Pharmacological characterisation of a cell line expressing GABA B1b and GABA B2 receptor subunits.
The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.
Hirst
Warren D
WD
Neurology and GI Centre of Excellence for Drug Discovery, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. warren.d.hirst@gsk.com
Babbs
Adam J
AJ
Green
Andrew
A
Minton
Jayne A L
JA
Shaw
Tracy E
TE
Wise
Alan
A
Rice
Simon Q
SQ
Pangalos
Menelas N
MN
Price
Gary W
GW
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D011869:Radioligand Assay; D018079:Receptors, GABA / Q000187:drug effects / Q000378:metabolism*; D018080:Receptors, GABA-B / Q000187:drug effects / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D018079:Receptors, GABA; D018080:Receptors, GABA-B; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP