Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy

Area of Science:

• Neuroscience
• Biophysics
• Pharmacology

Background:

• Conflicting data exists regarding ligand binding sites in neurotransmitter/sodium symporters (NSS).
• Understanding these sites is crucial for drug development and transporter function.
• Previous studies relied on indirect methods, lacking direct physical evidence.

Purpose of the Study:

• To investigate the number and functionality of ligand binding sites in NSS using a physiological approach.
• To directly measure single-molecule interactions between the serotonin transporter (SERT) and S-citalopram (S-CIT).
• To provide physical evidence for distinct binding sites and their allosteric coupling.

Main Methods:

• Development of novel atomic force microscopy (AFM) tools for single-molecule force measurements.
• Functionalization of AFM cantilever tips with S-citalopram (S-CIT) via a polyethylene glycol (PEG) linker.
• Validation of tip chemistry and measurement of S-CIT binding forces to SERT in different ionic conditions and a mutant transporter.

Main Results:

• Two distinct populations of S-CIT binding strengths to SERT were identified in Na+-containing buffer.
• SERT exhibited only low-force interactions in Li+-containing buffer, and a mutant SERT (SERT-G402H) showed only the high-force population.
• Dissociation rate constants were extracted, and competition experiments demonstrated allosteric coupling between the two binding sites.

Conclusions:

• Direct physical evidence supports the existence of two functionally distinct, allosterically coupled binding sites in SERT.
• AFM-based single-molecule force spectroscopy is a powerful tool for elucidating transporter-ligand interactions.
• Findings offer new insights into the mechanism of antidepressant action and transporter regulation.