Abstract
The GABA transporter GAT-1 mediates electrogenic transport of its substrate together with sodium and chloride. It is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Compared with all other neurotransmitter:sodium:symporters, GAT-1 and other members of the GABA transporter subfamily all contain an extra amino acid residue at or near a conserved glycine in transmembrane segment 10. Therefore, we studied the functional impact of deletion and replacement mutants of Gly-457 and its two adjacent residues in GAT-1. The glycine replacement mutants were devoid of transport activity, but remarkably the deletion mutant was active, as were mutants obtained by deleting positions on either side of Gly-457. However, the inward rectification of GABA-induced transport currents by all three deletion mutants was diminished, and the charge-to-flux ratio was increased by more than 2.5-fold, both of which indicate substantial uncoupled transport. These observations suggest that the deletions render the transporters less tightly packed. Consistent with this interpretation, the inactive G457A mutant was partially rescued by removing the adjacent serine residue. Moreover, the activity of several gating mutants was also partially rescued upon deletion of Gly-457. Structural modeling showed that the stretch surrounding Gly-457 is likely to form a π-helix. Our data indicate that the "extra" residue in transmembrane domain 10 of the GABA transporter GAT-1 provides extra bulk, probably in the form of a π-helix, which is required for stringent gating and tight coupling of ion and substrate fluxes in the GABA transporter family.
Highlights
The GABA transporter GABA transporter-1 (GAT-1) mediates electrogenic transport of its substrate together with sodium and chloride
Because the structural data on Mhp1, which belongs to a different family and yet shares the LeuT-fold, indicated that a glycine residue in TM10 could serve as a hinge involved in the occlusion of the substrate [16], we began our study with the idea that Gly-457 of GAT-1 would fulfill a similar role
It is not clear if Gly-457 is the “extra” residue, because the single residue insertion in TM10 could be aligned to either Gly-457 (60.0% of the sequences) or Ser-456 (39.7% of the sequences)
Summary
The GABA transporter GAT-1 mediates electrogenic transport of its substrate together with sodium and chloride. Our results indicate that the extra residue creates a -helix in TM10, which is required for stringent gating and tight coupling of ion and substrate fluxes in GABA transport. Radioactive Transport of Deletion and Replacement Mutants— Mutation of Gly-457 to various residues resulted in defective [3H]GABA transport as monitored in the HeLa cell expression system (Fig. 2A).
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