the occluded, to the inward-open states. This approach revealed low-energy alternative binding modes, suggesting high occupancy of the central site, yet considerable MDMA mobility within it, favouring the paroxetine-like orientation. Finally, we designed a pharmacophore that may be used to recognise hSERT-mediated serotonin releasers and uptake inhibitors of diverse chemical structure, identifying their active conformations and interacting residues. We conclude that the conserved amine-Asp98 ionic and edge-to-face π-π interactions are crucial to the mode of action of MDMA on the hSERT, underscoring the contributions of Tyr95 and gating residues Phe341, Tyr176 and Phe335. Amenable to experimental testing, our modelling may aid the rational design of novel entactogenic compounds and contribute to the understanding of an action mechanism, common and typical of psychotropic agents.

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The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux

Cited by 5 publications

References 52 publications

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

Post-translational mechanisms in psychostimulant-induced neurotransmitter efflux

Induced fit, ensemble binding space docking and Monte Carlo simulations of MDMA ‘ecstasy’ and 3D pharmacophore design of MDMA derivatives on the human serotonin transporter (hSERT)

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