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Updated: Aug 13, 2026
12:21
Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram
Published on: November 27, 2016
Accessibility and conformational coupling in serotonin transporter predicted internal domains
Andreas Androutsellis-Theotokis1, Gary Rudnick
1Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Summary
Investigating the serotonin transporter (SERT) structure, this study reveals key cytoplasmic domains. Cysteine modifications confirm SERT
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- The serotonin transporter (SERT) is crucial for regulating serotonin levels in the synaptic cleft.
- Understanding SERT's intracellular topology is essential for elucidating its transport mechanism and regulation.
- Previous topological models for SERT and related transporters have proposed different structural arrangements.
Purpose of the Study:
- To determine the intracellular topology of the serotonin transporter (SERT) using cysteine accessibility.
- To identify specific cysteine residues in the cytoplasmic domains of SERT that are accessible to modification.
- To validate the proposed topology of SERT against alternative models.
Main Methods:
- Utilized site-directed mutagenesis to introduce single cysteine residues into predicted cytoplasmic domains of SERT.
- Employed methanethiosulfonate (MTS) reagents to probe cysteine accessibility in intact cells and permeabilized membrane preparations.
- Assessed the functional impact of cysteine modifications by monitoring high-affinity serotonin binding activity and biotinylation.
Main Results:
- Cysteine residues in the NH2 terminus, COOH terminus, and internal loops (IL1-IL5) were accessible to MTS reagents upon membrane permeabilization.
- Modification of specific cysteine residues, including Cys-357 (IL3), Cys-137 (IL1), Cys-277 (IL2), and Cys-441 (IL4), resulted in loss of SERT activity.
- Cysteine accessibility and reactivity were influenced by ion and ligand binding, indicating conformational changes during transport.
Conclusions:
- The results support the originally proposed topology of SERT, with accessible cytoplasmic domains.
- The findings argue against alternative topological models proposed for related GABA and glycine transporters.
- Cytoplasmic domains of SERT undergo conformational changes during neurotransmitter transport, influenced by substrate binding and ion gradients.
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