Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1.
We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamate-101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.
Keshet
G I
GI
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Bendahan
A
A
Su
H
H
Mager
S
S
Lester
H A
HA
Kanner
B I
BI
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000502:physiology; D004553:Electric Conductivity; D050485:GABA Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D011487:Protein Conformation; D012964:Sodium / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D012964:Sodium