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A mechanism of uncompetitive inhibition of the serotonin transporter
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), partia...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9883013/ https://www.ncbi.nlm.nih.gov/pubmed/36648438 http://dx.doi.org/10.7554/eLife.82641 |
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author | Bhat, Shreyas El-Kasaby, Ali Kasture, Ameya Boytsov, Danila Reichelt, Julian B Hummel, Thomas Sucic, Sonja Pifl, Christian Freissmuth, Michael Sandtner, Walter |
author_facet | Bhat, Shreyas El-Kasaby, Ali Kasture, Ameya Boytsov, Danila Reichelt, Julian B Hummel, Thomas Sucic, Sonja Pifl, Christian Freissmuth, Michael Sandtner, Walter |
author_sort | Bhat, Shreyas |
collection | PubMed |
description | 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 K(M) 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-PG(601,602)AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG(601,602)AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG(601,602)AA 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. |
format | Online Article Text |
id | pubmed-9883013 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-98830132023-01-28 A mechanism of uncompetitive inhibition of the serotonin transporter Bhat, Shreyas El-Kasaby, Ali Kasture, Ameya Boytsov, Danila Reichelt, Julian B Hummel, Thomas Sucic, Sonja Pifl, Christian Freissmuth, Michael Sandtner, Walter eLife Structural Biology and Molecular Biophysics 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 K(M) 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-PG(601,602)AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG(601,602)AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG(601,602)AA 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. eLife Sciences Publications, Ltd 2023-01-17 /pmc/articles/PMC9883013/ /pubmed/36648438 http://dx.doi.org/10.7554/eLife.82641 Text en © 2023, Bhat et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Structural Biology and Molecular Biophysics Bhat, Shreyas El-Kasaby, Ali Kasture, Ameya Boytsov, Danila Reichelt, Julian B Hummel, Thomas Sucic, Sonja Pifl, Christian Freissmuth, Michael Sandtner, Walter A mechanism of uncompetitive inhibition of the serotonin transporter |
title | A mechanism of uncompetitive inhibition of the serotonin transporter |
title_full | A mechanism of uncompetitive inhibition of the serotonin transporter |
title_fullStr | A mechanism of uncompetitive inhibition of the serotonin transporter |
title_full_unstemmed | A mechanism of uncompetitive inhibition of the serotonin transporter |
title_short | A mechanism of uncompetitive inhibition of the serotonin transporter |
title_sort | mechanism of uncompetitive inhibition of the serotonin transporter |
topic | Structural Biology and Molecular Biophysics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9883013/ https://www.ncbi.nlm.nih.gov/pubmed/36648438 http://dx.doi.org/10.7554/eLife.82641 |
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