Recent data suggest that rare variants in the gene encoding DAT (DAT1) can play a hitherto unknown, but important role in the pathophysiology underlying both neuropsychiatric and neurodegenerative disorders. Despite that a number of mutations have been functionally characterized, it is still poorly understood how molecular perturbations in DAT are linked to distinct alterations in dopamine homeostasis in the brain of the patients. Interestingly, data suggest that the pathophysiological outcome might not only be a result of compromised DAT function but also involve “gain of disruptive functions” such as constitutive reverse transport of dopamine, enhanced ion permeability and ion channel-like functions. The overall goal of this project is to map specific structural perturbations, elicited by disease-associated DAT mutations found in a large Danish cohort of 13,000 psychiatric patients, and link these perturbations to functional molecular phenotypes that in turn will be responsible for distinct alterations in dopamine homeostasis.
The specific objectives of Project 1 are:
- to assess the basic function of the mutants in high-throughput uptake and efflux assays as well as radioligand binding assays upon expression of mutants in heterologous cells
- to determine cellular distribution using live imaging and fluorescently tagged cocaine analogues
- to screen mutants for ion-channel-like properties using fluorescent voltage sensors and investigate the precise electrophysiological properties of selected mutants using two-electrode voltage clamp in Xenopus oocytes
- Make purified preparations of selected mutants (using the purifiable drosophila DAT as model system) and employ HDX-MS to assess mutation-elicited global conformational changes and FRET based techniques to map discrete intramolecular structural rearrangements
- Analyze data in context of molecular dynamics simulations and computational algorithms in order to build predictive models of how distinct types of molecular perturbations can lead to converging functional alterations.