Ion Binding and Permeation at the GABA Transporter GAT1

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Abstract
This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed inXenopusoocytes and studied electrophysiologically as well as with [3H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3H]GABA and [3H]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 interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na+ions interact with the transporter even in the absence of GABA, and (2) that Cl−facilitates 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 saturating. However, two classes of manipulation—treatment with high-affinity uptake blockers and the W68L mutation—“lock” Na+onto the transporter by slowing or preventing the subsequent events that release the substrates 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 efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na+and Cl−, and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.