This Project of the PPG is devoted to the structural context of the mechanistic study of Na+/Cl- dependent biogenic amine transporters (NT) function. We will investigate - for DAT, SERT, and NET - the role of specific structural elements in the physiological processes underlying neurotransmission and mechanisms in drug addiction and abuse, with generalizations and comparisons to other NTs. The proposed studies use 3D modeling and computational analysis of dynamics and energetics to achieve, in close collaboration with the other PPG projects, a quantitative evaluation of the structural and energetic basis for: (i) the role of specific sets of both intra- and inter-molecular interactions of N-termini, C-termini and key loops of NTs in experimentally measured properties that underlie function, and the changes that are produced by phosphorylation (SA #1a), and (ii) the detailed mode of interaction and specificity for interactions with PDZ domains considered to be involved in internalization, recycling, sorting, stabilization of NT, and in regulation of NT function. We will investigate steric and dynamic properties of NT C-terminal interaction with PDZ domains (starting with DAT-PICK1 interactions), and the molecular determinants for regulation of interactions selectivity by phosphorylation (of either the peptide or the PDZ) (SA #1b). The considerable amounts and variety of data prompt us to build two modes of empowering representation: a) 3D models based on structure-informed working hypotheses, refined bioinformatics approaches and computational molecular biophysics algorithms, constraints from functional insights obtained in the PPG and the literature, and newly published structural information from x-ray and EM studies of transporter and membrane protein systems that can be used as guides for structural principles in 3D modeling and as probes for validation of approaches (SA #2), and b) a transporter-centric information management system (TIMS) to serve as an electronic catalog of quantitative data about NT interactions (with ligands: substrates, blockers and modulators, and cell components such as scaffolding proteins), as well as NT activities (e.g., substrate transport rates under various conditions and NT states) and other quantitative data obtained in the PPG Projects (SA #3).
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