Abstract
Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.
Highlights
␥-Aminobutyric acid (GABA)1 is the predominant inhibitory neurotransmitter in the central nervous system, and modulators of type A GABA (GABAA) receptors are used to treat anxiety, insomnia, muscle spasms, and epilepsy
When combined with ␣4 and 3, ␦ subunits, compared with ␥2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. ␣43␦ responses were markedly modulated by steroids and anesthetics
We examined receptor pharmacology by pretreating cells with compounds known to be active at ␣33␥2 GABAA receptors and applying a half-maximal concentration of GABA. ␣33␥2 responses were blocked by the antagonists bicuculline and picrotoxin, potentiated by the benzodiazepine agonist zolpidem, and partially inhibited by the benzodiazepine inverse agonist
Summary
␥-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system, and modulators of type A GABA (GABAA) receptors are used to treat anxiety, insomnia, muscle spasms, and epilepsy. GABAA receptors are pentameric ligand-gated chloride channels, mediating rapid inhibitory synaptic neurotransmission, and are composed of different combinations of subunits from a family including ␣1–6, 1–4, ␥1–3, ␦, ⑀, , and 1–2 [1] Elevated levels of ␣4 subunits are implicated in an animal model of alcohol dependence [29] and in steroid-withdrawal models of premenstrual syndrome and postpartum or postmenopausal dysphoria, the increased anxiety and incidence of seizures (30 –34) The association of these pathologies with changes in ␣4 and ␦ subunit expression and the observation that ligands with high affinity for ␣4␥2 GABAA receptors are amethystic [35, 36] suggest that novel selective modulators of these GABAA receptors may, as well as leading to a better understanding of the properties and physiological roles of these subunits in the brain, have great therapeutic benefit. We describe the use of this novel fluorescence technique to characterize the pharmacological activation and modulation of GABAA receptors with the subunit combinations ␣33␥2, ␣43␥2, and ␣43␦
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