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.
Maksay
Gábor
G
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
Fodor
László
L
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