This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The serotonin transporter (SERT) is an integral membrane protein responsible for clearance of the endogenous neurotransmitter 5-HT from the synaptic cleft after neuronal firing. Abnormalities in SERT structure-function and/or changes in SERT receptor densities have been implicated in a variety of significant neurological and mental health disorders. Thus, an enhanced understanding of SERT operation is needed. The exact details of SERT global tertiary structure and intimate three-dimensional (3D) representations of critical protein binding regions linked to the SERT transport phenomena remain ill defined. The SERT structure organization is thought to possess a ligand binding site that is composed by amino acids assembled in 3D space by the SERT tertiary protein structure. It is our hypothesis that the efficacious mapping of the amino acid constituents of the SERT binding domains will reveal critical features of the SERT tertiary structure. Our long-term objective is to significantly contribute to understanding of the commonalities and differences amongst key CNS transport proteins in terms of structure-function. The goal of this project is to map participating amino acid residues within critical SERT binding domains with custom designed, potent and selective, quipazine-based cross-linking probes. The following three specific aims will be accomplished: 1. To construct a 3D computational model of SERT that will enable accurate measurements of inter-residue secondary structure. 2. To synthesize and pharmacologically characterize a collection of mono- and homo-bifunctional probes defined by our computationally derived ligand design criteria. 3. To determine the amino acid cross-linked SERT 3D structure maps, first by the irreversible chemical tagging of the dual-headed cross-linking agents to expressed SERT, followed by mass spectrometry tagged SERT sequence analysis, and then correlating the labeled residue sites with the 3D SERT model.
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