Modeling and Simulation of hGAT1: A Mechanistic Investigation of the GABA Transport Process

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.

Assessment of Paroxetine Molecular Interactions with Selected Monoamine and γ-Aminobutyric Acid Transporters

“…The homology for each human transporter related to the others was sequence identified is approximately hDAT/hNET = 67%, hDAT/hSERT = 50%, hNET/hSERT = 53%. The evidence from these studies presents the lower sequence similarity for hNET/hDAT = 58% [34] compared with hGAT1/dDAT = 66% [35] of identity. All these values are quite similar to the sequence of proteins in all the transporters analyzed.…”

“…hBGT1 is only found in scarce amounts and hGAT2 is not found in the brain parenchyma at all [82]. hGAT1 is predominantly located on GABAergic nerve terminals [35,59,83], while hGAT3 and hBGT1 are commonly associated with perisynaptic and distal astrocytic sites [28,60]. The location and level of individual transporters in CNS seem to be crucial in the neurogenesis process.…”

“…Nevertheless, the analysis of structural similarities calculated as RMS determined by superimposing the structures of two proteins seems to be of key importance in understanding the interaction of various compounds with the analyzed objectives [37]. These results, in contrast to the data routinely presented in the literature based on the amino acid sequence identity technique [29,[32][33][34][35], better highlight the structural differences of the analyzed transporters. These differences are of particular importance within the ligand binding site.…”

Molecular Dynamic Simulations to Probe Stereoselectivity of Tiagabine Binding with Human GAT1

“…An in-house preprocessed hGAT1 model built in open-to-out conformation [ 35 ] was used to dock both R - and S -configured tiagabine. Briefly, homology modeling of hGAT1 (UniProt: P30531) in open-to-out conformation was based on the X-ray crystallographic structure of the Drosophila melanogaster dopamine transporter (dDAT) (Protein Data Bank ID: 4XP4) with the bound cocaine substrate and the co-transport di-sodium/chloride (2Na + /1Cl − ) ions within its structurally conserved ion/substrate binding sites.…”

“…Various reports highlighted the crucial role of Y140 in the GABA-induced translocation through hGAT1 by mediating hydrogen bond interaction with the –COOH group of GABA [ 35 , 42 ]. Therefore, constraint docking on Y140 residue has been performed previously to delineate the molecular basis of ligand–transporter interactions of hGAT1 antagonists [ 25 , 27 ].…”