RT Journal Article SR Electronic T1 Residues in Transmembrane Domains I and II Determine γ-Aminobutyric Acid Type AA Receptor Subtype-Selective Antagonism by Furosemide JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 993 OP 999 DO 10.1124/mol.55.6.993 VO 55 IS 6 A1 S.A. Thompson A1 S.A. Arden A1 G. Marshall A1 P.B. Wingrove A1 P.J. Whiting A1 K.A. Wafford YR 1999 UL http://molpharm.aspetjournals.org/content/55/6/993.abstract AB GABAA 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.