ION-BINDING AND PERMEATION AT THE GABA TRANSPORTER GAT1

This study addresses the binding of ions and the permeation of substra tes during function of the GABA transporter GAT1. GAT1 was expressed i n Xenopus oocytes and studied electrophysiologically as well as with [ H-3]GABA flux; GAT1 was also expressed in mammalian cells and studied with [H-3]GABA and [H-3]tiagabine binding. Voltage jumps, Na+ and Cl- concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive i nteractions among these three types of perturbations show that they al l measure the same population of charges. The concentration dependence s of the charge movements reveal (1) that two Na+ ions interact with t he transporter even in the absence of GABA, and (2) that Cl- facilitat es the binding of Na+. Comparison between the charge movements and the transport-associated current shows that this initial Na+-transporter interaction limits the overall transport rate when [GABA] is saturatin g. However, two classes of manipulation-treatment with high-affinity u ptake blockers and the W68L mutation-''lock'' Na+ onto the transporter by slowing or preventing the subsequent events that release the subst rates to the intracellular medium. The Na+ substitutes Li+ and Cs+ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that effi cient removal of synaptic transmitter by the GABA transporter GAT1 dep ends on the previous binding of Na+ and Cl-, and (2) indicate the impo rtant role of the conserved putative transmembrane domain 1 in interac tions with the permeant substrates.