Abstract
Human γ-Aminobutyric acid transporter 1 (hGAT1) is a Na(+)/Cl(-) dependent co-transporter that plays a key role in the inhibitory neurotransmission of GABA in the brain. Due to the lack of structural data, the exact co-transport mechanism of GABA reuptake by hGAT1 remains unclear. To examine the roles of the co-transport ions and the nature of their interactions with GABA, homology modeling and molecular dynamics simulations of the hGAT1 in the open-to-out conformation were carried out. Our study focused on the sequential preloading of Na(+) and Cl(-) ions, followed by GABA binding. Our simulations showed pre-loading of ions maintains the transport ready state of hGAT1 in the open-to-out conformation essential for GABA binding. Of the four putative preloaded states, GABA binding to the fully loaded state is most favored. Binding of Na(+) ion to the Na1 site helps to maintain the open-to-out conformation for GABA binding as compared to the Na2 site. GABA binding to the mono-sodium or the di-sodium loaded states leads to destabilization of Na(+) ions within their binding sites. The two most prominent interactions required for GABA binding include interaction between carboxylate group of GABA with the bound Na(+) ion in Na1 binding site and the hydroxyl group of Y140. Overall our results support the fully loaded state as the predominate state for GABA binding. Our study further illustrates that Na(+) ion within the Na1 site is crucial for GABA recognition. Therefore, a revised mechanism is proposed for the initial step of hGAT1 translocation cycle.