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Cyclic AMP-dependent protein kinase decreases gamma-aminobutyric acidA receptor-mediated 36Cl- uptake by brain
N J Leidenheimer1, T K Machu, S Endo
1Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Abstract:
The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- 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 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-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-alpha 1 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 GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.
Insights
Cyclic AMP (cAMP)-dependent protein kinase (PKA) inhibits gamma-aminobutyric acidA (GABAA) receptor function by increasing protein phosphorylation. This study shows PKA reduces chloride uptake mediated by GABAA receptors in mouse brain microsacs.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Gamma-aminobutyric acidA (GABAA) receptors are crucial inhibitory neurotransmitter receptors in the brain.
- Cyclic AMP (cAMP)-dependent protein kinase (PKA) is a key regulator of cellular signaling pathways.
- The precise role of PKA-mediated phosphorylation in regulating GABAA receptor function remains to be fully elucidated.
Purpose of the Study:
- To investigate the effect of cAMP-dependent protein phosphorylation on GABAA receptor function.
- To determine if PKA directly modulates GABAA receptor activity and phosphorylation status.
Main Methods:
- Utilized isolated mouse brain membrane vesicles (microsacs) for functional assays.
- Measured muscimol-stimulated 36Cl- uptake to assess GABAA receptor activity.
- Introduced catalytic subunit of PKA into microsacs to study phosphorylation effects.
- Employed immunoprecipitation with an anti-alpha 1 subunit antibody to identify associated proteins.
- Performed phosphopeptide mapping to analyze protein phosphorylation sites.
Main Results:
- PKA significantly inhibited muscimol-stimulated 36Cl- uptake by approximately 25%.
- PKA increased the phosphorylation of a 66-kDa polypeptide co-immunoprecipitated with the GABAA receptor.
- Phosphopeptide mapping confirmed the 66-kDa polypeptide's phosphorylation site is similar to the GABAA receptor.
Conclusions:
- The catalytic subunit of PKA inhibits the function of brain GABAA receptors.
- PKA-induced inhibition of GABAA receptor function is associated with increased protein phosphorylation.
- These findings highlight a novel regulatory mechanism for GABAA receptor activity.