Cyclic AMP‐Dependent Protein Kinase Decreases γ‐Aminobutyric AcidA Receptor‐Mediated 36Q1− Uptake by Brain Microsacs

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Abstract
The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on γ-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36C1− uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP- dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36C1− uptake by ∼25%. Jn parallel experiments, PKA and [γ-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitatc the entire GABAA receptor complex, under nondenaturing conditions, using an anti-α1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-α1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain G ABAAreceptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.