Regulation of GABA transporter 1 by the presynaptic release and trafficking machinery

Plasma membrane neurotransmitter transporters are critically involved in synaptic transmission through removal of neurotransmitter from the synaptic cleft. The release of neurotransmitter into the synaptic cleft involves numerous protein-protein interactions that allow for the trafficking of neurotransmitter-filled synaptic vesicles. I demonstrate here the importance of the presynaptic release and trafficking machinery for the regulation of the neurotransmitter reuptake protein gamma-aminobutyric acid (GABA) transporter 1 (GAT1). The aim of this dissertation has been threefold. First, reviews of current knowledge on the regulation and trafficking of neurotransmitter transporters are presented. Second, the data presented herein demonstrate a functional and physical interaction of GAT1 with syntaxin IA, a member of the synaptic vesicle release machinery. These interactions result in increased GAT1 surface levels and decreased transport rates shown by (1) GABA uptake studies, (2) glutathione S-transferase fusion protein pull-down assays, (3) cell surface biotinylation experiments, (4) GAT1-specific charge movements in oocytes, and (5) GAT1-specific GABA-induced currents in oocytes. Third, in studies focusing on the trafficking of GAT1, it is shown GAT1 traffics similarly yet distinctly from neurotransmitter-filled synaptic vesicles. The data indicate GAT1 is internalized via clathrin-mediated and dynamin-dependent mechanism, sorts through an endosomal system, resides on a distinct synaptic vesicle, and rapidly recycles on and off the plasma membrane. Several lines of evidence support this trafficking behavior and include (1) hypertonic media-induced increases in GABA uptake and surface expression, (2) increases in GAT1 surface expression with inactive dynamin expression, (3) flotation and fractionation assays, (4) organelle immunoisolations, and (5) temperature-dependent GAT1 recycling rates determined by modified biotinylation experiments. The removal of GABA from the synaptic cleft is primarily achieved through GABA reuptake and binding to GAT1. These studies have focused on how neurons use presynaptic proteins to regulate the activity of this transporter. This dissertation demonstrates the importance of presynaptic protein-protein interactions to regulate GAT1 transport rates and surface expression. GAT1 function and GABAergic signaling are altered in patients with temporal lobe epilepsy, and the work presented herein may help in the design of drugs that target GAT1 to better treat this disease.