Bm. Chronwall et al., Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cellsrequires both GABA(B(1)) and GABA(B(2)) genes, J NEUROCHEM, 77(5), 2001, pp. 1237-1247
Studies of gamma -aminobutyric acid (GABA)B receptor function in heterologo
us cell systems have suggested that expression of two distinct seven transm
embrane G-protein coupled receptor subunits is necessary for receptor activ
ation and signal transduction. Some results suggest that both receptor prot
eins must be inserted into the plasma membrane to create heterodimers; howe
ver, it is possible that subunit monomers or homodimers are functional in c
ells which constitutively express GABA(B) receptors. A new pituitary interm
ediate lobe melanotrope cell clone (mIL tsA58) has been isolated which cons
titutively expresses GABA(B), D-2 and corticotrophin releasing factor recep
tors, Here, we report on characterization of the GABA(B) receptors. Solutio
n hybridization-nuclease protection assays reveal the presence of GABA(B(1)
), and GABAB(B(2)) transcripts. Western blots show GABA(B(1a)), and one of
two GABA(B(2)) proteins. Addition of the GABA((B)) agonist baclofen to cult
ured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca2+ channels, a
s measured by agonist-induced inhibition of the K+-depolarization-stimulate
d increase in Ca2+ influx. CGP55845, a GABAs antagonist, blocks the respons
e to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with s
elective antisense oligodeoxynucleotides reduced GABA(B) protein levels and
completely abolished the GABA(B) receptor response in the mit cells. Taken
together, these results indicate that functionally active GABA(B) receptor
s in mit cells require the constitutive expression of both GABA(B) genes. T
his is a physiologic validation of results from recombinant overexpression
in naive cells and shows that the mil cell line is a useful model for study
ing GABA(B) receptor expression, regulation and function.