ABSTRACT
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.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Membrane Transport Proteins , Organic Anion Transporters , Protein Structure, Secondary , gamma-Aminobutyric Acid/metabolism , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Carrier Proteins/biosynthesis , Cloning, Molecular , Conserved Sequence , DNA Primers , Escherichia coli , Escherichia coli Proteins , GABA Plasma Membrane Transport Proteins , HeLa Cells , Humans , Liposomes , Membrane Proteins/biosynthesis , Molecular Sequence Data , Mutagenesis, Site-Directed , Proteolipids/metabolism , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Deletion , TransfectionABSTRACT
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.
