Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cellsrequires both GABA(B(1)) and GABA(B(2)) genes

Citation
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
Citations number
44
Categorie Soggetti
Neurosciences & Behavoir
Journal title
JOURNAL OF NEUROCHEMISTRY
ISSN journal
00223042 → ACNP
Volume
77
Issue
5
Year of publication
2001
Pages
1237 - 1247
Database
ISI
SICI code
0022-3042(200106)77:5<1237:CEOFGR>2.0.ZU;2-4
Abstract
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.