Residues in Transmembrane Domains I and II Determine γ-Aminobutyric Acid Type A_A Receptor Subtype-Selective Antagonism by Furosemide

Abstract

GABA_A receptors in cerebellar granule cells are unique in expressing a subtype containing the α6 subunit. This receptor subtype has high affinity for GABA and produces a degree of tonic inhibition on cerebellar granule cells, modulating the firing of these cells via spillover of GABA from GABAergic synapses. This receptor subtype also has selective affinity for the diuretic furosemide over receptors containing other α-subunits. Furosemide exhibits approximately 100-fold selectivity for α6-containing receptors over α1-containing receptors. By making α1/α6 chimeras we have identified a transmembrane region (209–279) responsible for the high furosemide sensitivity of α6β3γ2s receptors. Within the α1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the β-subunit selectivity of furosemide to be due to asparagine 265 in the β2 and β3 transmembrane-domain II similar to that observed with potentiation by the anticonvulsant loreclezole. We also show that Ile in transmembrane-domain I accounts for the increased GABA sensitivity observed at α6β3γ2s compared with α1β3γ2s receptors, but did not affect direct activation by pentobarbital or potentiation by the benzodiazepine flunitrazepam. Location of these residues within transmembrane domains leads to speculation that they may be involved in the channel-gating mechanism conferring increased receptor activation by GABA, in addition to conferring furosemide sensitivity.