title: Residues in transmembrane domains I and II determine gamma-aminobutyric acid type AA receptor subtype-selective antagonism by furosemide.
abstract: GABAA receptors in cerebellar granule cells are unique in expressing a subtype containing the alpha6 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 alpha-subunits. Furosemide exhibits approximately 100-fold selectivity for alpha6-containing receptors over alpha1-containing receptors. By making alpha1/alpha6 chimeras we have identified a transmembrane region (209-279) responsible for the high furosemide sensitivity of alpha6beta3gamma2s receptors. Within the alpha1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the beta-subunit selectivity of furosemide to be due to asparagine 265 in the beta2 and beta3 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 alpha6beta3gamma2s compared with alpha1beta3gamma2s 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.
authors.affiliation: Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, United Kingdom.
authors.forename: S A
authors.initials: SA
authors.lastname: Thompson
authors.forename: S A
authors.initials: SA
authors.lastname: Arden
authors.forename: G
authors.initials: G
authors.lastname: Marshall
authors.forename: P B
authors.initials: PB
authors.lastname: Wingrove
authors.forename: P J
authors.initials: PJ
authors.lastname: Whiting
authors.forename: K A
authors.initials: KA
authors.lastname: Wafford
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004232:Diuretics / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005665:Furosemide / Q000494:pharmacology*; D058787:GABA-A Receptor Antagonists; D006801:Humans; D007532:Isoleucine / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes; D017354:Point Mutation; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
chemical_list: D004232:Diuretics; D058787:GABA-A Receptor Antagonists; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D005665:Furosemide