Selective antagonist for the cerebellar granule cell-specific gamma-aminobutyric acid type A receptor.

Numerous ligands affect inhibitory gamma-aminobutyric acid (GABA)A receptors, none of them showing strict receptor subtype specificity. We report here that a cerebellar GABAA receptor subtype can be uniquely modulated by furosemide but not by bumetanide, another Cl-/cation transport blocker. Furosemide specifically reversed the inhibition by GABA of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in the cerebellar granule cell layer, as detected by autoradiography of rat brain sections. With recombinant receptors expressed in Xenopus oocytes, furosemide antagonized potently (IC50, about 10 microM), rapidly, and reversibly GABA-evoked currents of cerebellar granule cell-specific alpha 6 beta 2 gamma 2 receptors but not alpha 1 beta 2 gamma 2 receptors (IC50, > 3 mM). Furosemide reversed GABA inhibition of [35S]TBPS binding and elevated basal [35S]TBPS binding only with alpha 6 beta 2 gamma 2 and alpha 6 beta 3 gamma 2 receptors and not with alpha 6 beta 1 gamma 2 or alpha 1 beta 1/2/3 gamma 2 receptors. It appeared to interact with the receptor complex via a novel recognition site that allosterically regulates the Cl- ionophore. Furosemide is the first subtype-selective GABAA receptor (alpha 6 beta 2/3 gamma 2) antagonist and should facilitate studies on cerebellar physiology. It might serve as a prototypic structure for the development of additional subtype-selective GABAA ligands.