CLONING OF THE HUMAN GLYCINE TRANSPORTER TYPE-1 - MOLECULAR AND PHARMACOLOGICAL CHARACTERIZATION OF NOVEL ISOFORM VARIANTS AND CHROMOSOMAL LOCALIZATION OF THE GENE IN THE HUMAN AND MOUSE GENOMES

We report the molecular cloning of a cDNA encoding a high affinity hum an glycine transporter. An open reading frame of 1914 nucleotides enco des a 638-amino acid protein that transports glycine in a Na+/Cl--depe ndent manner. In common with other Na+/Cl--dependent transporters, it possesses 12 putative transmembrane domains, according to its hydropat hicity profile. This protein is the human homologue of a glycine trans porter previously isolated from rat [glycine transporter type 1b (GlyT -1b)]. In addition to the human GlyT-1b, we also characterized a novel functional isoform produced by alternative splicing. This isoform, Gl yT-1c, which is distinct from GlyT-2 recently characterized in rat, co ntains an additional exon encoding 54 amino acids in the amino-termina l part of GlyT-1b and is mainly expressed in brain. These two isoforms are products of the same gene and are localized on human chromosome 1 p31.3, as well as on mouse chromosome 4, close to the locus for the sp ontaneous mouse neuromuscular mutation clasper. When expressed in COS- 7 cells, both the human GlyT-1b and GlyT-1c display a time- and dose-d ependent uptake of glycine, which is abolished when either Na+ or Cl- is substituted with other ions. For both GlyT-1b and GlyT-1c the affin ities for glycine are similar, with K-m values of 70-90 mu M, and this uptake is inhibited by sarcosine with similar potencies. In addition to the three transporter isoforms present in the human genome, i.e., G lyT-1a, GlyT-1b, and GlyT-1c, point-mutated variants, which appear to be totally devoid of glycine uptake activity when expressed in COS-7 c ells, were obtained by polymerase chain reaction amplification of mRNA from human substantia nigra. These variants point to regions of the g lycine transporter that might be important in the processing or transp ort function of this protein.