The number of amino acid residues in hydrophilic loops connecting transmembrane domains of the GABA transporter GAT-1 is critical for its function.
Transporter proteins consist of multiple transmembrane domains connected by hydrophillic loops. As the importance of these loops in transport processes is poorly understood, we have studied this question using the cDNA coding for GAT-1, a Na+/Cl(-)-coupled gamma-aminobutyric acid transporter from rat brain. Deletions of randomly picked non-conserved single amino acids in the loops connecting helices 7 and 8 or 8 and 9 result in inactive transport upon expression in HeLa cells. However, transporters where these amino acids are replaced with glycine retain significant activity. The expression level of the inactive mutant transporters was similar to that of the wild-type, but one of these, delta Val-348, appears to be defectively targetted to the plasma membrane. Our data are compatible with the idea that a minimal length of the loops is required, presumably to enable the transmembrane domains to interact optimally with each other.
Kanner
B I
BI
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Bendahan
A
A
Pantanowitz
S
S
Su
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D011510:Proteolipids; D011994:Recombinant Proteins; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; C513601:gabP protein, E coli; C020485:proteoliposomes; D005680:gamma-Aminobutyric Acid