A mechanism of uncompetitive inhibition of the serotonin transporter

Shreyas Bhat; Ali El-Kasaby; Ameya Kasture; Danila Boytsov; Julian B Reichelt; Thomas Hummel; Sonja Sucic; Christian Pifl; Michael Freissmuth; Walter Sandtner

doi:10.7554/eLife.82641

Titel

A mechanism of uncompetitive inhibition of the serotonin transporter

Autor*in

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna

Ali El-Kasaby

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna

Ameya Kasture

Department für Neurobiologie, Fakultät für Lebenswissenschaften, Universität Wien

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Abstract

The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action – that is, the retrieval of serotonin from the extracellular space – SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives), and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

Stichwort

General Immunology and MicrobiologyGeneral Biochemistry, Genetics and Molecular BiologyGeneral MedicineGeneral Neuroscience

Objekt-Typ

journal article

Sprache

Englisch [eng]

Persistent identifier

phaidra.univie.ac.at/o:2046039

DOI

10.7554/eLife.82641

Erschienen in

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