Structural, mechanistic and pharmacological studies of inhibitory neurotransmitter transporters

Abstract

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). The synaptic levels of GABA are controlled by the activity of GABA transporters (GATs) present in the presynaptic and glial membranes. Impaired GABAergic signaling leads to hypersynchronous excitatory discharges that can result in epileptic conditions. Other than the roles in CNS, GABA plays an important role in hormone homeostasis, regulation of muscle tone, neural development and inflammation. GAT1, GAT2 and GAT3 are the most studied isoforms of GATs and 75% of the GABA reuptake is performed by GAT1 which is localized in the neurons. The antiepileptic drug tiagabine, NO-711 and SKF89976a are GAT1 specific inhibitors. GAT1 is a symporter that transport one molecule of GABA along with two sodium ions and one chloride ion resulting in the electrogenic movement of GABA across the membrane. The absence of a high-resolution structure of GAT1 limits the understanding of its pharmacology and the basis of GAT1 specific inhibition. The Drosophila melanogaster dopamine transporter (dDAT) can be used as a suitable model to study inhibitor interactions among the NSS members due to the availability of thermostabilizing mutants and an antibody chaperone for rapid crystallization. I have therefore used the dDAT, as a template to engineer the binding pocket of GAT1. Residues in the primary binding pocket were analyzed and identified residues which are divergent in GAT1. Ten residues in the primary binding pocket and one residue in the extracellular vestibule were mutated to GAT1 equivalent residues. The engineered dDAT termed as DATGAT showed interactions with GAT1 specific inhibitors including NO-711 and SKF89976a although it lacks GABA transport activity. The high-resolution crystal structures of DATGAT in complex with NO-711 and SKF89976a displayed an altered subsite architecture in the primary binding pocket revealing discrepancies in inhibitor interactions among GATs and biogenic amine transporters. DATGAT structures displayed that the L325 (L300 in rGAT1) side chain of subsite C’ undergoes a leftward shift in order to accommodate the aromatic moiety of GAT1 inhibitors. Interestingly, we observed an additional density for SKF89976a in the vestibule alongside that of the SKF89976a bound in the primary binding site. SKF89976a in the alternate site (allosteric site) forces the inward movement of the gating residue, F319, and resulted in the occlusion of the primary binding pocket of the transporter in DATGAT-SKF89976a bound structure. The residue S384 of DATGAT (S359 in rGAT1) interacts with the carboxylate group of SKF89976a via a water molecule in the vicinity. We also deciphered a crucial role of the extracellular loop 4 in influencing substrate gating in NSSs. The structural findings obtained from DATGAT structures were further validated using thorough biochemical analysis performed in a mammalian orthologue of GAT1.