TYROSINE-140 OF THE GAMMA-AMINOBUTYRIC-ACID TRANSPORTER GAT-1 PLAYS ACRITICAL ROLE IN NEUROTRANSMITTER RECOGNITION

The gamma-aminobutyric acid (GABA) transporter GAT-1 is located in ner ve terminals and catalyzes the electrogenic reuptake of the neurotrans mitter with two sodium ions and one chloride, We now identify a single tyrosine residue that is critical for GABA recognition and transport, It is completely conserved throughout the superfamily, and even subst itution to the other aromatic amino acids, phenylalanine (Y140F) and t ryptophan (Y140W), results in completely inactive transporters, Electr ophysiological characterization reveals that both mutant transporters exhibit the sodium-dependent transient currents associated with sodium binding as well as the chloride-dependent lithium leak currents chara cteristic of GAT-1. On the other hand, in both mutants GABA is neither able to induce a steady-state transport current nor to block their tr ansient currents. The nontransportable analog SKF 100330A potently inh ibits the sodium-dependent transient in the wild type GAT-1 but not in the Y140W transporter. It partly blocks the transient of Y140F. Thus, although sodium and chloride binding are unimpaired in the tyrosine m utants, they have a specific defect in the binding of GABA. The total conservation of the residue throughout the family suggests that tyrosi ne 140 may be involved in the liganding of the amino group, the moiety common to all of the neurotransmitters.