Constitutive expression of functional GABABreceptors in mIL‐tsA58 cells requires both GABAB(1)and GABAB(2)genes

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Abstract
Studies of γ-aminobutyric acid (GABA)B receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABAB receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABAB, D2 and corticotrophin releasing factor receptors. Here, we report on characterization of the GABAB receptors. Solution hybridization-nuclease protection assays reveal the presence of GABAB(1) and GABAB(2) transcripts. Western blots show GABAB(1a) and one of two GABAB(2) proteins. Addition of the GABAB agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca2+ channels, as measured by agonist-induced inhibition of the K+-depolarization-stimulated increase in Ca2+ influx. CGP55845, a GABAB antagonist, blocks the response to baclofen. Knockdown of either GABAB(1) or GABAB(2) subunits with selective antisense oligodeoxynucleotides reduced GABAB protein levels and completely abolished the GABAB receptor response in the mIL cells. Taken together, these results indicate that functionally active GABAB receptors in mIL cells require the constitutive expression of both GABAB genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABAB receptor expression, regulation and function.

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