Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter

Abstract

c-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.

Analysis of Different Binding Modes for Tiagabine within the GAT-1 Transporter

“…Firstly, as mentioned in the introduction, kinetics studies clearly indicate that tiagabine as well as its analogues exhibit a competitive/mixed type of inhibition, and its binding is dependent on sodium ions [ 32 , 33 , 34 , 37 , 38 ]. Ibogaine and bitopertin analogue, which were reported previously to bind in an inward-open state of SERT and GlyT-1, respectively, are fully non-competitive inhibitors [ 9 , 11 , 20 , 39 ].…”

“…While the nipecotic acid fragment of tiagabine was located within the S1 site, the aromatic fragments of the inhibitor were arranged within the intracellular release pathway, similarly to those from the bitopertin analogue in GlyT-1. To bind in this way, tiagabine needs to diffuse across the cell membrane and reach the binding site from the intracellular side, or to bind from the extracellular side and then squeeze through the middle of the transporter, as suggested by Motiwala et al Interestingly, in parallel with obtaining the cryo-electron microscopy structure of hGAT-1, Joseph et al released the crystal structure of DAT with modifications at the S1 site mimicking hGAT-1 (DAT GAT ), in complex with tiagabine analogues NO711 and SKF89976a (PDB codes: 7WGT, 7WLW) [ 34 ]. In the crystal structure of DAT GAT , compounds NO711 and SKF89976a are located entirely at the S1 site, blocking the transporter in an outward-open state.…”

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain

“…Initially, structures of bacterial homologues of the human SLC6 family, like LeuT in Aquifex aeolicus or MhsT transporter in Bacillus halodurans were resolved by crystallography followed by the first structures of transporter from eukaryotic organism: the biogenic amine transporter, the Drosophila melanogaster dopamine transporter (dDAT), and the human serotonin transporter (hSERT) ( Yamashita et al, 2005 ; Penmatsa et al, 2013 ; Coleman et al, 2016 ). More recently, using cryo-electron microscopy (cryo-EM), a number of mammalian transporters from the SLC6 family have been determined, including the substrate-bound SERT, the glycine transporter 1 GlyT1, the neutral amino acid transporter B 0 AT1 and the GABA transporter 1 GAT1 ( Coleman et al, 2019 ; Shahsavar et al, 2021 ; Joseph et al, 2022 ; Motiwala et al, 2022 ). While the structure of BGT-1 is still not known, we can obtain insights from these existing structural models, especially GAT1 given that they belong to the same sub-family.…”

“…We focus on the binding sites for Na + , Cl − , and substrates, and highlight specific molecular determinants of transporter functions and consequences for GABA homeostasis. We discuss the transport coupling mechanism, computational structural modelling, the recent cryo-EM structure ( Motiwala et al, 2022 ; Kanner and Dayan-Alon, 2023 ), and define the structure of the binding site for GAT1 ( Joseph et al, 2022 ). We also summarize information on GAT1 and BGT-1 specific inhibitors as well as the pharmacophore hypothesis of transporter substrates.…”

Clustering Heterogeneous Conformational Ensembles of Intrinsically Disordered Proteins with t-Distributed Stochastic Neighbor Embedding

“…In general, knowledge of the 3-dimensional structure of a protein is the first step toward a molecular-level mechanistic understanding of its biological function. This knowledge is also central to activities such as the rational design of drugs, inhibitors, and vaccines and in the broad area of protein engineering and biomolecular recognition. − With the advances made in structure determination techniques − and recent transformative leaps made in computationally predicting the structure from sequence, − the science of structural biology is going through paradigmatic changes where the knowledge of structure is not the biggest bottleneck anymore . However, outside the realm of these structured proteins exists a “dark” proteome of intrinsically disordered proteins (IDPs) that constitute more than 40% of all known proteins and play important roles in cellular physiology and diseases. − An IDP can populate a heterogeneous ensemble of conformations and is functional without taking on a unique structure.…”