Molecular determinants of ligand interaction with serotonin transporters

David Lance Roman, Purdue University

Abstract

The serotonin transporter (SERT) is a protein responsible for limiting the spatial and temporal actions of the neurotransmitter serotonin (5-HT) by regulating the concentration of 5-HT in the synapse through a reuptake mechanism. SERT is a target for a variety of drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), and the abused drugs cocaine and amphetamine. The two aims of this work are the investigation of how (1) antidepressants and (2) psychostimulants are recognized by SERT. Investigation focused on the recognition of antidepressants by SERT utilized Comparative Molecular Field Analysis (CoMFA) using data generated from rat brain synaptosomes and heterologous expression systems expressing rat SERT. Using these models, I have described the molecular requirements for the interactions of antidepressants with SERTs. In addition, molecular studies were performed using chimeric SERTs and SERT point mutants. These studies focused on identifying regions or discrete amino acids of SERT that may be responsible for recognizing antidepressants. Psychostimulant studies exploited pharmacological differences between the human and Drosophila species variants of SERT (hSERT and dSERT, respectively), to explore the molecular aspects of psychostimulant recognition by SERT. These studies entailed the screening of cocaine derivatives and a substituted-amphetamine library, followed by CoMFA analysis. These experiments revealed that a hSERT/dSERT chimera, H1–281D282–476 H477–638 exhibited pharmacology almost identical to the parental dSERT, indicating that transmembrane domains (TMDs) V–IX in SERT contained important determinants of psychostimulant recognition. Mutagenesis studies were focused on those residues that differ between BERT and BERT amino acid identity in TMDs V–IX. Two point mutants, F287K and Y289T, both localized to TMD V, were found to have altered recognition of some cocaine derivatives. A full cocaine derivative library screen was performed on these two point mutants, and CoMFA analysis was performed. The results of the CoMFA analysis indicate that F287K and Y289T may play a role in the recognition of cocaine derivatives. Furthermore, data from previous reports, as well as the data in this work, were used to generate models of the structural helical arrangement of SERT, as well as model of a proposed interaction site of cocaine derivatives on SERT. Grant support: NIH MH60221.

Degree

Ph.D.

Advisors

Barker, Purdue University.

Subject Area

Pharmacology|Neurology

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