Short external loops as potential substrate binding site of γ-aminobutyric acid transporters

While the γ-aminobutyric acid (GABA) transporter GAT1 exclusively transports GABA, GAT2, -3, and -4 also transport β-alanine. Cross-mutations in the external loops IV, V, and VI among the various GABA transporters were performed by site-directed mutagenesis. The affinity of GABA transport as well as inhibitor sensitivity of the modified transporters was analyzed. Kinetic analysis revealed that a cross-mutation in which loop IV of GAT1 was modified to resemble GAT4 resulted in increased affinity to GABA from K_m = 8.7 to 2.0 μM without changing the V_max. A cross-mutation in loop VI, which swapped the amino acid sequence of GAT2 for GAT1, decreased the affinity to GABA (K_m, 35 μM). These results suggest that loops IV and VI contribute to the binding affinity of GABA transporters. A substitution of three amino acids in loop V of GAT1 by the corresponding sequence of GAT3 resulted in β-alanine sensitivity of its GABA uptake activity. These three amino acids in loop V seem to participate in the β-alanine binding domain of GAT3. It is suggested that those three external loops (IV, V, and VI) form a pocket in which the substrate binds to the GABA transporters.