SHORT EXTERNAL LOOPS AS POTENTIAL SUBSTRATE-BINDING SITE OF GAMMA-AMINOBUTYRIC-ACID TRANSPORTERS

While the gamma-aminobutyric acid (GABA) transporter GAT1 exclusively transports GABA, GAT2, -3, and 4 also transport beta-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 mu M without changing the V-max. A cross mutation in loop VI, which swapped the amino acid sequence of GATE for GAT1, decreased the affinity to GABA (K-m, 35 mu M). These results suggest that loops TV 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 beta-alanine sensitivity of its GABA uptake activity, These three amino acids in loop V seem to participate in the beta-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.