No crystal or cyro-EM structures of a GABA transporter are available. Initially in this project, models of GABA transporters will be developed using the structures of the closely related serotonin transporters. Models, first evaluated using structure quality assessment methods, will be tested against modern force fields, which were shown to be able to detect also small structural deficiencies. Simulations will be carried out in close collaboration with Projects 1, 5, 6, 7, 8 and 11, which are experimentally studying the same transporters. Project 4 will make extensive use of advanced simulation methods on HPC clusters to investigate the transport cycle of GABA transporters with the goal to obtain a comprehensive understanding of GABA transporter function. Within the NeuroTrans consortium, simulations will serve as a hub for data integration for deriving a holistic model of GABA transporter function. The main objectives of Project 4 are:
- to collaborate with Projects 1, 5, 6, 7, 8 and 11 through model creation and computational validation of preliminary data and to evaluate hypothesis derived from experimental data. Interaction will be especially intense with Project 1 for evaluation of preliminary X-Ray and cryo-EM structures.
- to directly study ion and substrate binding under varying transmembrane voltage conditions using the computational electrophysiology approach and compare to experimental data of Project 5 and 6. In addition, a direct identification of the ion binding sites should be possible.
- to use a set of advanced sampling methods to simulate the transport cycle, thereby investigating the steps of substrate and ion binding, substrate occlusion, transition to the inward facing state and cargo release. Data validation will be carried out by comparison to experimental data obtained in Projects 1, 5, 6 and 11.
- to quantify the free energy profile of substrate transport to identify the interactions triggering transport, using at least 3 different approaches: Metadynamics, Accelerated Weight Histogram method and Potential of Mean Force calculations.