Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter

Abstract

While γ‐aminobutyric acid (GABA) neurotransmitter release is vital to inhibitory synapse function and GABA transporters (GATs) are targeted by antiepileptic drugs, the mechanistic basis for their unique pharmacology remains elusive. Combining structural and biochemical analysis of engineered transporters, this study uncovers distinct features of the GAT binding pocket that dictate inhibitor interaction specificities. Substitution of 11 binding site residues results in a chimeric Drosophila melanogaster dopamine transporter with rat GAT1‐like inhibitor binding site (dDAT GAT ). Substrate‐free dDAT GAT displays strongly altered binding site architecture with prominent subsites A and C′. GABA uptake inhibitors NO711 and SKF89976a bind dDAT GAT within the primary binding site in the subsites A and C′. SKF89976a shows additional binding in the vestibule of dDAT displaying both competitive and noncompetitive inhibition. Modification of SKF89976a binding residues in the dDAT GAT extracellular loop 4 affects neurotransmitter uptake activity.