Insights from molecular dynamics: the binding site of cocaine in the dopamine transporter and permeation pathways of substrates in the leucine and dopamine transporters.

The dopamine transporter (DAT) facilitates the regulation of synaptic neurotransmitter levels. As a target for therapeutic and illicit psycho-stimulant drugs like antidepressants and cocaine, DAT has been studied intensively. Despite a wealth of mutational and physiological data regarding DAT, the structure remains unsolved and details of the transport mechanism, binding sites and conformational changes remain debated. A bacterial homolog of DAT, the leucine transporter (LeuT(Aa)) has been used as a template and framework for modeling and understanding DAT. Free energy profiles obtained from Multi-Configuration Thermodynamic Integration simulations allowed us to correctly identify the primary and secondary binding pockets of LeuT(Aa). A comparison of free energy profiles for dopamine and cocaine in DAT suggests that the binding site of cocaine is located in a secondary pocket, not the primary substrate site. Two recurring primary pathways for intracellular substrate release from the primary pocket are identified in both transporters using the Random Acceleration Molecular Dynamics method. One pathway appears to follow transmembranes (TMs) 1a and 6b while the other pathway follows along TMs 6b and 8. Interestingly, we observe that a single sodium ion is co-transported with leucine during both simulation types.