Tyrosine 140 of the gamma-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition.
The gamma-aminobutyric acid (GABA) transporter GAT-1 is located in nerve terminals and catalyzes the electrogenic reuptake of the neurotransmitter 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 substitution to the other aromatic amino acids, phenylalanine (Y140F) and tryptophan (Y140W), results in completely inactive transporters. Electrophysiological 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 characteristic 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 transient currents. The nontransportable analog SKF 100330A potently inhibits 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 mutants, they have a specific defect in the binding of GABA. The total conservation of the residue throughout the family suggests that tyrosine 140 may be involved in the liganding of the amino group, the moiety common to all of the neurotransmitters.
Bismuth
Y
Y
Department of Biochemistry, Hadassah Medical School, the Hebrew University, Jerusalem 91120, Israel.
Kavanaugh
M P
MP
Kanner
B I
BI
D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology; D027361:Organic Anion Transporters; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; D014443:Tyrosine; D005680:gamma-Aminobutyric Acid; D012964:Sodium