Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function.
Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA(A)) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [(3)H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [(3)H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC(50)=150nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA(A) receptors containing α6 and δ subunits. In contrast, [(3)H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC(50) ~80nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [(3)H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [(3)H]EBOB binding with EC(50) ~0.5μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1μM GABA in cerebellar granule cells, for 5α-THDOC with EC(50,1) ~16nM and EC(50,2) ~1.3μM. Differences in peak current enhancements in the absence minus presence of 0.1mM furosemide corresponding to α6ßδ GABA(A) receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA(A) receptor populations.
Department of Molecular Pharmacology, Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17, H-1525 Budapest, Hungary. maksay@chemres.hu
Gábor
G
Maksay
László
L
Fodor
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism*; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism*; D003900:Desoxycorticosterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005665:Furosemide / Q000494:pharmacology; D058785:GABA-A Receptor Agonists / Q000494:pharmacology; D055786:Gene Knockout Techniques; D008297:Male; D051379:Mice; D018377:Neurotransmitter Agents / Q000494:pharmacology; D011280:Pregnanolone / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D058785:GABA-A Receptor Agonists; C546172:Gabra6 protein, mouse; D018377:Neurotransmitter Agents; D011963:Receptors, GABA-A; C091518:1-(4-ethynylphenyl)-4-propyl-2,6,7-trioxabicyclo(2.2.2)octane; D003900:Desoxycorticosterone; C009413:tetrahydrodeoxycorticosterone; D005665:Furosemide; D011280:Pregnanolone