# The Role of Cholesterol at the CHOL2 Site and the Dimeric Interface in the Regulation of Serotonin Transporter Function and Dimerization

## TL;DR

This study explores how cholesterol affects the serotonin transporter's function and dimerization, revealing its role in regulating transporter activity and conformation.

## Contribution

The study identifies specific cholesterol sites in SERT and their functional impact on transporter activity and dimerization.

## Key findings

- Mutations at the CHOL2 site reduced transport activity and stabilized the inward-facing conformation of SERT.
- Cholesterol at the dimeric interface mediates protomer interactions and affects transport activity.
- Residues at the interfacial site are conformationally sensitive to cholesterol presence.

## Abstract

Serotonin transporter (SERT) precisely regulates serotonin (5–HT) signaling in the central nervous system and is a major target of antidepressants for the treatment of major depressive disorder. Despite significant progress in characterizing its structure and transport mechanism, the regulation of SERT function by various modulators remains to be fully understood. In the present study, we focused on two potential cholesterol sites in human SERT to investigate cholesterol occupation at these sites and its functional relevance by biochemical approaches. Mutations of an intramolecular site (CHOL2) significantly decreased both specific transport activity and Km for 5–HT and stabilized the transporter in an inward-facing conformation. In addition, our NanoBiT luminescent assay for protein–protein proximity demonstrated that cholesterol mediated the protomer–protomer interactions by residing in a site at the dimeric interface. Mutations of the interfacial site remarkably reduced the interactions between SERT protomers and substantially impaired their transport activity. The structural analysis indicated that the residues participating in cholesterol residing in the interfacial site were conformationally sensitive. Thus, we have proposed that cholesterol at these sites could play a vital role in the regulation of SERT function by a conformational mechanism. Our study has provided new insights into the molecular mechanism by which cholesterol can regulate SERT function and dimerization.