Mutation of arginine 44 of GAT-1, a (Na++Cl-)-coupled gamma-aminobutyric acid transporter from rat brain, impairs net flux but not exchange

The gamma -aminobutyric acid (GABA) transporter GAT-1 is a prototype of a l arge family of neurotransmitter transporters that includes those of dopamin e and serotonin, GAT-1 maintains low synaptic concentrations of neurotransm itter by coupling GABA uptake to the fluxes of sodium and chloride. Here we identify a stretch of four amino acid residues predicted to lie in the jux tamembrane region prior to transmembrane domain 1 in the cytoplasmic amino- terminal tail of GAT-1, which is critical for its function. Two residues, a rginine 44 and tryptophan 47, are fully conserved within the transporter fa mily, and their deletion abolishes GABA transport in the HeLa cell expressi on system used. Tryptophan 47 can be replaced only by aromatic residues wit hout Toss of activity. Arginine 44 is essential for activity, Only when it is replaced by lysine, low activity levels (around 15% of those of the wild type) are observed. Using a reconstitution assay, we show that mutants in which this residue is replaced by lysine or histidine exhibit sodium- and c hloride-dependent GABA exchange similar to the wild type. This indicates th at these mutants are selectively impaired in the reorientation of the unloa ded transporter, a step in the translocation cycle by which net flux and ex change differ. The high degree of conservation in the consensus sequence RX XW suggests that this region may influence the reorientation step in relate d transporters as well.