CLONING, EXPRESSION AND PHARMACOLOGICAL CHARACTERIZATION OF A HUMAN GLUTAMATE-RECEPTOR - HGLUR4

A member of the ionotropic family of glutamate receptors, hGluR4, was isolated from a human cDNA library and characterized following express ion in mammalian cell lines. Human GluR4 possessed a 99% amino acid an d 92% nucleotide homology to that of its rat counterpart with sequence differences restricted to the carboxy and amino terminal regions of t he molecule. Transfection of simian kidney cells (COS-1) with an hGluR 4 expression plasmid resulted in the transient formation of a membrane protein that possessed high specific binding for lpha-amino-3-hydroxy -5-methylisoxazole-4-propionic acid ([H-3]AMPA) but not [H-3]kainate. Competition studies yielded a displacement profile of AMPA = quisquala te > glutamate > domoate > kainate much greater than N-methyl-D-aspart ate (NMDA) or dihydrokainate. Whole-cell, voltage-clamp recordings fro m a human embryonic kidney cell line (HEK 293) stably expressing hGluR 4 confirmed the presence of constitutively active, ligand-gated ion ch annels activated by AMPA, glutamate and kainate but not N-methyl-D-asp artate. Kainate-evoked currents were reversibly attenuated by 6-cyano- 7-nitro-quinoxaline-2,3-dione (CNQX) but not DL-2-amino-5-phosphonoval erate (DL-APS). Agonist-evoked currents exhibited inward rectification and ion substitution experiments indicated that hGluR4 receptor-linke d ion channels in their homomeric state are permeable to both Ca2+ and Na+ ions. In the same cell line antibody to rat GluR4 immunoprecipita ted a major protein band at similar to 108 kDa and a minor one at simi lar to 340 kDa. The immunoblot analysis of membranes chemically crossl inked with dithiobis(succinimidylpropionate) showed a broad band at 55 0-600 kDa suggesting that the GluR4 receptor forms a pentamer in situ. This is the first report of the cloning of hGluR4 receptor and its st able expression in a human cell line.