Only one of the charged amino acids located in the transmembrane alpha-helices of the gamma-aminobutyric acid transporter (subtype A) is essential for its activity.
The gamma-aminobutyric acid (GABA) transporter (subtype A) is located in nerve terminals and catalyses coupled electrogenic uptake of the neurotransmitter with two or three sodium and one chloride ions. It contains 599 amino acids and 12 putative membrane spanning alpha-helices and is the first described member of a neurotransmitter transporter superfamily. The membrane domain contains 5 charged amino acids which are basically conserved. Using site-directed mutagenesis, we show that only one of them, arginine 69, is absolutely essential for activity. It is located in a highly conserved region encompassing parts of helices 1 and 2. The three other positively charged amino acids and the only negative charged one, glutamate 467, are not critical. These results suggest that the translocation pathway of the sodium ions through the membrane does not involve charged amino acid residues and underline the importance of the highly conserved stretch between amino acids 66 and 86.
Pantanowitz
S
S
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Bendahan
A
A
Kanner
B I
BI
D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D027361:Organic Anion Transporters; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D027361:Organic Anion Transporters; D011994:Recombinant Proteins; C513601:gabP protein, E coli; D005680:gamma-Aminobutyric Acid