Mutation of arginine 44 of GAT-1, a (Na(+) + Cl(-))-coupled gamma-aminobutyric acid transporter from rat brain, impairs net flux but not exchange.
The gamma-aminobutyric acid (GABA) transporter GAT-1 is a prototype of a large family of neurotransmitter transporters that includes those of dopamine and serotonin. GAT-1 maintains low synaptic concentrations of neurotransmitter by coupling GABA uptake to the fluxes of sodium and chloride. Here we identify a stretch of four amino acid residues predicted to lie in the juxtamembrane region prior to transmembrane domain 1 in the cytoplasmic amino-terminal tail of GAT-1, which is critical for its function. Two residues, arginine 44 and tryptophan 47, are fully conserved within the transporter family, and their deletion abolishes GABA transport in the HeLa cell expression system used. Tryptophan 47 can be replaced only by aromatic residues without loss of activity. Arginine 44 is essential for activity. Only when it is replaced by lysine, low activity levels (around 15% of those of the wild type) are observed. Using a reconstitution assay, we show that mutants in which this residue is replaced by lysine or histidine exhibit sodium- and chloride-dependent GABA exchange similar to the wild type. This indicates that these mutants are selectively impaired in the reorientation of the unloaded transporter, a step in the translocation cycle by which net flux and exchange differ. The high degree of conservation in the consensus sequence RXXW suggests that this region may influence the reorientation step in related transporters as well.
Bennett
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Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem, Israel 91120.
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D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D051381:Rats; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; D014364:Tryptophan; D001120:Arginine