Exploration into the substrate permeation pathway of the serotonin transporter

Kellie Jo Novotny White, Purdue University

Abstract

The serotonin transporter (SERT) is the protein responsible for terminating serotonin (5-HT) neurotransmission by clearing 5-HT in the synapse through a reuptake mechanism. Selective serotonin reuptake inhibitors and tricyclic antidepressants target SERT for therapeutic benefits; conversely, the psychostimulants cocaine and amphetamines also elicit effects at SERT. Although information exists suggesting specific residues and areas of the protein important for ligand recognition, presently, little data exists detailing the structural arrangement of the twelve putative transmembrane helices (TMHs) within the membrane. The purpose of this study was to (1) explore substrate differences at Drosophila versus human SERT (dSERT and hSERT, respectively) and (2) delineate specific transmembrane helices of hSERT present in the permeation pathway. Analysis of numerous amphetamine analogs and their ability to generate currents at dSERT and hSERT in oocytes were compared to efflux data to study ligand recognition differences between the species variants. Although all the amphetamine analogs analyzed were substrates for hSERT as determined by their ability to generate current to an extent similar to 5-HT, only a portion of the analogs were substrates for dSERT. Analysis of the active versus inactive amphetamine analogs showed minor structural differences in the amphetamine's structure affected the ability of the amphetamines to induce current at dSERT. Additionally, electrophysiological studies were utilized to confirm the role of TMH XI residue hSERT/F556 in the permeation pathway of both 5-HT and the neurotoxin, MPP+. Interestingly, hSERT/F556 exhibits features of both hSERT and dSERT as 5-HT-induced currents were more related to dSERT, but MPP+-induced currents resembled hSERT. Thus, the 556 position could be important for gating 5-HT-induced currents. Further exploration into TMHs that form the permeation pathway confirmed that TMHs I and III are located in proximity to one another. I engineered potential Zn2+-binding sites between TMHs I and III, and identified that the TMH I mutation V 102C/hSERT could form a Zn2+-binding site with either 1179C/hSERT or MI80C/hSERT in TMH III when mutated simultaneously suggesting the orientation of the TMHs with respect to each other. This Zn 2+ inhibition of 5-HT uptake was found to be dose-dependent with mid-micromolar IC50 values. Grant support: NIH MH60221 and NIH Training in Biochemistry and Molecular Biology 5 T32 GM008737-05.

Degree

Ph.D.

Advisors

Barker, Purdue University.

Subject Area

Molecular biology|Neurosciences|Pharmacology

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