Stable expression of homomeric AMPA-selective glutamate receptors in BHK cells

• Published in: European journal of pharmacology Vol. 311; no. 1; pp. 95 - 100
• Main Authors: Andersen, Peter H., Tygesen, Charlotte K., Rasmussen, Jesper S., Søegaard-Nielsen, Lars, Hansen, Annette, Hansen, Kate, Kiemer, Anita, Stidsen, Carsten E.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 05-09-1996; Elsevier
• Subjects: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid - metabolism; Aminoacid receptors (glycine, glutamate, gaba); AMPA (α-amino-3-hydroxy-5-methyl-isoxalazole-4-propionate); Animals; BHK cell; Biological and medical sciences; Blotting, Western; cDNA; Cell Line; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Cricetinae; DNA - biosynthesis; Fundamental and applied biological sciences. Psychology; Glutamate receptor; Kidney - metabolism; Ligands; Molecular and cellular biology; Molecular Weight; Polymerase Chain Reaction; Proteins - metabolism; Rats; Receptors, AMPA - drug effects; Subunit; Glutamate receptor; cDNA; Subunit; AMPA (α-amino-3-hydroxy-5-methyl-isoxalazole-4-propionate); BHK cell; Cell culture; Ligand; Rodentia; Gene expression; In vitro; Kidney; Genetic transfer; Vertebrata; Biological fixation; AMPA receptor; Mammalia; Complementary DNA; Genetics; Molecular biology; Molecular cloning; Hamster
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/0014-2999(96)00399-8

cDNAs encoding glutamate receptor glu
1, glu
2 (Q and R) or glu
4 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]α-amino-3-hydroxy-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 glu
1, glu
2 (Q), and glu
4 receptors displayed a single site in Scatchard analysis with
K
d
values of 12, 15.7 and 21 nM, respectively. However, the Ca
2+ impermeable variant of the glu
2 receptor, glu
2 (R) displayed a curvilinear Scatchard plot. Computer resolution suggested the presence of a high and low affinity state (
K
H> = 2.9 nM;
K
L = 40.7 nM). The pharmacological rofile 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.