A Glutamine Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Interaction of Chloride with the GABA Transporter GAT-1

Abstract

Neurotransmitter:sodium symporters are crucial for efficient synaptic transmission. The transporter GAT-1 mediates electrogenic cotransport of GABA, sodium, and chloride. The presence of chloride enables the transporter to couple the transport of the neurotransmitter to multiple sodium ions, thereby enabling its accumulation against steep concentration gradients. Here we study the functional impact of mutations of the putative chloride-binding residues on transport by GAT-1, with the emphasis on a conserved glutamine residue. In contrast to another putative chloride coordinating residue, Ser-331, where mutation to glutamate led to chloride-independent GABA transport, the Q291E mutant was devoid of any transport activity, despite substantial expression at the plasma membrane. Low but significant transport activity was observed with substitution mutants with small side chains such as Q291S/A/G. Remarkably, when these mutations were combined with the S331E mutation, transport was increased significantly, even though the activity of the S331E single mutant was only ϳ25% of that of wild type GAT-1. Transport by these double mutants was largely chloride-independent. Like mutants of other putative chloride coordinating residues, the apparent affinity of the active Gln-291 single mutants for chloride was markedly reduced along with a change their anion selectivity. In addition to the interaction of the transporter with chloride, Gln-291 is also required at an additional step during transport. Electrophysiological analysis of the Q291N and Q291S mutants, expressed in Xenopus laevis oocytes, is consistent with the idea that this additional step is associated with the gating of the transporter.

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

“…3, f and g). It is also noteworthy that the recent discovery (27)(28)(29) of residues involved in Cl Ϫ binding in GAT-1 and SERT suggests that residues Tyr-233 (TM2), Gln-473, and Ser-477 (TM6) together with Asn-509 and Ser-513 (TM7) are likely to perform equivalent roles in GlyT2 (Fig. 4a).…”

“…4b), which lacks the hydroxyl necessary for the coordination of the Cl Ϫ ion and may prevent Cl Ϫ binding. Because a hydroxyl at this relative position is highly conserved in SLC6 transporters (27)(28)(29), the S513I substitution is predicted to interfere with glycine transport by disrupting the both Cl Ϫ (directly) and Na ϩ (indirectly) interactions.…”

“…For modeling the GlyT2 chloride ion-binding site, Cl Ϫ was placed into our model at the coordinates of the carboxylate carbon of the LeuT Glu-290 side chain (27), based on the assumption that this position may be occupied by Cl Ϫ in homologous transporters (27)(28)(29). The residues involved in the coordination of Cl Ϫ ion are generally conserved within the SLC6 transporter family.…”

Functional Defects in the External and Internal Thin Gates of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Can Compensate Each Other

“…Cell Surface Biotinylation-Labeling of wild type and mutant transporters at the cell surface, using Sulfo-NHS-SS-Biotin (Pierce), quenching the reaction, cell lysis, and isolation of the biotinylated proteins by streptavidin-agarose beads (Pierce) were done as described (17). After SDS-PAGE (10% gel) and transfer to nitrocellulose, the GAT-1 protein was detected with an affinity-purified antibody, directed against an epitope from the cytoplasmic C-terminal tail of GAT-1, at a 1:500 dilution, with horseradish peroxidase-conjugated secondary antibody at a 1:40,000 dilution, and with ECL.…”

“…eral of the homologous residues are critical for transport in GAT-1 (12)(13)(14)(15)(16)(17), it is likely that the formation of the thin gate is an obligate intermediate during substrate translocation: the transition from the outward-to the inward-facing conformation. We have recently shown that perturbation of this thin gate of GAT-1 by mutation increases the proportion of outwardfacing transporters (6).…”