Relationship of GABA and diazepam receptors in cerebellar Purkinje cells

Receptor studies are routinely carried out on crude heterogeneous membrane fractions. We have attempted to prepare a fraction containing a single cell type and analyze the properties of various receptors on this preparation. The preparation we developed consists of isolated Purkinje cell bodies prepared in greater than 90% purity. Using this preparation, GABA receptors and diazepam receptors were measured and their interaction was studied. An analysis of GABA receptor binding using either (³H)-GABA or (³H)-muscimol reveals two binding sites with K_D's of 1.4 and 26.7 nM respectively. These two sites are clearly present on Purkinje cells and do not represent two morphologically distinct receptors, such as presynaptic and postsynaptic receptors. Diazepam binding studies carried out on these cells reveals specific binding with a K_D of 2.6 nM. The specific density of GABA receptors is 0.6 pmol/mg for the higher affinity site and 1 pmol/mg for the lower, while the diazepam sites have 0.55 pmol/mg maximum binding. This suggests that the receptor densities of these compounds are nearly equivalent, with the combined GABA sites being about three times more prevalent than diazepam sites. GABA at concentrations as low as 3 μM nearly doubles the apparent diazepam binding.