Exploring the entrance to the human serotonin transporter permeation pathway

Melissa I Torres Altoro, Purdue University

Abstract

The serotonin transporter (SERT) is a member of the Na+/Cl --dependent family of transporters that share a common topology of 12-transmembrane α-helices, intracellular N- and C-termini, and a large extracellular loop between transmembrane helix (TMHs) III and IV with putative glycosylation sites. SERT regulates the concentration of serotonin (5-hydroxytryptamine, 5-HT) at the synaptic cleft through a reuptake mechanism. These transporters are highly modulated by therapeutic compounds such as the tricyclic antidepressants and selective serotonin reuptake inhibitors as well as many abused drugs as cocaine and amphetamines. Extensive efforts have been made to elucidate the preferred conformation of these transporters at the plasma membrane. However, no high-resolution crystal structure for members of the eukaryotic neurotransmitter transporters exists, thus impairing the study of these valuable drug targets. Conversely, a groundbreaking discovery came when the crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters was determined, providing a pathway to explore structural features of transporter function. The purpose of this study was to (1) use a homology model of SERT based on the Aquifex aeolicus LeuTAa structure to identify the residues that reside at the entrance to the permeation pathway; and (2) define the proximity relationships between the helices forming the protein pore. The model predicts that the extracellular entrance to the substrate permeation pathway is formed by TMHs I, III, VI, X, and XI as well as extracellular loop 4 (EL4). Residues predicted to be at the entrance to the pore were subjected to the substituted cysteine accessibility method (SCAM) to explore their role in SERT function. Analysis of a novel SCAM reagent derived from the transporter substrate MPP+ identified residues at the extracellular surface that became accessible as a result of functional changes associated with substrate binding. These positions were characterized by protection studies where SERT substrates and antagonists were used to protect against MTS sensitivity. Particularly, TMH VI contains important sites for transporter-ligand interaction that may be conformationally sensitive to substrate transport. The model provides the opportunity to predict the distances between residues, thus I defined the proximity relationships between residues by cross-linking studies using bifunctional MTS reagents. The data suggest that TMHs I-VI and VI-XI are in close proximity to each other. The results are consistent with our SERT model, providing a valuable tool to expand SERT characterization and develop more refined models of SERT or other neurotransmitter transporters. This research was supported by NIH grant R01DA018682.

Degree

Ph.D.

Advisors

Barker, Purdue University.

Subject Area

Molecular biology|Neurosciences|Pharmacology

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