Exploration of ligand recognition sites on the serotonin transporter using molecular modeling

Crystal Carla Walline, Purdue University

Abstract

The serotonin transporter (SERT) is a member of the neurotransmitter sodium-symporter family composed of the monoamine, amino acid, and other transporter subfamilies. SERT regulates the temporal and spatial action of serotonin (5-HT) on pre- and post-synaptic receptors by transporting 5-HT into the presynaptic neuron. SERT is the molecular target of several drugs of abuse, including cocaine and amphetamine, as well as the selective serotonin reuptake inhibitors and the tricyclic antidepressants. Until recently, no tertiary structural information existed to detail the arrangement of the twelve transmembrane helices (TMHs). Therefore, the purpose of this study was to (1) use species-scanning mutagenesis to determine the role of TMH III in antagonist recognition and substrate translocation, and (2) to use a recently developed homology model and existing mutagenesis data to determine an antagonist binding site. Investigation focused on the recognition of antidepressants, cocaine analogs, and amphetamine analogs by SERT by utilizing Comparative Molecular Field Analysis (CoMFA) selectivity fields using data generated from heterologous expression systems expressing human SERT. Using a novel structure-activity-relationship analysis, CoMFA selectivity fields were generated and I determined that residue I172 is likely to be a direct contact site for the cocaine analogs. This analysis is a useful tool to distinguish between general conformational changes and potential direct protein-ligand interactions. Additionally, residue A169 was determined to be near the sodium binding site due to alterations in sodium requirements. This result was verified by the human SERT homology model constructed from the leucine transporter LeuTAa from Aquifex aeolicus. Additionally, I used the human SERT homology model and existing data as a rational approach for determining an antagonist binding site using transport inhibition assays. The results indicate that the putative antagonist binding site located at the junction of extracellular loops 2, 4, and 5, and TMHs X, and XII is not likely to be a binding site for cocaine. However, this site may represent a contact site for some antidepressants as there was a loss of potency for several antidepressants at some of the mutants constructed. Additionally, these data corroborate the results from two recent antidepressant-LeuTAa co-crystal structures which suggest that some of the residues involved in my putative antagonist binding site compose a tricyclic antidepressant binding site. Grant support: NIH grant R01DA018682

Degree

Ph.D.

Advisors

Barker, Purdue University.

Subject Area

Molecular biology|Neurosciences

COinS