Stable expression of homomeric AMPA-selective glutamate receptors in BHK cells.
cDNAs encoding glutamate receptor glu1, glu2 (Q and R) or glu4 under control of a constitutively active metallothionine promoter, were transfected into baby hamster kidney cells. Following the addition of selection agent, transfectants expressing high levels of glutamate receptor as measured by [3H]alpha-amino-3-hydroxyl-5-methyl-isoxalazole-4-propionate (AMPA) binding, were selected for further studies. Using glutamate receptor antibodies, the receptor proteins were visualized in Western blotting as having a molecular weight of approximately 100 kDa. [3H]AMPA binding to the glutamate receptor expressing cell lines revealed that glu1, glu2 (Q), and glu4 receptors displayed a single site in Scatchard analysis with Kd values of 12, 15.7 and 21 nM, respectively. However, the Ca2+ impermeable variant of the glu2 receptor, glu2 (R) displayed a curvilinear Scatchard plot. Computer resolution suggested the presence of a high and low affinity state (KH = 2.9 nM; KL = 40.7 nM). The pharmacological profile of the [3H]AMPA binding to these recombinant receptors resembled the high affinity [3H]AMPA binding site in rat brain showing high affinity for glutamate, quisqualate, and medium affinity for 6-cyano-7-nitro-quinoxaline-2,3-dione, CNQX; 6,7-dinitro-quinoxaline-2,3-dione, DNQX; and 6-nitro-7-sulphanyl-benzo(f)quinoxaline-2,3,dione, NBQX. Kainate displayed low affinity and N-methyl-D-aspartate (NMDA), was inactive in inhibiting specific [3H]AMPA binding. These cell lines will prove to be important tools in the study of glutamate receptors.
Andersen
P H
PH
Department of Molecular and Cellular Biology I, Novo Nordisk Drug Discovery, Bagsvaerd, Denmark. pha@novo.dk
Tygesen
C K
CK
Rasmussen
J S
JS
Søegaard-Nielsen
L
L
Hansen
A
A
Hansen
K
K
Kiemer
A
A
Stidsen
C E
CE
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D007668:Kidney / Q000378:metabolism; D008024:Ligands; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D051381:Rats; D018091:Receptors, AMPA / Q000187:drug effects*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism*
D008024:Ligands; D011506:Proteins; D018091:Receptors, AMPA; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D004247:DNA