Ecophysiology of neuronal metabolism in transiently oxygen-depleted environments: evidence that GABA is accumulated pre-synaptically in the cerebellum.
Biochemical, molecular, and electrophysiological studies suggest that phosphorylation of beta subunits of the GABAA receptor (GaR) by exogenous protein kinase A inactivates the receptor channels. We have developed a method which for the first time allows the study of GaR phosphorylation in brain tissues by endogenous PKA. Desalted homogenates or crude synaptic membranes from mouse cerebral cortex were incubated with [gamma-32P]ATP and 8-Br-cAMP or chlorophenylthio-cAMP. Extracts from these incubations were immunoprecipitated by polyclonal antibodies against native GaR and analyzed by SDS-gel electrophoresis and autoradiography. In both homogenates and membranes, cAMP-dependent incorporation of 32P was observed for a 57-kDa peptide, and to a lesser extent 51- to 53-kDa peptides. Phosphorylation of affinity-purified GaR by the catalytic subunit of PKA also produced a major 57-kDa phosphopeptide and a minor 51-kDa phosphopeptide. Limited digestion by S. aureus V-8 protease of the 57-kDa phosphopeptide from the desalted homogenates or from purified receptors produced a major 32P-labeled fragment of 11 kDa, suggesting that the phosphorylation site is similar to that shown previously to reduce GaR function. The phosphorylation of GaRs in homogenates was time dependent and blocked by H-89 or protein kinase inhibitor 5-24, specific inhibitors of protein kinase A. Prolonged incubations resulted in dephosphorylation of the 57-kDa phosphoprotein by a microcystin-LR sensitive phosphatase. In cortical homogenates the level of cAMP-dependent phosphorylation of the 57-kDa GaR peptide was more than 5 times that obtained with washed synaptic membranes. However, assays of PKA using the heptamer kemptide as substrate showed that the specific activity in the particulate fraction was 57% that of the homogenate. This suggests that GaRs on synaptic membranes are preferentially phosphorylated by a cytoplasmic form of protein kinase A. By comparing the [3H]flunitrazepam-photolabeled 53-kDa GaR subunit with the 51-57 kDa [32P]peptides from cortical homogenates, the molar ratio of [32P]/[3H] was estimated at 0.43, suggesting that a substantial fraction of the GaR pool is phosphorylated under these conditions.