Identification of tryptophan residues critical for the function and targeting of the gamma-aminobutyric acid transporter (subtype A).

The gamma-aminobutyric acid transporter is localized in nerve terminals. It catalyzes coupled electrogenic translocation of the neurotransmitter with two or three sodium ions and one chloride ion. The transporter contains 599 amino acids and 12 putative membrane spanning alpha-helices. It is the first described member of a neurotransmitter transporter superfamily. Using site-directed mutagenesis we have investigated the role of all 10 tryptophan residues predicted to reside in these helices. All 10 have been changed to serine as well as to leucine residues. Expression of mutant cDNAs in which the tryptophans, located in positions 68, 222, and 230, are replaced by either of these two amino acids reveals that they are severely impaired in gamma-aminobutyric acid transport. Mutants in which a phenylalanine or a tyrosine residue is introduced, at either position 68 or 230, are active. On the other hand, at the 222 position replacement of the tryptophan by the aromatic amino acids results in inactive transport. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of mutant transporters indicates that their expression levels are similar to those of the wild type. Reconstitution experiments, aimed to reveal the activity of transporter molecules not apparent in the plasma membrane, indicate that the lack of activity of the W230S transporter in intact cells is by and large due to its inefficient targeting to the plasma membrane. Tryptophan residues 68 and 222 appear to be required for the intrinsic activity of the transporter. Based on several observations, including one that tryptophan residue 222 is conserved in all amino acid transporter members of the superfamily, but not in those transporting biogenic amines, we hypothesize that the pi electrons of this tryptophan could be involved in the binding of the amino group of these neurotransmitters.