Natural point mutation reveals a behavioural correlate for a cerebellar GABAA receptor

THE selectively outbred alcohol-non-tolerant (ANT) rat line1 is highly susceptible to impairment of postural reflexes by benzodiazepine agonists2 such as diazepam. ANT cerebella are generally devoid3 of diazepam-insensitive high-affinity binding of the benzodiazepine [3H]Ro 15-4513 (refs 4, 5), whereas in non-selected strains such binding marks a granule-cell-specific GABAA (γ-aminobutyric acid) receptor containing the α6 subunit5,6. A critical determinant for diazepam insensitivity of this ‘wild-type’ cerebeilar GABAA receptor is an arginine residue7 in α6 position 100, where other a subunits carry a histidine8. Here we report that the ct6 gene of ANT rats is expressed at wild-type levels but carries a point mutation generating an arginine-to-glutamine substitution at position 100. In consequence, α6(Q 100)β2γ2 receptors show diazepam-mediated potentiation of GABA-activated currents and diazepam-sensitive binding of [3H]Rol5-4513. Our results suggest that cerebeilar motor control may be a distinct behavioural correlate of the a6-subunit-containing GABAA receptor subtype

Inverse but not full benzodiazepine agonists modulate recombinant α6β2γ2 GABAA receptors in transfected human embryonic kidney cells

We compared the modulation of GABA (γ-aminobutyric acid)-activated currents by benzodiazepines in recombinant (GABAA receptors containing either one of two α subunits, α1 or α6. Lüddens et al. (Nature, 346 (1990) 648–651) have previously demonstrated that the α6 subunit is part of a cerebellar receptor subtype which selectively binds Ro15–4513, an antagonist of alcohol-induced motor ataxia. Here we report that the imidazobenzodiazepine Ro15–4513 (ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-(1,5-a) (1,4)benzodiazepine-3-carboxylate) reduced GABA-activated currents in recombinant α6β2γ2 and α1β2γ2 receptors, thus acting consistently as an inverse agonist. Moreover, another well characterized negative modulator, DMCM (methyl-4-ethyl-6,7-dimethoxy-β-carboline-3-carboxylate), also reduces GABA activated-currents in both receptors. In contrast, flunitrazepam (FNZM), a benzodiazepine agonist, increases GABA-activated currents in α1β2γ2 receptors, but not in α6β2γ2 receptors. This study lends further support to the hypothesis that the binding sites of full and partial inverse agonists are different

Current potentiation by diazepam but not GABA sensitivity is determined by a single histidine residue

The GABAA/benzodiazepine receptor is the principal inhibitory neurotransmitter receptor in the mammalian brain and is assembled from sequence-related subunits, such as alpha 1 beta 2 gamma 2. In contrast to alpha 1 beta 2 gamma 2 receptors, alpha 6 beta 2 gamma 2 receptors fail to exhibit high-affinity binding of allosteric positive modulators of GABA-activated chloride currents. The critical determinant responsible for this difference in ligand binding was previously traced to a position in the extracellular domain of the two alpha subunits (alpha 1 His100 and alpha 6 Arg 101). We now show by patch clamp analysis that this amino acid exchange also determines the diazepam potentiation. Thus, alpha 1(Arg101)beta 2 gamma 2 receptors do not, but alpha 6(His100)beta 2 gamma 2 receptors do exhibit diazepam potentiation. However, the same extracellular determinant is not responsible for the increased GABA sensitivity of alpha 6 beta 2 gamma 2 receptors relative to alpha 1 beta 2 gamma 2 receptors as revealed by electrophysiological analysis and by differential GABA sensitivity of [35S]TBPS binding

Current potentiation by diazepam but not GABA sensitivity is determined by a single histidine residue

AB THE GABAA/benzodiazepine receptor is the principal inhibitory neurotransmitter receptor in the mammalian brain and is assembled from sequence-related subunits, such as [alpha]1[beta]2[gamma]2* In contrast to [alpha]1[beta]2[gamma]2 receptors, [alpha]6[beta]2[gamma]2 receptors fail to exhibit high-affinity binding of allosteric positive modulators of GABA-activated chloride currents. The critical determinant responsible for this difference in ligand binding was previously traced to a position in the extracellular domain of the two [alpha] subunits ([alpha]1His100 and [alpha]6Arg101). We now show by patch clamp analysis that this amino acid exchange also determines the diazepam potentiation. Thus, [alpha]1(Arg101)[beta]2[gamma]2 receptors do not, but [alpha]6(His100)[beta]2[gamma]2 receptors do exhibit diazepam potentiation. However, the same extracellular determinant is not responsible for the increased GABA sensitivity of [alpha]6[beta]2[gamma]2 receptors relative to [alpha]1[beta]2[gamma]2 receptors as revealed by electrophysiological analysis and by differential GABA sensitivity of [35S]TBPS binding. (C) Lippincott-Raven Publishers

Natural point mutation reveals a behavioural correlate for a cerebellar GABAA receptor

THE selectively outbred alcohol-non-tolerant (ANT) rat line1 is highly susceptible to impairment of postural reflexes by benzodiazepine agonists2 such as diazepam. ANT cerebella are generally devoid3 of diazepam-insensitive high-affinity binding of the benzodiazepine [3H]Ro 15-4513 (refs 4, 5), whereas in non-selected strains such binding marks a granule-cell-specific GABAA (γ-aminobutyric acid) receptor containing the α6 subunit5,6. A critical determinant for diazepam insensitivity of this ‘wild-type’ cerebeilar GABAA receptor is an arginine residue7 in α6 position 100, where other a subunits carry a histidine8. Here we report that the ct6 gene of ANT rats is expressed at wild-type levels but carries a point mutation generating an arginine-to-glutamine substitution at position 100. In consequence, α6(Q 100)β2γ2 receptors show diazepam-mediated potentiation of GABA-activated currents and diazepam-sensitive binding of [3H]Rol5-4513. Our results suggest that cerebeilar motor control may be a distinct behavioural correlate of the a6-subunit-containing GABAA receptor subtype

GABAA receptor subtypes: current potentiation by diazepam but not GABA sensitivity is determined by single histidine residue

AB THE GABAA/benzodiazepine receptor is the principal inhibitory neurotransmitter receptor in the mammalian brain and is assembled from sequence-related subunits, such as [alpha]1[beta]2[gamma]2* In contrast to [alpha]1[beta]2[gamma]2 receptors, [alpha]6[beta]2[gamma]2 receptors fail to exhibit high-affinity binding of allosteric positive modulators of GABA-activated chloride currents. The critical determinant responsible for this difference in ligand binding was previously traced to a position in the extracellular domain of the two [alpha] subunits ([alpha]1His100 and [alpha]6Arg101). We now show by patch clamp analysis that this amino acid exchange also determines the diazepam potentiation. Thus, [alpha]1(Arg101)[beta]2[gamma]2 receptors do not, but [alpha]6(His100)[beta]2[gamma]2 receptors do exhibit diazepam potentiation. However, the same extracellular determinant is not responsible for the increased GABA sensitivity of [alpha]6[beta]2[gamma]2 receptors relative to [alpha]1[beta]2[gamma]2 receptors as revealed by electrophysiological analysis and by differential GABA sensitivity of [35S]TBPS binding. (C) Lippincott-Raven Publishers