Abstract
The GABA transporter GAT-1 belongs to the neurotransmitter:sodium:symporters which are crucial for synaptic transmission. GAT-1 mediates electrogenic transport of GABA together with sodium and chloride. Structure-function studies indicate that the bacterial homologue LeuT, which possess extra- and intracellular thin gates, is an excellent model for this class of neurotransmitter transporters. We recently showed that a conserved aspartate residue of GAT-1, Asp-451, whose LeuT equivalent participates in its thin extracellular gate, is functionally irreplaceable in GAT-1. Only the D451E mutant exhibited residual transport activity but with an elevated apparent sodium affinity as a consequence of an increased proportion of outward-facing transporters. Because during transport the opening and closing of external and internal gates should be tightly coupled, we have addressed the question of whether mutations of the intracellular thin gate residues Arg-44 and Asp-410 can compensate for the effects of their extracellular counterparts. Mutation of Asp-410 to glutamate resulted in impaired transport activity and a reduced apparent affinity for sodium. However, the transport activity of the double mutant D410E/D451E was increased by approximately 10-fold of that of each of the single mutants. Similar compensatory effects were also seen when other combinations of intra- and extracellular thin gate mutants were analyzed. Moreover, the introduction of D410E into the D451E background resulted in lower apparent sodium affinity than that of D451E alone. Our results indicate that a functional interaction of the external and internal gates of GAT-1 is essential for transport.
Highlights
Mutations in “thin” extra- and intracellular gates of GABA transporter-1 (GAT-1) impair gating and transport
The extracellular gate mutation D451E resulted in a transport activity of 1.4 Ϯ 0.3% of that of WT-GAT-1, in agreement with previously published results [6] (Fig. 2)
Whereas the mutation R44K led to ϳ70% decrease in transport activity, the double mutant R44K/D451E had a transport activity of 2.61 Ϯ 0.3% of WT-GAT-1 (Fig. 2), which is somewhat higher than that by D451E
Summary
Mutations in “thin” extra- and intracellular gates of GAT-1 impair gating and transport. We have recently shown that perturbation of this thin gate of GAT-1 by mutation increases the proportion of outwardfacing transporters [6] Another conserved charge pair, Arg-44 and Asp-410 in GAT-1, is located near the cytoplasmic surface and probably acts as a thin intracellular gate [7] (Fig. 1B). Arg-44 and Asp-410 in GAT-1, is located near the cytoplasmic surface and probably acts as a thin intracellular gate [7] (Fig. 1B) Mutation of these residues in the dopamine transporter DAT, as well as of residues interacting with this charge pair, resulted in an increase of the proportion of inward-facing transporters [18]. Our results indicate that the functional interaction of the external and internal gates of GAT-1 is essential for transport
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
