Bridging Project 4: Transport Cycle in Neurotransmitter Uptake Systems

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DAT, NET, and SERT are well established targets for many pharmacological agents that affect brain function [16]. These biogenic amine transporters terminate synapfic transmission by reuptake of the released neurotransmitters from the synaptic cleft back to the presynaptic neuron, coupled to the movement of Na+ down its electrochemical gradient. Drugs that interfere with reuptake profoundly influence behavior and mood. For example, DAT is the primary target for the psychosfimulants cocaine, amphetamine, and methylphenidate [17] whereas inhibitors of SERT are antidepressants (imipramine, fluoxefine) [18]. However, our understanding of the molecular mechanisms whereby these inhibitors exert their effects is sfill at a primifive stage. Our analysis of the LeuT structure [2] has shown intriguingly strong consistency between the structural and funcfional characteristics and our current understanding of the mammalian homologues DAT, SERT, and NET [1]. This is important from a modeling perspective, because computational simulation results are strongly influenced by the quality of the homology models, which in turn depends on the degree of conservafion and similarity between the template and target. Advantages to working with bacterial membrane proteins and bacterial expression systems include easier scale up and increased levels of protein expression, and more limited posttranslational modificafion and thus more homogeneous material as compared to their eukaryotic counterparts. Thus, we propose to use LeuT as an established model system [13] for the proposed studies. To monitor protein conformational changes under native-like conditions, i.e. in proteoliposomes, without the conformational selectivity of crystal lattice forces, we will use the established 8DSL-EPR technique (see [15]). This technique requires site-directed mutafion of native residues to cysteine for the incorporation of a sulfhydryl-specific nitroxide spin label. EPR analysis of the spin labeled proteins yields spectroscopic constraints describing the local environment of a nitroxide probe incorporated at select sites in a protein sequence. These structural constraints are generated from observables such as spin label solvent accessibility, which describes the collisional frequency of the probe with other paramagnetic reagents. Furthermore, dipolar coupling between two spin labels has been shown to be an effective spectroscopic ruler for the determination of global spatial constraints that can provide details of packing interactions and domain movements[19]. Changes in the pattern of these spectroscopic signatures have been shown to correlate with conformafional changes in protein structure [20]. When the labeling sites are selected based on specific hypotheses generated from computafional analyses of dynamics in structurally defined or cognate systems, the approach becomes an incisive tool for determining key functional characteristics in a structure-dynamic context that is interpretable, in turn, in the frame of the computational analysis and simulation and the experimental data for functional properties of the system. The proposed studies aim at a major challenge in structure-function studies of NSS or any transporter family, namely the characterization of the conformational states that constitute the substrate translocation cycle (e.g., see [11]). The absence of crystal structures for multiple conformational states (see above) calls for elucidation of the dynamic nature of transport through the type of combined approach of computational and experimental studies we propose here. In this respect, the integration of the proposed study in this glue grant offers major advantages as it will take advantage of the capabilities and resources in the Cores (see section 4.4, below) and the cognate studies on other membrane protein systems as described throughout this application.