ABSTRACT
We have recently identified a novel class of allosteric modulators of GABA(A) receptors, the ROD compounds that are structurally related to bicuculline. Here, the relationship of their site of action relative to other known modulatory sites of this receptor was investigated. Two types of ROD compounds, R1 (ROD164A, ROD185) and R2 (ROD222 and ROD259) could be differentiated. R1 compounds competitively inhibited binding of benzodiazepines in alpha1beta2gamma2 receptors, and their functional effects were partially inhibited by the benzodiazepine antagonist Ro15-1788 in a noncompetitive manner. The enhancement by an R1 compound was not additive with that by diazepam. R2 compounds in contrast failed to inhibit binding of benzodiazepines; the R2 compounds' functional effects were not inhibited by the benzodiazepine antagonist. The enhancement by an R2 compound was additive with that by diazepam. In contrast to benzodiazepines, both R1 and R2 type compounds were still able to enhance alpha1beta2 receptors. ROD164A in alpha1beta2gamma2 receptors was found to be partially antagonized by Ro15-1788 in a noncompetitive way. ROD178B did not affect gamma-aminobutyric acid induced currents, but was able to inhibit both enhancement by R1 and R2 type compounds as well as enhancement by diazepam. R1 and R2 type compounds as well as diazepam enhanced pentobarbital-induced currents in a Ro15-1788-sensitive way. We conclude that R1 type compounds act at the benzodiazepine binding site and additionally at a different R1 site, and that the R1, but not the R2 site is allosterically coupled to the benzodiazepine binding site. ROD178B is a competitive antagonist at the R1 site in that it shows allosteric interaction with the benzodiazepine binding site and displacement of benzodiazepines, and a negative allosteric modulator at the R2 site.
Subject(s)
Alkaloids/pharmacology , Benzodiazepines/pharmacology , Receptors, GABA-A/metabolism , Allosteric Regulation , Animals , Bicuculline/pharmacology , Binding Sites , Cells, Cultured , GABA Modulators/pharmacology , Humans , Pentobarbital/pharmacology , Rats , Receptor Cross-TalkABSTRACT
Analogues of bicuculline devoid of the benzo ring fused to the lactone moiety were prepared by reacting 2-(tert-butyl-dimethylsiloxy)furans with 3,4-dihydroisoquinolinium salts. Some of these compounds (e.g., ROD185, 8) acted as modulators of the GABAA receptor, displacing ligands of the benzodiazepine binding site. They also strongly stimulated GABA currents mediated by recombinant GABA(A) receptors expressed in Xenopus oocytes.
Subject(s)
Bicuculline/pharmacology , GABA Antagonists/pharmacology , Receptors, GABA-A/drug effects , Allosteric Regulation , Ligands , Receptors, GABA-A/metabolismSubject(s)
Furans/chemical synthesis , GABA Modulators/chemical synthesis , Isoquinolines/chemical synthesis , Receptors, GABA-A/drug effects , Tetrahydroisoquinolines , Allosteric Regulation , Animals , Brain/metabolism , Electrophysiology , Furans/chemistry , Furans/pharmacology , GABA Modulators/chemistry , GABA Modulators/pharmacology , In Vitro Techniques , Isoquinolines/chemistry , Isoquinolines/pharmacology , Molecular Conformation , Oocytes/metabolism , Oocytes/physiology , Radioligand Assay , Rats , Receptors, GABA-A/genetics , Receptors, GABA-A/physiology , Stereoisomerism , Structure-Activity Relationship , XenopusABSTRACT
(+)-ROD188 was synthesized in the search for novel ligands of the GABA binding site. It shares some structural similarity with bicuculline. (+)-ROD188 failed to displace [(3)H]-muscimol in binding studies and failed to induce channel opening in recombinant rat alpha1beta2gamma2 GABA(A) receptors functionally expressed in Xenopus oocytes. (+)-ROD188 allosterically stimulated GABA induced currents. Displacement of [(3)H]-Ro15-1788 indicated a low affinity action at the benzodiazepine binding site. In functional studies, stimulation by (+)-ROD188 was little sensitive to the presence of 1 microM of the benzodiazepine antagonist Ro 15-1788, and (+)-ROD188 also stimulated currents mediated by alpha1beta2, indicating a major mechanism of action different from that of benzodiazepines. Allosteric stimulation by (+)-ROD188 was similar in alpha1beta2N265S as in unmutated alpha1beta2, while that by loreclezole was strongly reduced. (+)-ROD188 also strongly stimulated currents elicited by either pentobarbital or 5alpha-pregnan-3alpha-ol-20-one (3alpha-OH-DHP), in line with a mode of action different from that of barbiturates or neurosteroids as channel agonists. Stimulation by (+)-ROD188 was largest in alpha6beta2gamma2 (alpha6beta2gamma2>>alpha1beta2gamma2=alpha5beta2gamma2++ +>alpha2beta2ga mma2= alpha3beta2gamma2), indicating a unique subunit isoform specificity. Miniature inhibitory postsynaptic currents (mIPSC) in cultures of rat hippocampal neurons, caused by spontaneous release of GABA showed a prolonged decay time in the presence of 30 microM (+)-ROD188, indicating an enhanced synaptic inhibitory transmission.
