Regional gamma-aminobutyric acid sensitivity of t-butylbicyclophosphoro[35S]thionate binding depends on gamma-aminobutyric acidA receptor alpha subunit.

gamma-Aminobutyric acid (GABA) modulates the convulsant binding site on GABAA receptors labeled by t-butylbicyclophosphoro[35S] thionate ([35S]TBPS). The modulation varies between different brain regions, reflecting the molecular heterogeneity of the GABAA receptors. In rat brain cryostat sections, the main sensitivity difference to GABA between brain regions was observed within the cerebellum. [35S]TBPS binding in the granule cell layer was more sensitive to GABA than was that in the molecular layer and was detected only after blockade of the GABA agonist sites by the specific GABAA antagonists SR 95531, RU 5135, and bicuculline. This indicates that the [35S]TBPS binding sites in cerebellar granule cells were blocked by endogenous GABA. In contrast, the internal rim of the granule cell layer had a small amount of binding that was largely insensitive to 50 microM GABA. The molecular basis for the sensitivity difference could be traced to the alpha subunits of the GABAA receptor. Expression in human embryonic kidney 293 cells of alpha 6 beta 2 gamma 2 receptors produced [35S] TBPS binding sites that were about 10-fold more sensitive to inhibition by GABA than were those inherent to alpha 1 beta 2 gamma 2 receptors. Coexpression of alpha 6 and beta 2 subunits produced [35S]TBPS binding sites that were largely insensitive to GABA inhibition, resembling in their pharmacological profile the sites in the internal granule cell layer. Furthermore, the differences between alpha 6 beta 2 and alpha 6 beta 2 gamma 2 receptors stress the importance of the gamma 2 subunit for the proper pharmacological fingerprint of the rest of the granule cell layer. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one affected the binding in both alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptors, but inhibition was greater in alpha 6-containing than in alpha 1-containing receptors, suggesting differential coupling of both GABA and neurosteroid sites with the convulsant site. These data might serve as a platform for additional studies to assess the amino acid residues in the two alpha subunits that are critically involved in the allosteric interactions between the GABAA agonist/antagonist or neurosteroid domains and the convulsant site.